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Book of Abstracts
Albany 2011: The 17th Conversation
June 14-18 2011
Journal of Biomolecular Structure &
Dynamics
Volume 28, Issue # 6 June 2011
Schedule of 17th Conversation ......................................................................... ii-iv
Book of Abstracts: The 17th Conversation ....................................................975- 1164
Index to Authors ........................................................................................ 1165 - 1170
Registration form: The 17th Conversation ............................................... 1171 - 1172
Sponsored by:
University at Albany
Department of Chemistry
Department of Biology
Office of the Dean, Arts and Sciences
Vice President for Research
National Institutes of Health (pending)
JBSD
Adenine Press
Tuesday, June 14: You are arriving Today
1:00-11:00 pm
Mount your posters around LC-18 concourse
4:00-7:00
Dinner-Campus Center Cafe; you pay cash & buy
6:00-8:00 JBSD + Organizing Cmte Dinner, Sitar
8:30-12:00 am
Wine & Cheese Reception, Empire Commons, Wednesday, June 15
7:00-8:30 am
Breakfast, LC-18 Concourse
Thursday, June 16
6:15-9:15 am
Breakfast, Hospitality Suites, Empire Commons
8:00-9:55
8:00-8:05
8:05-8:25
8:25-8:45
8:45-9:05
9:05-9:25
9:25-9:35
9:35-9:55 9:55-10:05 Session 7: Prebiotic Evolution
Chair: Nobel Laureate Jack Szostak, Harvard
Remarks by the Chair
Edward Trifonov, Haifa, Israel
Uwe Meierhenrich, Univ. of Nice, France
Ernesto Di Mauro, Univ. of Rome, Italy
Chair: Jiri Sponer, IBP, Czech Republic
James Ferris, RPI
Christopher D. McFarland, Harvard
Nick Hud, Georgia Tech
Irena Mamajanov, Georgia Tech
10:05-11:00
Coffee and Poster Session III
11:00-12:30
11:00-11:20
11:20-11:30
11:30-11:50
11:50-12:00
12:00-12:10
12:10-12:30
Session 8: Protein Evolution
Chair: Anna Panchenko, NCBI
Igor Berezovsky, Univ. of Bergen, Norway
Kosuke Hashimoto, NCBI
William Duax, Univ. at Buffalo
Chair: Dan Fabris, Univ. at Albany
Ivanka Besseva, IBP, Czech Republic
Brandon Tolbert, Miami Univ. Oxford, OH
Loren Williams, Georgia Tech
8:00-8:10 am
Welcome by VPR Dias, and Chairs Paul Toscano &
Richard Zitomer
8:10-10:25
8:10-8:15
8:15-8:35
8:35-8:45
8:45-8:55
8:55-9:05
9:05-9:25
9:25-9:45
9:45-9:55 9:55-10:05
10:05-10:15
10:15-10:35
Session 1: Proteins-I: New in Dynamics & Folding
Chair: Harold Scheraga, Cornell Univ.
Remarks by the Chair.
Dave Thirumalai, UMD
Menahem Pirchi, Weizmann, Israel
Amrita Dasgupta, NCBS, India
Ghosh Dastdar, Bose Insti, India
Angel Garcia, RPI
Chair: Aditya Mittal, IIT Delhi, India
Ken Dill, Stony Brook
Sanjeev Singh, Alagappa Univ, India
Priya Banerjee, Univ. at Albany
Adeleh Divsalar, Tarbiat Moallem Univ., Iran
B. Jayaram, IIT, Delhi, India
10:35-11:25
Coffee and Poster Session I
12:30-1:40
11:25-12:30
11:25-11:30
11:30-11:50
11:50-12:00
12:00-12:10
12:10-12:30
Session 2. Proteins - II: New in Design & Detection
Chair: Neville Kallenbach, NYU
Irit Sagi, Weizmann, Israel
Hridoy Bairagya, NIT-Durgapur , India
Traaseth Nate, NYU,
David Cowburn, Albert Einstein
Lunch, Campus Center Cafeteria & Barbique Pit
(open 11 am- 2 pm) you pay cash and buy
1:40-2:30
1:40-2:00
2:00-2:20
2:20-2:30
Session 9: Artficial DNA
Chair: S.Wijmenga, Radboud Univ., The Netherlands
Peter Nielsen, Univ. of Copenhagen, Denmark
Maxim Frank Kamenetskii, BU
Marcus Wilhelmsson, Chalmers Univ., Sweden
12:30-1:40
Lunch, Campus Center Cafeteria & Barbique Pit
(open 11 am- 2 pm) you pay cash and buy
1:40-3:10
1:40-2:00
2:00-2:20
2:20-2:30
2:30-2:40
2:40-3:00
3:00-3:10
Session 3: Proteins-III: New Dynamics & Allostery
Chair: Miroslav Fojta, IBP, Czech Republic
Richard Bryce, Univ. of Manchester, UK
Amnon Horovitz, Weizmann, Israel
Stan George, Univ. of Cincinnati
Moitrayee Bhattacharyya, IISc, Bangalore, India
Ivet Bahar, Univ. of Pittsburgh
Sefica Banu Ozkan, ASU, Tempe, AZ
2:30-3:15
2:30-2:50
2:50-3:10
Session 10: What is New in Z-DNA
Chair: Wolfram Saenger, Frie Univ. Berlin, Germany
Alex Rich, MIT
Alpana Ray, Univ. of Missouri, Columbia MO
3:10-3:40
3:10-3:20
3:20-3:30
3:30-3:40
Session 11 (Mini): Telomeres and Quadruplexes
Chair: Nick Ulyanov, UCSF
Katie Castor, McGill Univ.
Saptarpani Ghosh, Saha Insti, Calcutta, India
Lim Kah Wai, Nanyang Univ., Singapore
3:40-4:25
Coffee & Poster Session IV
3:10-4:25
3:10-3:30
3:30-3:50
3:50-4:00
4:00-4:20
Session 4: Proteins-IV: Neurodegeneration
Chair: Volodya Uversky, IUPUI
Gary Daughdrill, Univ South Florida
David Eliezer, Cornell Univ
Sai P. Srinivasan, RPI
Akihiko Takashima, RIKEN, Japan
4:20-5:15
Coffee & Poster Session II
5:15-6:35
5:15-5:35
5:35-5:55
5:55-6:15
6:15-6:25
6:25-6:35
Session 5: DNA Nanotechnology
Chair:Ned Seeman, NYU
Ned Seeman, NYU
Kurt Vesterager Gothelf, Aarhus Univ
Friedrich Simmel, TU Munchen
Hari KK Subramaniam NYU
Graham Hamblin, McGill Univ.
4:25-6:20
4:25-4:30
4:30-4:50
4:50-5:10
5:10-5:20
5:20-5:40
5:40-6:00
6:00-6:20
Session 12: DNA Repair
Chair: Rick Cunningham, SUNY at Albany.
John A. Tainer, Scripts
Dmitry Zharkov, ICB, Novosibirsk, Russia
Sarah Delaney, Brown Univ.
Chair: Krystyna Zakrzewska, IBCP, France
Reuben S. Harris, Univ. of Minnesota, Minneapolis
V. Enrico Avvedimento, Univ. Federico II, Italy
Mike Fried, Univ. of Kentucky
6:20-7:40
Dinner, Campus Center Cafeteria (open 4:00-7:10
pm) you pay cash and buy
6:35-8:00
Dinner, Empire Commons, (open 5:30-7:45 pm)
8:00-9:10
8:00-8:05
8:05-9:05
Session 6: Nobel Laureate Evening Lecture
Chair & Introduction: Alex Rich MIT
Jack Szostak, Harvard
7:40-9:05
7:40-7:45
7:45-8:05
8:05-8:15
8:15-8:25
8:25-8:45
8:45-9:05
Session 13: Chromosomes
Chair: Wilma Olson, Rutgers
Remarks by the Chair,
Thomas Cremer, LMU, Germany
Ekaterina Khrameeva, IITP, Moscow, Russia
Khushhall Menaria, ANIT, Bhopal, India
Kazuhiro Maeshima, NIG, Mishima, Japan
Jonathan Widom, Northwestern Univ.
9:30- 12:00 am
Reception for Jack Szostak, Empire Commons
9:05- 12:00 am
Trifonov & Maxim host their Russian Party
in their apartments
-ii-
Friday, June 17
6:15-9:15 am
Saturday, June 18: You are going home today after lunch
7:15-10:15 am
8:00-9:50
8:00-8:20
8:20-8:40
8:40-9:00
9:00-9:10
9:10-9:30
9:30-9:50
9:50-10:00
Session 14: Replication Meets Transcription
Chair: Sergei Mirkin, Tufts Univ.
Sergei Mirkin, Tufts Univ.
Benedicte Michel, CNRS, Gif-sur-Yvette, France
Jorge B. Schvartzman, CIB, Madrid, Spain
Boris Belotserkovskii, Stanford
Sue Jinks-Robertson, Duke Univ.
Michael O'Donnel, Rockefeller Univ.
Yayan Zhou, Wesleyan
9:00-10:35
9:00-9:05
9:05-9:25
9:25-9:45
9:45-9:55
9:55-10:05
10:05-10:15
10:15-10:35
Session 18: DNA-Protein-I
Chair: Tom Tullius, BU
Christoph W. Müller, EMBL, Heidelberg, Germany
Koby Levy, Weizmann, Israel
Lydia-Ann Harris, Univ. at Buffalo
Steve Parker, NIH
Racca Joe, Case Wesrwern Reserve
Scot Wolfe, U Mass., Worcester
10:00-11:00
Coffee & Poster Session V
11:00-12:30
11:00-11:20
11:20-11:40
11:40-12:00
12:00-12:10
12:10-12:20
12:20-12:30
Session 15: Cis Regulatory Modules; DNA Stuff
Chair: Mikhail Gelfand, IITP, Moscow, Russia
Martha Bulyk, Harvard
Vsevolod Makeev, GosNIIGenetika, Moscow, Russia
Olga Ozoline, RAS, Pushchino, Russia
Ivan Kulakovsky, EIMB, Moscow, Russia
Alexander Lomzov, ICB, Novosibirsk, Russia
Alexander Ivanov, ICBCRC, Moscow, Russia
10:35-10:50
Coffee
10:50-11:55
10:50-10:55
10:55-11:15
11:15-11:25
11:25-11:35
11:35-11:55
Session 19: DNA-Protein-II
Chair: Richard Mann, Columbia
Remo Rohs, USC
Matt Slattery, Univ. of Chicago
Todd Riley, Columbia
Udo Heinemann, MDC, Berlin, Germany
12:30-1:40
Lunch, Campus Center Cafeteria & Barbique Pit (open 11 am- 2 pm) you pay cash and buy
1:40-3:40
1:40-1:45
1:45-2:05
2:05-2:15
2:15-2:25
2;30-2;50
2:50-3:00
3:00-3:20
3:20-3:30
3:30-3:35
3:35-3:55
Session 16: Molecular Simulation: Breakthroughs
Chair: Valeri Barsegov, Univ. of Mass, Lowell
Remarks by Chair
Klaus Schulten, UIUC
Ilya Kovalenko, Moscow Sate Univ.
Lilian Chong, Univ. of Pittsburgh
Ruxandra Dima, Univ. of Cincinnati
Peyel Das, IBM
Gianni De Fabritiis, Univ Pompeu Fabra, Spain
Rajib Mukherjee, Tulane Univ.
Chair: Tom Cheatham, Univ. of Utah
Remarks by Chair
David Beveridge, Wesleyan
11:55-12:45
11:55-12:00
12:00-12:20
12:20-12:40
Session 20: DNA-Protein-III
Chair: Barry Honing, Columbia
Victor Zhurkin, NIH
Zippi Shakked, Weizmann, Israel
12:40-12:45
Barry Honing Closes the Conversation
12:45-2:00
Farewell Lunch Empire Commons
Go Home After Lunch
3:55-5:00
Coffee & Poster Session VI
5:00-7:00
5:00-5:05
5:05-5:25
5:25-5:45
5:45-5:55
5:55-6:00
6:00-6:20
6:20-6:30
6:30-6:40
6:40-7:00
Session 17: Beveridge Celebrations
Chair: B. Jayaram, IIT Delhi, India
Mihaly Mezei, Mount Sinai
Ishita Mukerji. Wesleyan Univ.
Elizabeth Wheatly, Wesleyan
Chair: Manju Hingorani, Wesleyan
Matthew Young, Univ. of Michigan
Na Le Dang, Wesleyan
Tom Bishop, Tulane Univ.
David Beveridge
7:30-12 am
Big Feast, Campus Center Ball Room
-iii-
Albany 2011
The 17th Conversation
State University of New York
Albany NY USA
June 14-18 2011
Director
Prof. Dr. Ramaswamy H. Sarma
Chemistry Department
State University of New York
Albany NY 12222 USA
ph: 518-456-9362; fx: 518-452-4955
email: [email protected]
Hi Folks:
In behalf of the University at Albany, State University of New York, I have the great pleasure of
welcoming all of you to our uptown Albany campus.
Have a great time, enjoy your stay, above all let us have a memorable Conversation in biological
structure, dynamics, interactions and expression.
Sincerely yours
Senior Organizing Committee
Paul Agris. Univ. at Albay
David. L. Beveridge, Wesleyan
Tom Bishop, Tulane Univ.
Maxim Frank-Kamenetskii, Boston U.
Robert Jernigan. Iowa State Univ.
Thomas Cheatham, Utah
Dan Fabris, Univ. at Albany
Udo Heinemann, Berlin, Germany
Richard Lavery, IBCP, France
David Lilley Dundee, UK
Sergei Mirkin, Tufts
Dino Moras, Strasbourg, France
Bengt Norden, Nobel Cmte, Sweden
Wilma Olson, Rutgers
Alex Rich, MIT
Wolfram Saenger, Berlin, Germany
Ned Seeman, New York Univ.
Zippi Shakked , Weizmann, Israel
Jiri Sponer, Czech Republic
Ed Trifonov , Uni. of Haifa, Israel
Volodya Uversky, IUPUI
Sybrren Wijmenga, Univ. Nijmegen
Krystyna Zakrzewska, IBCP, France
Victor Zhurkin, NIH
Prof. Dr. Ramaswamy H. Sarma
Chemistry,
University at Albany -SUNY
Albany NY 12222
Ph: 518-456-9362; fx: 518-452-4955
Email: [email protected]
April 2 2011
Junior Organizing Committee
Elena Bichenkova, Univ. of Manchester, UK; Calvin Yu-Chian Chen, China Medical Univ., Taiwan;
Miroslav Fojta, Institute of Biophysics, Czech Republic; Yaakov (Koby) Levy, Weizmann Institute of
Science, Israel; Vsevolod J. Makeev, Genetika, Moscow, Russia; Aditya Mittal, IIT-Delhi, India; Anna
Panchenko, NCBI, NLM, NIH, USA; Remo Rohs, HHMI, Columbia Univ., USA; Dmitry O. Zharkov, SB RAS
Institute of Chemical Biology, Russia.
Dynamics, ISSN 0739-1102
Volume 28, Issue Number 6, (2011)
©Adenine Press (2011)
Book of Abstracts
Albany 2011:17th Conversation
Resolving Fibrinogen Nanomechanics Using Dynamic
Force Measurements In Vitro and In Silico
Mechanical functions of protein fibers are important in cytoskeletal support and
cell motility (1), cell adhesion and formation of extracellular matrix (2), and
blood clotting (3). Due to their complexity (103−105 amino acids) and large size
(~40−200 nm), experimental force measurements (4, 5) of their physical properties
yield results that are impossible to interpret without some input from the computerbased modeling. Fibrinogen, the precursor of fibrin, provides building blocks for
fibrin polymers, a scaffold of blood clots and thrombi. The mechanical properties
of fibrin(ogen), which control how clots and thrombi respond to external mechanical factors, are essential for hemostasis. Yet, the complexity of fibrin(ogen) structure makes it difficult to uncover the unfolding mechanism using dynamic force
measurements in vitro alone. We carried out combined experimental-theoretical
studies of the mechanical properties of fibrinogen, using AFM assays and Langevin
simulations on Graphics Processing Units (GPUs). A combination of the SelfOrganized Polymer (SOP) model (6) and simulations on GPUs (7) makes it possible
to characterize the fibrinogen nanomechanics in the experimental 0.1-1s timescale.
The mechanical unraveling of fibrinogen is determined by the microscopic transitions that couple reversible extension-contraction of the coiled-coils and unfolding
of the terminal γ C-domains. The coiled-coils play a role of the biomolecular storage of mechanical energy to amortize an external perturbation and to transmit and
distribute tension among the γ C-domains. Unfolding of the γ C-domains, stabilized
by domain interactions with the βC-domains, result in three force signals, which
are characterized by the average force of ~100 pN and peak-to-peak distance of
~30 nm. The results obtained provide important quantitative characteristics of the
fibrinogen nanomechanics necessary to understand fibrin viscoelasticity at the fiber
and whole clot levels.
Artem Zhmurov1,2
Andre Brown3
Rustem I. Litvinov3
Ruxandra I. Dima4
John W. Weisel3
Valeri Barsegov1,2*
1Department
1
of Chemistry, University of
Massachusetts, Lowell, MA 01854
2Moscow
Institute of Physics and
Technology, Moscow, Russia 141700
3Department
of Cell and
Developmental Biology, University of
Pennsylvania School of Medicine
Philadelphia, PA 19104
4Department
Cincinnati, Cincinnati, OH 45221
*[email protected]
975
976
This work has been supported by the American Heart Association grant
(09SDG2460023) and by the Russian Ministry of Education grant (02−740−
11−5126).
References
1. T. P. Stossel, J. Condeelis, L. Cooley, J. H. Hartwig, A. Noegel, M. Schleicher, and
S. S. Shapiro. Nat Rev Mol Cell Biol 2, 138-145 (2001).
2. V. Barsegov and D. Thirumalai. Proc Natl Acad Sci USA 102, 1835-1839 (2005).
3. J. W. Weisel. Biophys Chem 112, 267-276 (2004).
4. I. Schwaiger, C. Sattler, D. R. Hostetter, and M. Rief. Nature Mat 1, 232-235 (2002).
5. W. Liu, L. M. Jawerth, E. Sparks, M. R. Falvo, R. R. Hantgan, R. Superfine, S. T. Lord, and
M. Guthold. Science 313, 634 (2006).
6. R. I. Dima and H. Joshi. Proc Natl Acad Sci USA 105, 15743-15748 (2008).
7. A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010).
2
Phase Transition from a-Helices to b-Sheets in
Fibrinogen Coiled Coils
Artem Zhmurov1,2
Andre Brown3
Rustem I. Litvinov3
Ruxandra I. Dima4
John W. Weisel3
Valeri Barsegov1,2*
1Department
of Chemistry, University
of Massachusetts, Lowell, MA 01854
2Moscow
Technology, Moscow, Russia 141700
3Department
of Cell and
Developmental
Biology, University
of Pennsylvania School
of Medicine,
Philadelphia, PA 19104
4Department
of
Chemistry, University
of Cincinnati
Cincinnati, OH 45221
*valeri_barsegov@
uml.edu
Mechanical functions of fibrin fibers are essential for hemostasis and wound healing (1). Fibrinogen, the precursor of fibrin, is a branched polymer that provides the
scaffold for a thrombus in vertebrates. The physical properties of fibrin(ogen) are
essential for the ability of fibrin clots to accomplish hemostasis and are an important determinant of the pathological properties of thrombi. Despite such critical
importance, the structural basis of fibrin clot mechanics is not well understood (2).
Graphics Processing Units (GPUs) are being used in a variety of scientific applications, including the biological N-body problem (3). We carried out theoretical studies of the mechanical properties of fibrinogen molecule, using all-atom Molecular
Dynamics (MD) simulations in implicit water fully implemented on a GPU. When
the α-helical regions in the coiled-coils are subject to an external mechanical perturbation, they undergo reversible phase transition to form the extended β-sheets
(4). The D-regions of the molecule make several turns around the direction of force
application to accommodate the mechanical unraveling of the coiled coils. As a
result, the hydrophobic side-chains buried inside the α-helices in the fibrin(ogen)
977
folded state become exposed to solvent. We argue that the observed increase in
the free energy of solvation might lead to protein aggregation, and hypothesize
that these transitions provide the molecular mechanism for the negative compressibility observed in experiments on whole blood clots (5). These results provide
important quantitative characteristics of the fibrinogen nanomechanics necessary
to understand the viscoelastic properties of fibrin polymers at the fiber and whole
clot levels.
This work has been supported by the American Heart Association grant
(09SDG2460023) and by the Russian Ministry of Education grant (02−
740−11−5126).
References
1. J. W. Weisel. Science 320, 456-457 (2008).
2. A. E. X. Brown, R. I. Litvinov, D. E. Discher, and J. W. Weisel. Biophys J 92, L39-L41
(2007).
3. A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010).
4. A. Zhmurov, A. E. X. Brown, R. I. Litvinov, R. I. Dima, J. W. Weisel, and V. Barsegov
(manuscript in preparation).
5. A. E. X. Brown, R. I. Litvinov, D. E. Discher, P. K. Purohit, and J. W. Weisel. Science 325,
741-744 (2009).
Computer Simulations of Protein–Protein Complex
Formation on Graphics Processing Units
Protein-protein interactions create new properties to the interacting components
and to the whole system, and it is an important aspect of the biological machinery
(1, 2). We present a new method for computer simulations of formation of proteinprotein complexes in a cell environment. The method is based on Brownian dynamics, which makes it possible to simulate association reactions of several hundreds
of protein pairs in sub-cellular compartments, and to obtain the real-time dynamics
of protein-protein interactions. The method allows us to explore the effect of electrostatic forces on the protein-protein complex formation, evaluate the kinetic rate
constants, and to unmask the molecular interactions (diffusion, electrostatic interactions) underlying the dynamics of processes in a cell. Biomolecular simulations
were used to study the kinetics of protein-protein interactions between the electron
transport proteins involved in photosynthesis, plastocyanin–cytochrome f complex,
both for wild-type and mutant plastocyanins. The method describes accurately binding interactions for different values of ionic strength in the solution (3) and in the
chloroplast thylakoid lumen (4), while taking into account (b) electrostatic interactions between proteins and in the thylakoid membrane (5), (c) kinetic characteristics
of ferredoxin and ferredoxin:NADP–reductase complex formation in solution (6),
and (d) complex formation of the transmembrane pigment-protein complex Photosystem I and proteins plastocyanin and ferredoxin (7). The developed method can
also be used as a predictive tool to resolve the binding sites and to describe complex
structures for a range of protein (8). Biomolecular simulation was accelerated on a
Graphics Processing Unit (GPU) to describe interactions among a large number of
proteins. The 10-100-fold computational acceleration, attained on the GPU device,
enabled us calculate the kinetic rates of protein-protein complex formation under
the physiologically relevant conditions of sub-cellular environment.
References
1. L. K. Chang, J. H. Zhao, H. L. Liu, J. W. Wu, C. K. Chuang, K. T. Liu, J. T. Chen,
W. B. Tsai, and Y. Ho. J Biomol Struct Dyn 28, 39-50 (2010).
2. M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12
(2010).
Ilya Kovalenko1*
Alexandra Diakonova1
Sergey Khrushchev1
Anna Abaturova1
Galina Riznichenko1
Andrei Rubin1
Sergey Trifonov2
Ivan Morozov2
Yaroslav Kholodov2
Valeri Barsegov3
1Biology
3
Department, Moscow State
University, Moscow, Russia, 119992
2Moscow
Technology, Moscow Region
Russia, 141700
3Department
of Massachusetts, Lowell, MA 01854
*[email protected]
978
4
Ilya Kovalenko*
Alexandra Diakonova
Olga Knyazeva
Anna Abaturova
Galina Riznichenko
Andrei Rubin
Biophysics Department, Biological
Faculty, Moscow State University,
Leninskie Gory,
Moscow 119992, Russia
*[email protected]
3. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, E. A. Grachev,
G. Y. Riznichenko, and A. B. Rubin. Phys Biol 3, 121-129 (2006).
4. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, G. Y. Riznichenko,
E. A. Grachevo, and A. B. Rubin. Biophysics 53, 140-146 (2008).
5. O. S. Knyazeva, I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, E. A. Grachev, and
A. B. Rubin. Biophysics 55, 221-227 (2010).
6. I. B. Kovalenko, A. N. Diakonova, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin.
Phys Biol 7, 026001 (2010).
7. I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. BioSystems, doi:
10.1016/j.biosystems.2010.09.013 (2010).
8. I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. Dokl Biochem
Biophys 427, 215-217 (2009).
Computer Simulation of Plastocyanin Diffusion and
Interaction with Its Reaction Partners
We present a Langevin dynamics computer model of limited diffusion of protein
plastocyanin and its interaction with transmembrane protein complexes photosystem 1 and cytochrome bf in the narrow chloroplast thylakoid lumen. The model
is multiparticle, it considers many plastocyanin molecules that compete to form
complexes with numerous photosystem 1 and cytochrome bf complexes embedded
in the photosynthetic membranes. The model takes into account the geometry of
the luminal space packed with many protein molecules and considers electrostatic
interactions of plastocyanin with its reaction partners in the thylakoid membrane.
The model uses a continuum electrostatic approach that describes molecules at the
atomic level using a macroscopic description. The Poisson-Boltzmann formalism
was used to determine the electrostatic potentials of the electron carrier proteins
and the thylakoid membrane at different ionic strengths. This work uses the model
parameters of protein-protein association estimated in our papers (1-4).
Calculations correctly reproduce the experimentally registered kinetic curves of
redox changes of the reaction center P700 of photosystem 1 and cytochrome f. The
model demonstrates non-monotonic dependences of complex formation rates on
the ionic strength as the result of long-range electrostatic interactions. The simulation method presented in this work can be applied for the description of diffusion
and functioning of many macromolecules that interact in the heterogeneous interior
of subcellular systems.
References
1. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, E. A. Grachev,
G. Y. Riznichenko, and A. B. Rubin. Phys Biol 3, 121-129 (2006).
2. I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, G. Y. Riznichenko,
E. A. Grachev, and A. B. Rubin. Biophysics 53, 140-146 (2008).
3. I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, and A. B. Rubin. BioSystems, doi:
10.1016/j.biosystems.2010.09.013 (2010).
4. O. S. Knyazeva, I. B. Kovalenko, A. M. Abaturova, G. Y. Riznichenko, E. A. Grachev, and
A. B. Rubin. Biophysics 55, 221-227 (2010).
5
Klaus Schulten
Department of Physics,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA
[email protected]
Computational Microscopy Biomolecular modeling, taking advantage of ever increasing computer power, has
dramatically improved in accuracy as well as in time and size scale covered. For
example, low resolution single molecule force measurements are imaged at atomic
resolution through steered molecular dynamics simulations; crystallography and
electron microscopy data, combined through molecular dynamics flexible fitting calculations, are interpreted through atomic level structures of functional intermediates of large cellular machines; simulations at multiple length scales show movies
of peripheral membrane proteins sculpting cellular membranes. The lecture will
illustrate for the three examples atomic resolution images and movies provided
979
by means of biomolecular modeling as a result of new simulation concepts, algorithms, and technology.
A computational-experimental collaboration (with H. Gaub, Munich) discovered a
possibly fundamental epigenetic mechanism of methylated DNA. Highly sampled
chip-based stretching of double stranded DNA combined with simulation revealed
that strand separation mechanics is strongly affected by epigenetic modification of
DNA. The study, involving long time scale and large size simulations, resolved and
explained the observed effect of DNA methylation.
Atomic resolution crystallographic structures and electron microscopy density
maps at better than 10 Angstrom resolution (from R. Beckmann, Munich) were
combined in a computational analysis employing a new simulation method, molecular dynamics flexible fitting, to construct an atomic resolution structure of a ribosome seen in the process of threading a nascent protein through a translocon into a
biological membrane. The simulations revealed also in great detail the interaction
between nascent protein and ribosomal exit channel as well as interaction with the
translocon, for example the binding of the signaling element to the translocon.
The shape of cellular membranes can be induced by peripheral proteins for example N-BAR and F-BAR domains. The latter proteins have been observed in vitro
to form tubular membranes from vesicles and a similar behavior has been seen in
simulations. Employing a combination of coarse-grained and all atom molecular
dynamics simulations, calculations offer movies revealing how the peripheral proteins, in forming regular lattices as observed by electron microscopy (Unger, Yale U.),
bend flat membranes into tubes, the latter remaining stable after removal of protein.
The simulations shed light on the protein-lipid interactions responsible for membrane bending. Exploring the Role of Protein-Protein Interactions in
the Mechanical Unfolding of Protein Assemblies
Dynamic force spectroscopy methods provide unique opportunities for directly
probing the free energy landscape of complex biomolecules. However, even if they
provide information on the chain extension of the molecule, they cannot supply
full details of the populated structures. This is especially true in the case of multidomain or multi-chain proteins for which the complexity of the fold translates into
many unfolding scenarios. An assumption employed by the majority of experiments
for the interpretation of the force-unfolding of such proteins is that the behavior of
the ensemble is the sum of its parts. While this assumption is likely to apply to
tandems of identical units, for hetero-protein tandems or whenever units are connected by interfaces as in multi-domain or multi-chain complexes this assumption
needs to be revisited.
I will present our investigations into the role of protein-protein interactions through
simulations of the biomechanical unfolding reactions of multi-domain and multichain protein complexes covering a range of fundamental cellular functions from
fusion to cytoskeletal support in real (experimental) time (1, 2). To obtain the
force extension curves using experimental pulling speeds, we employed a coarsegrained minimalist model (SOP model) (3) of proteins to carry out overdamped
Langevin simulations implemented on Graphics Processing Units (GPUs). GPUs
have unleashed tremendous computational power that has been utilized in a wide
range of scientific applications. The system size dependent 10-90-fold computational speedup on a GPU, compared to an optimized CPU program, enabled us to
follow the dynamics in the centisecond timescale (4). While our model reproduces
Ruxandra I. Dima
Department of Chemistry,
University of Cincinnati,
[email protected]
6
980
the experiments, we find that the independence assumption needs to be critically
assessed. I will discuss the signature of the protein-protein interactions as a function of the applied vector force, and the connection between the shape of the force
peaks and the degree of cooperativity in the protein complex. Remarkably, we find
that the degree of stabilization conferred by the interactions between units is determined by a combination between the stability of the interface and the internal fluctuations of a module.
This research has been supported by NSF CAREER Award MCB-0845002.
References
1.
2.
3.
4.
7
Matthew C. Zwier
Joseph W. Kaus
Lillian T. Chong*
University of Pittsburgh,
Pittsburgh, PA 15260
*[email protected]
R. I. Dima and H. Joshi. Proc Natl Acad Sci USA105, 15743-15748 (2008).
J. Y. Lee, T. Iverson, and R. I. Dima. J Phys Chem B 115, 186-195 (2011).
C. Hyeon, R. I. Dima, and D. Thirumalai. Structure 14, 1633-1645 (2006).
A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010).
Efficient Explicit Solvent Simulations
of Molecular Association Kinetics
Atomically detailed views of molecular association events are of great interest to a
variety of research areas in biology and chemistry. A natural approach to providing
such views is to use molecular dynamics (MD) simulations in explicit solvent which
are quite routine for tens of nanoseconds in certain systems (1-4). However, it
has been computationally prohibitive to perform MD simulations for a sufficiently
long time (by “brute force”) to capture more complicated association events, e.g.
protein-protein associations, that require microseconds or beyond (5). Fortunately,
the long timescales required are not necessarily because the actual events take a long
time; instead the events may be fast but infrequent, separated by long waiting times.
Path sampling approaches aim to capture rare events by minimizing the simulation
of long waiting times between events [as reviewed by Zwier and Chong (6)]. In this
work, we have combined the “weighted ensemble” path sampling approach (7) with
explicit solvent MD simulations. This approach allows us to obtain accurate kinetics as well as ensembles of molecular association pathways. We have determined
the efficiency of this approach relative to brute force simulations in sampling the
molecular association events for a range of well-studied systems: methane/methane, Na+/Cl-, methane/benzene, and K+/18-crown-6 ether (pictured below from left
to right). Relative to brute force simulation, we obtain efficiency gains of at least
1,100-fold for the most challenging system, K+/18-crown-6 ether, in terms of sampling the distribution of molecular association pathways. Our results indicate that
weighted ensemble sampling is likely to allow for even greater efficiencies for
more complex systems with higher barriers to molecular association. Applications
of weighted ensemble sampling to explicit solvent MD simulations of protein binding events will also be discussed.
This research has been supported by NSF CAREER Award MCB-0845216.
981
References
1. P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010).
(2010).
3. C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010).
4. Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010).
5. K. A. Henzler-Wildman and D. Kern. Nature 450, 964-972 (2007).
6. M. C. Zwier and L. T. Chong. Curr Opin Pharmacol 10, 745-752 (2010).
7. G. A. Huber and S. Kim. Biophys J 70, 97-110 (1996).
Molecular Simulations Unravel Key Amino
Acid Interactions Regulating Stability and
Aggregation of Human Lens gD-crystallin
The prevalent eye disease age-onset cataract is associated with aggregation of
human γD-crystallins, one of the longest-lived proteins of the body. The molecular determinants of the unusually high stability and (un)folding/aggregation of
γD-crystallin remain to be unidentified. Determination of complex dynamics during protein (un)folding and aggregation events require molecular simulations at
long time scales (micro to milliseconds). Even though simulations that are tens of
nanoseconds long for certain systems (1-4) are routine, it remains challenging to
perform them at longer time scales for atomistic models of biologically relevant
proteins. In this study, we perform extensive atomistic molecular dynamics simulations using massively parallel IBM Blue Gene/L supercomputer (5) to characterize
unfolding (6) and oligomerization (7) of human gamma D crystallin to advance our
current understanding of cataract.
Using large-scale atomistic simulations (6), we have shown that the isolated
N-terminal domain (N-td) of γD-crystallin is less stable than its isolated C-terminal
domain (C-td), in addition to being the less stable domain in the full-length protein
(Figure 1A and B), in agreement with biochemical experiments. Sequential unfolding of individual Greek key motifs was revealed within each isolated domain. Our
simulations strongly indicate that the stability and the folding mechanism of the
Figure 1: Schematic summary of human γ D-crystallin polymerization.
Payel Das1*
Jonathan A. King2
Ruhong Zhou1,3
1IBM
8
Thomas J. Watson Research
Center, Yorktown Heights, NY 10598
2Department
of Biology,
Massachusetts Institute of Technology,
Cambridge, MA 02139
3Department
of Chemistry,
Columbia University, New York,
NY 10027
982
N-td are regulated by the interdomain interactions, consistent with experimental
observations. We also found that the a and b strands from the Greek Key motif 4
comprising the interdomain interface are the most stable structures within the full
protein. Detailed analysis uncovers a surprising Glu-Arg salt-bridge at the topologically equivalent positions of residues E135 and R142 that plays a significant
role in determining the stability of a Greek Key motif (see Figure 1A). Disrupting the E135-R142 salt-bridge in silico resulted in destabilizing the inter-domain
interface and facilitated the N-td unfolding. These findings (6) indicate that certain
highly conserved charged residues, that is, Glu135 and Arg142, of γD-crystallin are
crucial for stabilizing its hydrophobic domain interface in native conformation, and
disruption of charges on the γD-crystallin surface might lead to unfolding.
Identification of the aggregation precursors of γ-crystallins is extremely crucial for
developing strategies to prevent and reverse cataract. We have used large-scale simulations to determine the structural basis of the pathogenic monomeric state and the
intermolecular association of γD-crystallin. Our microseconds of atomistic molecular dynamics simulations (7) uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native
γ D-crystallin with a largely folded C-terminal domain and a mostly unfolded Nterminal domain (see Figure 1B). About 30 residues including a, b, and c strands from
the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure
of hydrophobic residues as well as partial unstructuring upon N-terminal domain
unfolding. Those strands comprise the domain-domain interface that is crucial for
the unusually high stability of γ D-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors (7), which reveals domainswapped dimeric structures (Figures 1C and D). In the simulated dimeric structure,
the N-terminal domain of one monomer is frequently found in contact with residues
135-164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second
molecule. The present results suggest that γD-crystallin polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability (Figure 1). These findings (7)
thus provide critical molecular insights onto the initial stages of age-onset cataract
formation, which is important toward understanding protein aggregation diseases.
References
1.
2.
3.
4.
5.
6.
7.
9
Gianni De Fabritiis
Research Group of biomedical
Informatics (GRIB-IMIM)
Universitat Pompeu Fabra
Barcelona Biomedical Research
Park (PRBB), C/ Dr. Aiguader 88
08003, Barcelona, Spain
[email protected]
P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010).
M. J. Aman, H. Karauzum, M. G. Bowden, and T. L. Nguyen. J Biomol Struct Dyn 28, 1-12 (2010).
C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010).
Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010).
S. Kumar, et al. IBM J Res Dev 52, 177-188 (2008).
P. Das, J. A. King, and R. Zhou. Prot Sci 19, 131-140 (2010).
P. Das, J. A. King, and R. Zhou. Proc Natl Acad Sci, USA, under review (2011).
Reconstructing an Enzyme-Inhibitor Binding Process
by Molecular Dynamics Simulations
The understanding of protein-ligand binding is of critical importance for biomedical research, yet the process itself has been very difficult to study due to its
intrinsically dynamic character. In this talk, we will go through the quantitative
reconstruction of the complete binding process of the enzyme-inhibitor complex
Trypsin-Benzamidine performed with molecular dynamics simulations of free
inhibitor binding. The binding events obtained are able to capture the kinetic pathway of the inhibitor diffusing from solvent to bound passing for few metastable
intermediate states. Unexpectedly, rather than directly entering the binding pocket,
the inhibitor appears to roll on the surface of the protein to the final binding pocket.
The trajectories are analysed via a Markov state model-based analysis which additionally yields the kinetic parameters and binding affinity of the interaction. These
983
results show an impressive predictive power for unconventional high-throughput
molecular simulations. At the same time, the general methodology is easily applicable to other molecular systems becoming of interest to biomedical and pharmaceutical research.
The Nucleosome Simulator: 100 Nucleosomes;
2 Microseconds and Counting
Molecular simulation is an effective tool to study structure function relationships in
biomolecules. A modest molecular dynamics simulation today includes ~100,000
atoms and represents over 100 ns of time. The computational effort requires nearly
one hundred processors in order to complete in less than one week. Today’s supercomputers contain up to 200,000 processors, too many for a single simulation of
modest size. However, it is reasonable to simulate 10’s to 100’s of structures simultaneously on a single supercomputer or to distribute them to any suitable computing resources as they become available. Such high throughput high performance
simulations require careful coordination and strategy. The ManyJobs and BigJobs
tools provide this functionality.
Rajib Mukherjee1*
Hideki Fujioka1
Abhinav Thota2
Shantenu Jha2
Thomas C. Bishop1
Our present efforts are directed toward the investigation of nucleosome positioning and stability as a function of DNA sequence using all atom molecular dynamics simulation. Nucleosome positioning is one of the current ‘hot’ issues and the
various approaches and hypotheses on what positions nucleosomes are discussed
extensively in recent publications, particularly in an issue of this Journal devoted
exclusively to this subject (1-14). One of the major factors affecting the nucleosome
positioning is DNA sequence. Nucleosomes consist of 147 base pair (bp) of DNA
wrapped ~1.7 left handed superhelical turns around a histone core. This study
requires simulations of hundreds of nucleosomes with different sequences. Our chosen sequences are divided into four broad categories: naturally occurring positioning sequences, artificial positioning sequences, sequences from the Saccharomyces
cerevisiae genome and sequences used for control purposes, e.g. homopolymers.
For the S. cerevisiae derived sequences, we model 336 nucleosomes. The collection represents 16 of the most well positioned nucleosomes and their immediate
neighbors in sequence space. Each neighborhood spans two turns of the DNA, one
upstream turn and one downstream turn. The 21 individual neighbors contain only
147 bp, each created by threading the appropriate sequence onto the histone core.
Thus each neighborhood has a common segment of 126 bp located at 21 successive
positions on the histone core. To date, we have simulated over 100 nucleosomes,
including 4 separate neighborhoods, and accumulated over 2 microseconds of
nucleosome dynamics. Our high throughput approach requires significant computational power, constant scheduling, monitoring, and efficient utilization of resources
in order to achieve the shortest time to completion.
LA 70118, USA
To manage the workflow we have utilized two scheduling tools: ManyJobs and
BigJobs. ManyJobs is a portable tool written in Python. ManyJobs maintains a
database of all compute tasks and the dependencies between tasks. At the beginning of a run, ManyJobs submits requests for resources to all computers listed by
the user. Once a resource is allocated and a job starts. ManyJobs assigns a task
to the resource and requests additional resources in anticipation of the next task.
Upon job completion, the task is marked complete in the ManyJobs database. The
process repeats until all tasks in the database are completed. The current version of
ManyJobs uses secure shell for communications between the machine maintaining
the task database and the various compute resources. BigJobs is a Simple API for
Grid Applications (SAGA) based implementation of the pilot job concept. SAGA
provides alternate methods for authentication and communication than secure shell.
Another distinction of BigJobs is the ability to dynamically bundle individual tasks
into a container with multiple tasks, the pilot job.
1Center
10
for Computational Science,
Tulane University, New Orleans,
2Center
for Computation & Technology,
Johnston Hall, Louisiana State
University, Baton Rouge,
LA 70803, USA
*[email protected]
984
We will discuss implementation and proper utilization of these tools and their pros
and cons. A meta-analysis of simulation results is conducted to identify features
of nucleosome positioning and stability. We focus attention on DNA structural
deformations, in the form of kinks, their location, sequence dependencies, and the
timescale associated with kink formation and healing.
References
1. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. J Biomol Struct Dyn 27,
713-724 (2010).
2. F. Xu and W. K. Olson. J Biomol Struct Dyn 27, 725-739 (2010).
3. E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010).
4. P. De Santis, S. Morosetti, and A. Scipioni. J Biomol Struct Dyn 27, 747-764 (2010).
5. G. A. Babbitt, M. Y. Tolstorukov, and Y. Kim. J Biomol Struct Dyn 27, 765-780 (2010).
6. D. J. Clark. J Biomol Struct Dyn 27, 781-793 (2010).
7. S. M. Johnson. J Biomol Struct Dyn 27, 795-802 (2010).
8. G. Arya, A. Maitra, and S. A. Grigoryev. J Biomol Struct Dyn 27, 803-820 (2010).
9. F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010).
10. D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010).
11. S. M. West, R. Rohs, R. S. Mann, and B. Honing. J Biomol Struct Dyn 27, 861-866 (2010).
12. Y. V. Sereda and T. C. Bishop. J Biomol Struct Dyn 27, 867-887 (2010).
13. I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 26, 403-411 (2009).
11
Vinod Jani
Shruti Koulgi
Uddhavesh Sonavane
Rajendra Joshi*
Bioinformatics Team, SECG
Centre for Development of Advanced
Exploring Fast Protein Folding Funnel Using
Replica Exchange Molecular Dynamics at
Different Temperature Ranges
Protein folding is a biological process through which one dimensional sequence
acquires its three dimensional structure. During past four decades various aspects
of protein folding have been explored experimentally and theoretically. There have
been some success in predicting 3D structure of proteins using statistical potentials
(for example see references 1, 2). Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. With the development of
advanced Molecular Dynamics (MD) techniques like Replica Exchange Molecular
Dynamics (REMD), simulations can possibly reach to the experimental timescales.
Computing,
Pune University Campus,
Pune – 411 007, India
*[email protected]
Figure 1: NMR structure showing the three helices along with the amino acid sequence of same
(pdb code 1VII).
985
The major difficulty in experimental studies of protein folding lies in capturing
of transient intermediates and events, which is possible via performing very long
simulations. Here an attempt has been made to reach the multi-microsecond simulation time scale by carrying out folding simulation on a fast folding three helix
bundle protein using REMD. REMD based folding simulation has been carried
out on Villin headpiece (PDB code 1VII, Figure 1), a 36-residue small protein (3)
and Engrailed Homeodomain (PDB ID: 1ENH), a 54 residue protein (4) starting
from its extended structure. The multiple REMD simulations were carried out
at moderate and broader temperature range. The population landscape has been
built using segment wise Root Mean Square Deviation (RMSD), Principal Component Analysis (PCA) as reaction coordinates. The REMD has helped to carry
out very long time scales simulations where results are close to the experimental
findings. Also the effect of temperature range on the population landscape has
been discussed in detail.
References
(2010).
3. V. Jani, U. B. Sonavane, and R. Joshi. J. Biomol Struct Dyn 28, 845-860 (2011).
4. S. Koulgi, U. B. Sonavane, and R. Joshi. J Mol Graph Model, 29, 481-491 (2010).
Exploring Folding Funnel of Villin Headpiece
Using Replica Exchange Molecular Dynamics
and Amber United Atom Model
Understanding protein folding has been a scientifically and computationally challenging task till date. The question, “How does an amino acid sequence dictate
the structure of a protein?” has been of major interest and different theories have
been put forward to answer it. Despite the success of deriving the 3 D structure of
a protein (for example see 1, 2) from our conventional understanding of the preferential interactions between certain amino acids, recently questions have been raised
regarding the use of statistical potentials and preferential interactions (3). Reaching
the experimental time scale of millisecond is a grand challenge for protein folding
simulations. The major challenges involved in the use of molecular dynamics (MD)
simulations are to explore folding landscape for fast folding proteins and give an
atomic level understanding of the folding process (4). The advanced methodologies
like REMD (5) and Coarse-grained MD and computational power enable one to
carry out long simulations. The advanced methods like REMD have significantly
contributed to the understanding of the folding landscape of various ultrafast folding proteins. The advancement of force field to handle Coarse Grained models over
all atom model system may be needed in achieving the goal of very long time scale
folding simulation. Here an attempt has been made to reach the multi-microsecond
simulation time scale by carrying out folding simulation on a fast folding three
Helix bundle protein. A combination of REMD and Amber United atom model (6)
has been employed. Two folding simulations based on REMD have been carried
out on Villin headpiece (PDB code 1VII), a 36-residue small three Helix bundle
protein (7) starting with an extended conformation. The protein folding funnel
has been explored using segment wise Root Mean Square Deviation (RMSD) and
Principal Component Analysis (PCA) as reaction coordinates. The combination of
REMD and CGMD has helped to carry out very long time scales simulations where
the results are close to the experimental findings. The present study is targeted to
explore the folding funnel as well as to study the effect of temperature range in
REMD simulations.
Vinod Jani
Uddhavesh Sonavane
Rajendra Joshi*
12
Bioinformatics Group
Computing, Pune University Campus,
*[email protected]
986
References
(2010).
3. A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142
(2010).
4. K. A. Dill, S. B. Ozkan, T. R. Weikl, J. D. Chodera, and V. A. Voelz. Current Opinion in
Structural Biology 342-346 (2007).
5. Y. Sugita, and Y. Okamoto. Chem Phys Lett 329, 261-270 (1999).
6. L. Yang, C. H. Tan, M. J. Hsieh, J. Wang , Y. Duan, P. Cieplak, J. Caldwell, P. A. Kollman,
and R. Luo. J Phys Chem B, 110, 13166-76 (2006).
7. V. Jani, U. Sonavane, and R, Joshi. J Biomol Struct Dyn 28, 845-860 (2011).
13
Computational Investigation of the Free Energy
Landscape of the) of the Four Stereomers of
Ac-L-Pro-c3Phe-NHMe (c3Phe=2,
3-Methanophenylalanine) in Explicit
and Implicit Solvent
Juan J. Perez*
Alex Rodriguez
Francesc Corcho
Department of Chemical Engineering,
Technical University of Catalonia,
Barcelona, Spain
*[email protected]
The prediction capabilities of atomistic simulations of peptides are hampered by
different difficulties including, the reliability of force fields, the treatment of the
solvent or the adequate sampling of the conformational space. The present report
regards a computational study aimed at assessing the conformational profile of
the four stereoisomers of the peptide Ace-Pro-c3Phe-NMe, previously reported to
exhibit β-turn structures in dichloromethane with different type I/type II β-turn
profiles [1]. For this purpose, we carried out a thorough sampling of the conformational space of the four peptides in explicit solvent using the replica exchange
molecular dynamics method as a sampling technique and compared the results
with simulations of the system modeled using the analytical linearized PoissonBoltzmann (ALPB) method with two different AMBER force fields: parm96, and
parm99SB.
The free energy landscapes of the different peptides computed in explicit solvent
show two minima separated by high barriers and agree well with the published
experimental results. The calculations carried out in implicit solvent do not describe
the system in the same manner. Moreover, it is shown that implicit solvent calculations carried out with the parm96 force field agree better with those obtained with
the parm99SB force field in explicit solvent [2,3]. The results of the simulations
suggest that the balance between intra- and intermolecular interactions is the cause
of the differences between implicit and explicit solvent simulations in this system,
stressing the role of the environment to define properly the conformational profile
of a peptide in solution.
References
1. A. I. Jimenez, C. Cativiela, A. Aubry, and M. Marraud. J Am Chem Soc 120, 9452-9459
(1998).
2. A. Rodriguez, J. Canto, F. Corcho, and J. J. Perez. Biopolymers 92, 518-524 (2009).
3. A. Rodriguez, P. Mokoema, F. Corcho, K. Bisetty, and J. J. Perez. J Phys Chem B, 115,
1440-1449 (2011).
987
Folding Globular Proteins: Collapse Kinetics
and Chevron Plots
Quantitative description of how proteins fold under experimental conditions remains
a challenging problem. Experiments often use urea and Guanidinium Chloride
(GdmCl) to study folding whereas the natural variable in simulations is temperature.
To bridge the gap, we use the Molecular Transfer Model that combines measured
denaturant-dependent transfer free energies for the peptide group and amino acid
residues, and a coarse-grained model for polypeptide chains to simulate the folding
mechanism of src SH3. Stability of the native state decreases linearly as [C] (the
concentration of GdmCl) increases with the slope that is in excellent agreement with
experiments. We show that lnkobs (kobs is the sum of folding and unfolding rates) as a
function of [C] has the characteristic V (Chevron) shape. In the dominant transition
state, which does not vary significantly at low [C], the core of the protein and certain
loops are structured. Besides solving the long-standing problem of computing the
Chevron plot, our work lays the foundation for incorporating denaturant effects in a
physically transparent manner either in all atom or coarse-grained simulations.
D. Thirumalai
Institute for Physical Science and
Technology University of Maryland,
College Park,
MD 20742, USA
[email protected]
Single-molecule Fluorescence Maps the Folding
Landscape of a Large Protein
A substantial body of information, both theoretical and experimental, has accumulated over the last years on protein folding, including a provocative recent idea that
it is driven by amino acid stoichiometry (1), as opposed to preferred interactions
(e.g., hydrophobic interactions) between specific amino acids (2, 3). Particularly
advanced is our understanding of the principles that govern the folding of small,
single-domain proteins (4). But can we apply the same principles in order to decipher the folding mechanisms of larger, multi-domain proteins? More than 70% of
the eukaryotic proteins belong to this group, yet rather little is known about their
folding reactions (5).
14
Menahem Pirchi
15
Israel Perlman Chemical Sciences
Building 603, Weizmann Institute
of Science,
P.O.Box 26, Rehovot 76100, Israel
[email protected] We propose here that high-throughput single-molecule fluorescence spectroscopy,
combined with statistical analysis, can be used to study folding dynamics of large
proteins (6). As a proof-of-concept, we studied the folding landscape of adenylate
kinase (AK), a 214-residue protein from Escherichia coli. AK molecules
were double-labeled for FRET at two sites in their CORE domain. The molecules were individually trapped within surface-tethered lipid vesicles (7, 8)
in the presence of different concentrations of guanidinium chloride (GdmCl),
a chemical denaturant. The trapped molecules were examined using an automated single-molecule fluorescence microscope, which allowed us to obtain
data sets consisting of many thousands of short FRET trajectories of individual molecules. The availability of this massive amount of data enabled us to
construct a detailed map of the folding landscape of AK, using hidden Markov
modeling (HMM) (9). We found that the folding dynamics of AK could be
described in terms of transitions between six quasi-stable states, one of which
is probably misfolded. The folding reaction involves many parallel pathways
connecting these states in jumps of various sizes (See Figure). This folding
pattern differs markedly from the well-known two-state model of small proteins. Future work, involving both experiments and computation, will attempt
Figure: 1D projection of the energy landscape of adenylate
to structurally characterize the intermediate states discovered here.
kinase molecules, based on analysis of single-molecule fluoresReferences
1. A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142
(2010).
2. B. W. Matthews. J Biomol Struct Dyn 28, 589-591 (2011).
cence trajectories measured at 0.5 M GdmCl. Shown are the
relative stabilities of the observed states, as well as free energy
barriers corresponding to transitions between them. The amount
of flux carried by each transition is represented by its line width.
The figure represents transitions which carry at least 10% of the
folding flux.
988
16
Amrita Dasgupta
Jayant B. Udgaonkar
National Centre for Biological Sciences,
Tata Institute of Fundamental Research,
GKVK Campus, Bellary Road,
Bangalore 560065, India
[email protected]
[email protected]
3. S. Rackovsky and H. Scheraga. J Biomol Struct Dyn 28, 593-594 (2011).
4. E. Shakhnovich. Chem Rev 106, 1559-1588 (2006).
5. J. H. Han, S. Batey, A. A. Nickson, S. A. Teichmann, and J. Clarke. Nat Rev Mol Cell Biol
8, 319-330 (2007).
6. V. Ratner, D. Amir, E. Kahana, and E. Haas. J Mol Biol 352, 683-699 (2005).
7. E. Boukobza, A. Sonnenfeld, and G. Haran. J Phys Chem B 105, 12165-12170 (2001).
8. E. Rhoades, E. Gussakovsky, and G. Haran. Proc Natl Acad Sci USA 100, 3197-3202
(2003).
9. L. R. A. Rabiner. Proc IEEE 77, 257-286 (1989).
Evidence for Initial Non-specific Polypeptide
Chain Collapse During the Refolding of the SH3
Domain of PI3 Kinase
Proteins are evolutionarily selected heteropolymers, but their response to solvent
change appears to be very similar to that of simple homopolymers (1, 2). The
unfolded protein chains undergo global contraction when transferred from a good
to a bad solvent (3, 4). This ultrafast compaction of the unfolded state of a protein
(collapse) channels it to the unique native structure by reducing its conformational
space. The collapse reaction of any polypeptide chain has been assumed to be
driven by non-specific hydrophobic interactions (3, 5). Alternatively, the driving
force of the collapse reaction could also be the formation of backbone hydrogen
bonds at low concentration of denaturant (6, 7). For some recent provocative discussion on protein folding, see Mittal et al. (8), Matthews (9) Scheraga (10) and
others published in the February 2011 issue of this Journal.
In the present study, the refolding of the PI3K SH3 from the guanidine hydrochloride (GdnHCl)-unfolded state was probed with millisecond (stopped flow) and
sub-millisecond (continuous flow) measurements of the change in tyrosine fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance
energy transfer (FRET) efficiency (11). Previously, the refolding of this protein
appeared two-state (12). Presently, our studies show that the folding of the protein commences via the rapid (complete within 150 μs) formation of a collapsed
ensemble in a transition that is gradual and without any significant accumulation
of secondary structure. All three intra-molecular distances collapsed to the same
extent indicating that the compaction was synchronous as that expected for a coil
to globule transition of a simple homopolymer as a consequence of solvent change
(13). These results highlight the homopolymer nature of the unfolded polypeptide
chain of the PI3K SH3. Furthermore to investigate the role of intra-chain hydrogen
bonding in the collapse reaction, the folding was initiated by dilution of the ureaunfolded state (11). The extent of compaction seen for one of the intra-molecular
distance was similar to that observed for the GdnHCl induced unfolded state.
To elucidate the importance of a non-specific collapsed ensemble in the subsequent
structure formation of the PI3K SH3, an attempt was made to tune folding conditions such that a specific structured component of the collapsed ensemble could be
preferentially populated. With the above objective, the effect of 500 mM sodium
sulphate on the refolding of the PI3K SH3 was studied using multiple spectroscopic
probes. Results indicate the formation of a specifically collapsed intermediate that
has greater ANS binding than the previously reported non-specific ensemble (8).
Two intra-molecular distances in this collapsed ensemble show greater contraction
than just a solvent induced compaction, indicating the formation of a specific structured ensemble, before the rate limiting step of folding. Interestingly, one of the
FRET pairs indicates the formation of native-like distance in the first millisecond
of the refolding reaction.
989
Abbreviations: FRET: Fluorescence resonance energy transfer; PI3K SH3: SH3
domain of PI3 kinase; ANS: 1-anilino-naphthalene-8-sulfonate.
References
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2.
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13.
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D. W. Bolen, G. D. Rose. Annu Rev Biochem 77, 339-362 (2008).
L. M. Holthauzen, J. Rosgen, and D. W. Bolen. Biochemistry 49, 1310-1318 (2010).
A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142
(2010).
B. W. Matthews. J Biomol Struct Dyn 28, 589-591 (2011).
S. Rackovsky and H. Scheraga. J Biomol Struct Dyn 28, 593-594 (2011).
A. Dasgupta and J. B. Udgaonkar. J Mol Biol 403, 430-445 (2010).
J. I. Guijarro, C. J. Morton, K. W. Plaxco, I. D. Campbell, C. M. Dobson. J Mol Biol 276,
657-667 (1981).
C. Williams, F. Brochard, H. L. Frisch, Annu. Rev. Phys. Chem. 32, 433-451 (1981).
17
Simulations of the Folding Unfolding of Proteins
Under Different Solvent Condition
Proteins exhibit marginal stability, determined by the balance of many competing
effects. This stability can be perturbed by changes in temperature, pH, pressure, and
other solvent conditions. Osmolytes are small organic compounds that modulate the
conformational equilibrium, folded (F) and unfolded (U), of proteins as cosolvents.
Protecting osmolytes such as trimethylamine N-oxide (TMAO), glycerol, and sugars that push the equilibrium toward F play a crucial role in maintaining the function
of intracellular proteins in extreme environmental conditions. Urea is a denaturing
osmolyte that shifts the equilibrium toward U. Here we report the reversible folding/unfolding equilibrium, under various solution conditions that include urea, high
pressure, and different charge states of the Trp-cage miniprotein (1-4). The folding/
unfolding equilibrium is studied using all-atom Replica exchange MD simulations.
For urea, the simulations capture the experimentally observed linear dependence of
unfolding free energy on urea concentration. We find that the denaturation is driven
by favorable direct interaction of urea with the protein through both electrostatic
and van der Waals forces and quantify their contribution. Though the magnitude of
direct electrostatic interaction of urea is larger than van der Waals, the difference
between unfolded and folded ensembles is dominated by the van der Waals interaction. We also find that hydrogen bonding of urea to the peptide backbone does not
play a dominant role in denaturation. The unfolded ensemble sampled depends on
Deepak R. Canchi1,2
Camilo Jimenez-Cruz1,3
Angel E. Garcia1,2,3*
1 Center
for Biotechnology and
Interdisciplinary Studies
2 Department
of Chemical and
Biological Engineering
3 Department
of Physics,
Applied Physics and Astronomy,
Rensselaer Polytechnic Institute,
Troy, NY 12180, USA
*[email protected]
Figure 1: Preferential binding of urea to the
protein side chains over the backbone. The left
figure shows the binding of urea around the
Trp-cage protein. Red spheres represent urea
molecules closer to the backbone atoms, while
blue spheres represent molecules closer to the
sidechains. The figure on the left shows the
preferential interaction of urea to the side chains
and backbone.
990
urea concentration, with greater urea concentration favoring conformations with
greater solvent exposure. The m-value is predicted to increase with temperature
and more strongly so with pressure (3, 4).
We also explored the effect of protonation of charged groups in protein and
found that the unfolded state ensemble changes little as a function of protonation. However, charge-charge interactions in the folded state ensemble are
responsible for the change in stability of the protein. Our results show how
atomic level simulations with explicit solvent models can be used to characterize the stability of proteins.
This research has been supported by grants from NSF MCB-0543769 and
MCB-1050966.
References
1. D. Paschek, S. Hempel, and A. E. Garcia. Procs Natl Acad Sci (USA) 105, 17754-17759
(2008).
2. R. Day, D. Paschek, and A. E. Garcia. Proteins 78, 1889-1899 (2010).
3. D. Canchi, D. Paschek, and A. E. Garcia. J Amer Chem Soc 132, 2238-2244 (2010).
4. D. Canchi and A. E. Garcia. Biophys J 100, (2011) doi:10.1016/j.bpj.2011.01.028.
18
Shubhra Ghosh Dastidar
Bose Institute, Kolkata 700054, India
[email protected]
Flexibility and Modulations in Protein-Protein
Interactions: Mechanistic Insights from Molecular
Dynamics Simulations of MDM2 and P53
The p53 protein provides a protection mechanism to cells against cancer (1). In normal cells p53 is complexed and down-regulated by another protein MDM2. In damaged cells the process is interrupted by stress signals that activate p53’s network,
whose dysfunction is associated with cancers. Inhibiting over-expressed MDM2 to
up-regulate p53’s function is a promising therapeutic strategy which will involve
design of ligands to inhibit MDM2. In order to accomplish this one can employ
modeling and molecular dynamics (2-5) approaches.
In this presentation, it will be shown how modeling and MD simulations have
extracted the dynamics of interactions between MDM2-p53 in atomistic detail and
have revealed the mechanisms that yield tight binding peptides to inhibit MDM2
(6). The simulations have attributed the flexibility and the conformational modulations to be key players which lead to the multiple mechanisms of binding associated
with varying thermodynamic origins (6). Switching between electrostatics and van
dar Waals components of interactions bring the uncomplexed MDM2 and p53 to
each other to encounter, leading to transition to the complexed state (7, 8). Taking
this system as an example, MD studies suggest that consideration of the detailed
dynamics is essential to gain mechanistic insights about the plasticity of such systems and to yield improved strategies for ligand design. Atomistic insight into the
effect of post-translational modifications of MDM2 on p53 regulations will also be
discussed (9).
References
1. B. Vogelstein, D. Lane, and A. Levine. Nature 408, 307-310 (2000).
(2010).
4.
5.
6.
7.
8.
9.
991
C. Koshy, M. Parthiban, and R. Sowdhamini. J Biomol Struct Dyn 28, 71-83 (2010).
Y. Tao, Z. H. Rao, and S. Q. Liu. J Biomol Struct Dyn 28, 143-157 (2010).
S. G. Dastidar, D. P. Lane, and C. S. Verma. J Am Chem Soc 130, 13514-13515 (2008).
S. G. Dastidar, D. P. Lane, and C. S. Verma. BMC Bioinformatics 10 (Suppl 15):S6 (2009).
S. G. Dastidar et al. Theor Chem Acc 125, 621 (2010).
S. G. Dastidar et al. Cell Cycle (In Press).
Homology Modeling and Molecular Dynamics
Simulations of RNA Polymerases
Although transcription is of central importance to cell survival, only few antimicrobial agents have been directed towards the RNA polymerase (RNAP) enzyme.
Rifampicin, one of the most potent and broad spectrum antibiotics and a key component of anti-tuberculosis therapy, binds in a pocket of the RNAP deep within the
DNA/RNA channel, but more than 12 Å away from the active site. Unfortunately,
binding of Rifampicin can be easily disturbed by enzyme mutations. Therefore, we
are interested in blocking of active sites of bacterial RNAPs directly using analogs
of NTPs. Such approach was found as very potent in the case of viral infections.
Here, we present results of homology modeling (using the MODELLER software
package), ab initio and QMMM calculations (GAUSSIAN03, ONIOM) and classical molecular dynamics simulations (AMBER and NAMD software packages).
RNAPs in complex with nucleic acids (template DNA strand, RNA transcript,
NTPs – either natural or chemically modified) were investigate in detail.
Ivan Barvik*
Vlastimil Zima
Kamil Malac
19
Charles University,
Faculty of Mathematics and Physics,
Institute of Physics,
Ke Karlovu 5,
Prague 2, 121 16, Czech Republic,
*[email protected]
Support from the Ministry of Education, Youth and Sports of the Czech Republic
(Project No. MSM 0021620835 and Project No. NPVII 2B06065) is gratefully
acknowledged.
Method of Molecular Dynamics for Proteins
in the Ionization-Conformation Phase Space at
Equilibrium Conditions at Constant pH
In view of the extensive interest in molecular dynamics simulations (1-4), a new
realization of the constant-pH molecular dynamics simulation method (5) is proposed. Molecular dynamics simulation is performed in the potential of mean force
of protein molecule in the water-proton bath at equilibrium titration conditions
(MD-pH-ET). It is shown that: i) the algorithm of MD-pH-ET which delivers the
highest numerical accuracy of the MD-pH-ET method, is the simulation of protein
in its instant most probable physical ionization microstate with additional potential
of mean force of equilibrium titration, which depends on a deviation of the most
probable physical ionization microstate from an equilibrium ensemble of ionization microstates for a given protein conformation; ii) the new method MD-pH-ET
allows one to carry out the optimization of protein structure and the total free
energy of a protein in the aqueous solution at constant pH, and the calculation of the
pH-dependent properties. Method MD-pH-ET possesses unique features such as: i)
it uses precise and computationally effective realization of calculation of ionization
equilibrium and electrostatic energy and atomic forces for protein in water solution by the model of continuous dielectric media with Poisson equation solved by
the Fast Adaptive Multigrid Boundary Element (FAMBE) method (6). Coupling
of FAMBE method with Generalized Born method by definition of the Poisson
“ideal” Born atomic radii allows one to accelerate an accurate calculation of forces
and energies in the MD-pH-ET method; ii) it uses the same model of the potential
energy surface in the ionization-conformational phase space, both for the calculation
Y. N. Vorobjev
20
Institute of Chemical Biology and
Fundamental Medicine,
Siberian Division, Russian Academy
of Sciences, Novosibirsk, 630090, Russia
[email protected]
992
of the potential energy of the protein and atomic forces and for determining the
ionization states; iii) it calculates the total free energy of the protein in the aqueous
solution in proton reservoir under the conditions of equilibrium titration. The workability of the new method MD-pH-ET is demonstrated on a set of proteins.
This research has been supported by grant of Russian Fond of Basic Research
#09-04-00136 and by projects #09-26 and # 09-119 of the Siberian Brunch of
the RAS.
References
(2010).
5. S. L.Williams, C. A. F. Oliveira, and J. A. McCammon. J Chem Theory Comput 6, 560-568
(2010).
6. Y. N. Vorobjev, J. A. Vila, and H. A. Scheraga. J Phys Chem 112, 11122-11136 (2008).
7. A. Onufriev, D. A. Case, and D. Bashford. J Comput Chem 23, 1297-1304 (2002).
21
Jihua Wang
Liling Zhao
Zanxia Cao
Key Lab of Biophysics in Shandong
(Dezhou University),
Dezhou, China, 253023
[email protected]
Molecular Dynamics Simulations on Structural
Heterogeneity States of Protein
Well-defined structure proteins can turn into all kinds of structural heterogeneity
states such as intermediate state, transition state or unfolded state under different circumstance. Another type of protein, namely intrinsically disordered protein
(IDPs), is lacking independently folded states at physiological condition. These
states have highly structural heterogeneity (1) and they play central roles in a range
of important biological process. It is a formidable challenge to reveal structural
character of the flexible and conformatically highly heterogeneous states. Some
effective sampling methods such as replica exchange molecular dynamics (REMD)
and restraint molecular dynamics simulation (restraint MD) are used to study the
disorder states. Here, we gave two cases to study IDPs and protein intermediate
state by REMD and restraint MD.
Case 1: Human α-synuclein protein, a presynaptic protein of 140 amino acid residues, is the major component of Lewy bodies (LBs) deposited in the brains of
patients with Parkinson’s disease, and it usually has extensive intrinsically disordered
regions (IDRs). The N-terminal 12 residues peptide of the α-synuclein (α-syn12)
was choosen. The structural and thermodynamics character of α-syn12 peptide in
aqueous solution have been investigated by temperature replica exchange molecular
dynamics (T-REMD) simulations. The structural and thermodynamic characters of
α-syn12 peptide at different pH and temperatures have also been studied by temperature replica exchange molecular dynamics (T-REMD) simulations (2).
Case 2: The other case is to study the thermal intermediate state of high mobility
group (HMG) box 5 of human upstream binding factor using the restraint MD
simulations. The thermal intermediate state of high mobility group (HMG) box 5
of human upstream binding factor was detected at 55ºC by experimental technique
(3), but could not be defined for the sparse data. We performed ensemble-averaged
MD simulations to study the intermediate state ensemble of HMG box-5 at 328K
with forty-eight replicas for 2.88 ms. 421 inter-atomic distances derived from NOE
and PRE were used as restraints. The results indicated that the intermediate state
ensemble presented the structural characteristic of box-5 protein intermediate state
and were consistent with experimental results.
993
The two cases show that molecular dynamics simulation is a powerful route to
study structural and thermodynamics characters of the conformational heterogeneous states of protein.
Financial support from the Chinese Natural Science Foundation and Shandong
Natural Science Foundation (30970561and 31000324) are acknowledged.
References
1. M. Vendruscolo. Current Opinion in Structural Biology 17, 15-20 (2007).
2. Z. Cao, L. Liu, and J. Wang. Journal of Biomolecular Structure and Dynamics 28, 343-353
(2010).
3. Z. Liu, J. Zhang, X. Wang, Y. Ding, J. Wu, and Y. Shi. Proteins: Structure, Function
and Bioinformatics 77, 432-447 (2009).
Swarm Intelligence: Cooperative Replica Methods for
Prediction of Protein Structure
The use of atomistic simulation techniques to directly resolve protein tertiary
structure from primary amino acid sequence is hindered by the rough topology of
the protein free energy surface and the resulting simulation timescales required.
An interesting approach to improve the exploration of this surface, termed the
SWARM-MD method, was proposed by Huber and van Gunsteren (1). Their
approach is inspired by particle swarm methods. Introduced in 1995 and originally applied to electrical circuit design, the particle swarm algorithm mimics the
social behaviour of bird flocking or fish schooling. The efficiency of the algorithm’s search behaviour, which exhibits remarkable organization and planning,
can be traced to cooperativity of individual members of the swarm, influenced by
their memory and that of their peers. In SWARM-MD, the free energy surface is
smoothed through the use of a swarm of multiple interacting simulation replicas
that are driven towards the average conformation of the swarm members. We have
successfully applied this approach to prediction of the native states of a series of
model peptides (2), including Trp-cage miniprotein in aqueous solvent. In each
case, the cooperation between replicas was found to improve the convergence of
the simulations towards the native state. Limitations and future directions of this
method will be discussed.
Neil J. Bruce
Richard A. Bryce*
22
School of Pharmacy and
Pharmaceutical Science,
University of Manchester,
Manchester, M13 9PT, UK
*[email protected]
This research has been supported by the EPSRC.
References
1. T. Huber and W. F. van Gunsteren. J Phys Chem A 102, 5937-5943 (1998).
2. N. J. Bruce and R. A. Bryce. J Chem Theory Comput 6, 1925-1930 (2010).
Thermodynamics of Waters Sequestered
Inside Macromolecules
The talk will discuss application of concepts developed while working in the
Beveridge Laboratory. Grand-canonical ensemble simulations (1, 2) were shown to
be well suited for the exploration of solvent occupancy of isolated pockets inside
macromolecules (3). From the ensemble of configurations thus generated, the concept of generic sites (4) serves to identify distinct water sites inside proteins. The
thermodynamics if different waters will be characterized both using the technique
developed in the Lazaridis Laboratory (5, 6) and by comparison of occupancies
from simulations run at different chemical potentials. The work is done in collaboration with Roman Osman.
Mihaly Mezei
23
Department of Structural and
Chemical Biology,
Mount Sinai School of Medicine,
NY 10029, New York,
[email protected]
994
References
1.
2.
3.
4.
5.
6.
24
Lian-Ming Liang1
Ke-Qin Zhang1
Shu-Qun Liu1,2*
1 Laboratory
for Conservation and
Utilization of Bio-Resources & Key
Laboratory for Microbial Resources of
the Ministry of Education, Yunnan
University, Kunming 650091,
P. R. China
2 Sino-Dutch
Biomedial and Information
Engineering School, Northeastern
University, Shenyang 110003,
P. R. China
*[email protected]
D. J. Adams. Molec Phys 29, 307-311 (1975).
M. Mezei. Mol Phys 61, 565-582 (1987); Erratum, 67, 1207-1208 (1989).
H. Resat and M. Mezei. J Am Chem Soc 116, 7451-7452 (1994).
M. Mezei and D. L. Beveridge. J Comp Chem 6, 523-527 (1984).
Z. Li and T. Lazaridis. J Phys Chem B 109, 662-670 (2005).
Z. Li and T. Lazaridis. J Phys Chem B 110, 1464-1475 (2006).
Role of Disulfide Bonds in Structural Stability and
Flexibility of Cuticle-degrading Proteases from
Nematophagous Fungi—A Molecular Dynamics
Simulation Study
It has been shown that disulfide bonds play an important role in the stability of some
proteins by an entropic effect (1), usually the globular proteins secreted to extracellular medium (2). Two cuticle-degrading proteases, Ver112 and PII, which were
derived respectively from nematode-parasitic and nematode-trapping fungi, belong
to the subtilisin family sharing relatively high sequence identity (45.7%). Ver112
is an alkaline protease and has two disulfide bonds, C35-C124 and C179-C250 (3);
PII is a neutral protease and has no disulfide bond. Despite the minor structural
difference between them (root mean square deviation (RMSD) is ~0.6 Å), Ver112
displays higher thermal stability and stronger nematicidal/catalytic activity than PII
does (4).
In order to investigate how the disulfide bonds influence structural stability
and flexibility of these two proteases, molecular dynamics simulations on their
structures of wild-type and disulfide bond-disrupted mutant (Ver112_124C/A,
Ver112_179C/A, and Ver112_124C/A_179C/A) were performed at temperatures
300 K and 400 K, respectively. Analyses of the geometrical properties along the
300 K MD trajectories indicate that PII has higher average values of Cα RMSD and
solvent accessible surface area (SASA) while lower average values of number of
native hydrogen bonds (NNH) and number of native contacts (NNC), suggesting
a higher flexibility and less compact equilibrium structure of PII in comparison
with Ver112. This may be caused by the lack of equivalent disulfide bonds in
PII. The geometrical properties of Ver112 are similar on average to those of its
three mutants during simulations at 300 K, while at 400 K the wild-type Ver112
presents more NHB and NNC and less SASA than its mutants suggesting that
disulfide bonds contribute to the global stability of Ver112 at high temperature.
Additionally, the stability of local structures within 5 Å of the two disulfide bonds
C35-C124 and C179-C250 was also enhanced, as indicated by their increased
RMSF and decreased NNC values upon disulfide bond breaking. Analyses of the
average RMSF values of the S1 and S4 substrate-binding pockets show that upon
disruption of C35-C124, RMSF of S1 pocket decreased by 21.2% while that of
S4 pocket showed almost no change; upon disruption of C179-C250, the relatively large reduction in flexibility of both S1 and S4 pockets was observed; and
the most pronounced reduction (30.7% and 17.2%) occurred when both disulfide
bonds were broken. According to these results, we can conclude that i) the presence of disulfide bonds enhances not only the local but also the global stability of
the protease, thus explaining the higher thermal stability of the alkaline protease
Ver112 compared to that of the neutral protease PII; ii) the presence of disulfide
bonds increases the flexibility of substrate-binding pockets located relatively far
from disulfide bonds, thus explaining why alkaline proteases have higher substrate
affinity (5, 6) and catalytic activity than neutral proteases.
995
This research was supported by grants from NSFC (No. 30860011) and Yunnan
province (2007PY-22), and foundation for Key Teacher of Yunnan University.
References
1. M. Matsumura, G. Signor, and B. W. Matthews. Nature 342, 291-293 (1989).
2. C. S. Sevier, and C. A. Kaiser. Nat Rev Mol Cell Biol 3, 836-847 (2002).
3. L. M. Liang, Z. Y. Lou, F. P. Ye, J. K. Yang, S. Q. Liu, Y. N. Sun, Y. Guo, Q. L. Mi,
X. W. Huang, C. G. Zou, Z. H. Meng, Z. H. Rao, and K. Q. Zhang. FASEB J 24,
1391-1400 (2010).
4. L. M. Liang, S. Q. Liu, J. K. Yang, Z. H. Meng, and K. Q. Zhang. FASEB J (2011) (in
press).
5. S. Q. Liu, Z. H. Meng, Y. X. Fu, and K. Q. Zhang. J Mol Model 17, 289-300 (2011).
Thinking into Mechanism of Protein Folding and
Molecular Binding
Protein folding and molecular binding provide the basis for life on earth. The native
3D structure of a protein is a prerequisite for its function; and the molecular binding
is the fundamental principle of all biological processes (1). Therefore unraveling
the mechanisms of protein folding and binding is fundamental to describing life at
molecular level. Of particular interest is that protein folding and binding are similar
processes because the only difference between them is the presence and absence
of the chain connectivity. Among many models (such as diffusion-collision (2),
hydrophobic collapse (3) and stoichiometry (4) models) proposed to describe the
mechanism of these two processes, the “folding funnel” (5) model (Figure 1) is
most widely accepted. In this model, protein folding can be viewed as going down
the free energy hill through multiple parallel pathways towards the bottom of the
funnel (6); and molecular binding can occur along rough free energy surface around
the funnel bottom, especially for binding between flexible proteins/molecules.
These are essentially thermodynamically controlled processes involving various
types of driving forces, including the enthalpic contribution of noncovalent bond
formations, entropic effects such as solvent release and burial of apolar surface
area (hydrophobic effect), restrictions of degrees of freedom of protein/ligand, and
loss of rotational and translational freedom of interacting partners. Briefly, these
two processes, which are driven by a decrease in total Gibbs free energy (ΔG), are
dictated by the mechanism of a delicate balance of the opposing effects of
enthalpic (ΔH) and entropic (ΔS) contributions (equation 1).
DG 5 DH2TDS
Xing-Lai Ji1,2
Shu-Qun Liu1,2*
1Laboratory
25
Laboratory for Microbial Resources
of the Ministry of Education,
Yunnan University, Kunming 650091,
P. R. China
2 Sino-Dutch
P. R. China
*[email protected]
1
Here we emphasize that it is the thermodynamically driven subtle enthalpyentropy compensation that leads to the global free energy minimum of the
protein/ligand-solvent system (7), and that the specific inter-atomic interactions observed in the folded or complexed structure are to large extent the
consequence of thermodynamic equilibrium but can not fully define the driving forces for folding and binding interactions.
Interestingly, we speculate that many other processes can be explained by
thermodynamic enthalpy-entropy compensation, i.e., the Yin and Yang balance in traditional Chinese medicine theory could correspond to the enthalpy
and entropy compensation of the second law of thermodynamics; global
warming can be considered as the consequence of excessive production of
positive entropy (carbon dioxide) from chemically ordered fossil fuel, urging people to slow resource consumption to delay the inevitable death by
entropy.
Figure 1: Schematic 2D funnel of protein folding and
binding (modified from (6)).
996
A deeper understanding of mechanism of biological processes from thermodynamic point of view can facilitate greatly the understanding of life and rational
drug design in the post-genomic times.
province (2007PY-22), and foundation for Key Teacher of Yunnan University and
SRF for ROCS, SEM.
References
1.
2.
3.
4.
R. Perozzo, G. Folkers, and L. Scapozza. J Recept Signal Transduct Res 24, 1-52 (2004).
M. Karplus and D. L. Weaver. Protein Sci 3, 650-668 (1994).
V. R. Agashe, M. C. Shastry, and J. B. Udgaonkar. Nature 377, 754-757 (1995).
A. Mittal, B. Jayaram, S. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142
(2010).
5. P. E. Leopold, M. Montal, and J. N. Onuchic. Proc Natl Acad Sci USA 89, 8721-8725
(1992).
6. J. M. Yon. J Cell Mol Med 6, 307-327 (2002).
7. X. L. Ji and S. Q. Liu. J Biomol Struct Dyn 28, 621-623 (2011).
26
Molecular Motions of Proteins Play Crucial Role in
their Function
Shu-Qun Liu1,2*
Shi-Xi Liu3
Zhao-Hui Meng1,4
Yan Tao1
Ke-Qin Zhang1
Yun-Xin Fu1,5
1 Laboratory
Laboratory for Microbial Resources of
the Ministry of Education, Yunnan
University, Kunming 650091, P. R. China
2 Sino-Dutch
P. R. China
3
School of Chemical Science and
Technology, Yunnan University,
Kunming 650091, P. R. China
4
Department of Cardiology, No. 1
Affiliated Hospital, Kunming Medical
College, Kunming 650032, P. R. China
5
Human Genetics Center, The University
of Texas Health Science Center, Houston,
TX 77030, USA
*[email protected]
Proteins, which are the materials central to cellular function, should not be regarded
simply as static pictures as determined by X-ray crystallography. They are dynamic
entities in cellular solution with functions governed ultimately by their dynamic
character (1). Therefore a complete understanding of the structure-function relationship of a protein requires an analysis of its dynamic behavior and molecular
motion.
Using molecular dynamics (MD) simulation or CONCOORD (2) approach, the
dynamic behaviors of HIV-1 gp120 envelope glycoprotein and serine protease proteinase K were investigated. Apart from analyses of the conventional structural
properties during simulations, the essential dynamics analysis method was used to
study the large concerted motions of these two proteins, including the influence of
ligand bindings or residue mutations on molecular motions. The results revealed
that i) the proteinase K shows relatively rigid internal core with some highly flexible surface loops forming the substrate-binding region, supporting the induce-fit
or conformational selection mechanism of substrate binding (3); ii) the removal
of Ca2+ cations from proteinase K increases the global conformational flexibility,
decreases the local flexibility of substrate-binding region and does not influence the
thermal motion of catalytic triad, thus explaining the experimentally determined
decreased thermal stability, reduced substrate affinity and almost unchanged catalytic activity upon Ca2+ removal (4); iii) the substrate binding affects the large
concerted motions and flexibility behavior of proteinase K suggesting that the variations in substrate-pocket motions can be connected to substrate binding, catalysis and product release (5); amino acid mutations 375 S/W and 423 I/P of HIV-1
gp120 have distinct effects on molecular motions of gp120 (6), facilitating 375 S/W
mutant to adopt the CD4-bound conformation while 423 I/P mutant to prefer for
CD4-unliganded state (7). Analyzing the dynamic character of proteins not only is
important for the characterization of the functional properties of proteins but also
facilitates the reasonable interpretation of experimentally determined structural,
biochemical and biological data.
province (2007PY-22), and foundation for Key Teacher of Yunnan University.
997
References
1. K. Henzler-Wildman and D. Kern. Nature 450, 964-972 (2007).
2. B. L. de Groot, D. M. F. van Aalten, R. M. Scheek, A. Amadei, G. Vriend, and
H. J. C. Berendsen. Proteins 29, 240-251 (1997).
6. S. Q. Liu, S. X. Liu, and Y. X. Fu. J Mol Model 14, 857-870 (2008).
7. S. Q. Liu, C. Q. Liu, and Y. X. Fu. J Mol Graphics Modell 26, 306-318 (2007).
Origins of the Mechanical Stability of the C2 Domains
in Human Synaptotagmin 1
Synaptotagmin 1 (Syt1) induces the buckling of plasma membrane during neurotransmitter release at the synapse (1). Therefore, elucidating the mechanical
properties of Syt1 is essential for understanding its biological function in synaptic
response. Syt1 contains two homologous cytoplasmic domains, C2A and C2B. We
employed a self-organized polymer (SOP) model of a protein chain (2) to carry out
molecular simulations, implemented on a CPU and on a GPU (Graphics Processing
Unit) (3), using experimental pulling speeds. The forced unfolding of isolated C2A
and C2B domains occurs under comparable forces starting from their C-terminal
ends, but according to different pathways. Our results for the behavior of the C2A
domain correlate very well with dynamic spectroscopy experimental studies (4, 5),
but no direct measurements of the mechanical behavior of the isolated C2B domain
exist to date. Thus, to confirm the presence of the pathways generated with the
SOP model, we also carried out implicit solvent model simulations. Atomic force
microscopy (AFM) experiments found an increase in the critical unfolding force
of C2B when joined with C2A in the Syt1 molecule (4), which was proposed to
result from the contribution of the C2A-C2B interface. However, our simulations
reveal that the presence of an intact interface does not lead to the unfolding of Syt1
according to the AFM experiments. In contrast, we discovered that the presence of
linkers used in the experimental set-up plays a crucial role in the behavior of this
synaptic protein complex and, their inclusion in simulations as well leads to data
that fully matches the experiments. Interestingly, we found that the stabilization
effect of the linker on the C2B domain alters not only the critical force, but also
the unfolding pathways of both C2 domains. Our findings provide insights into the
relative conformation variability of the C2 domains and the origins of stability of
the Syt1 protein.
Li Duan1*
Artem Zhmurov2
Valeri Barsegov2
Ruxandra I. Dima1
1
27
OH 45221, Cincinnati,
2Department
of Chemistry,
University of Massachusetts,
Lowell MA 01854, Lowell,
*[email protected]
998
References
1.
2.
3.
4.
5.
28
B. Jayaram
A. Mittal
Department of Chemistry & School of
Biological Sciences,
Indian Institute of Technology,
Hauz Khas, New Delhi-110016, India
[email protected]
[email protected]
S. Martens, M. M. Kozlov, and H. T. McMahon. Science 316, 1205-1208 (2007).
C. Hyeon, R. I. Dima, and D. Thirumalai. Structure 14, 1633-1645 (2006).
A. Zhmurov, R. I. Dima, Y. Kholodov, and V. Barsegov. Proteins 78, 2984-2999 (2010).
K. L. Fuson, L. Ma, R. B. Sutton, and A. F. Oberhauser. Biophys J 96, 1083-1090 (2009).
M. Carrion-Vazquez, P. E. Marszalek, A. F. Oberhauser, and J. M. Fernandez. PNAS 96,
11288-11292 (1999).
Universality of the Spatial Distribution of the
Backbones and a Narrow Band of Amino Acid
Stoichiometries Amidst the Structural and Functional
Diversity of Folded Proteins
Is there a universal principle guiding protein folding besides the thermodynamic
hypothesis of Anfinsen? Rigorous analyses of several thousand crystal structures of
folded proteins reveal a surprisingly simple unifying principle of backbone organization (1, 2). We find that protein folding is a direct consequence of a narrow band
of stoichiometric occurrences of amino-acids in primary sequences, regardless of
the size and the fold of the protein. Furthermore, the amino acid residues in folded
proteins display novel and invariant neighborhoods, independent of their locations
(e.g. center vs. periphery) in contrast to anticipations from preferential interaction
theories (3). These findings present a compelling case for a newer view of protein
folding which takes into account solvent mediated and amino acid shape and size
assisted optimization of the tertiary structure of the polypeptide chain to make a
functional protein.
References
1. A. Mittal, B. Jayaram, S. R. Shenoy, and T. S. Bawa. J Biomol Struct Dyn 28, 133-142
(2010).
2. A. Mittal and B. Jayaram. J Biomol Struct Dyn 28, 669-674 (2011) and references therein.
3. A. Mittal and B. Jayaram. J Biomol Struct Dyn 28, 443-454 (2011).
999
Largescale Protein Properties: Crystallization,
Amyloid Formation, and the Stability of the Proteome
We are interested in largescale protein behaviors. By largescale, we mean processes such as amyloid aggregation or protein crystallization that involve multiple
proteins associating into complexes, or collective properties of the thousands of
proteins in whole proteomes. There are many experimentally measurable properties
for which some basic insights do not require large computer simulations of atomically detailed models.
Protein crystallization. Why do proteins crystallize? Under typical crystallization
conditions, two protein molecules have charges of the same sign, so they should
repel. So, the usual explanation for protein crystallization is that proteins also have
a neutral sticking energy that overcomes the charge-charge repulsion. However,
more is needed in order to account for the strong observed dependence of crystal
stability on salt, which is of practical importance for crystallizing proteins. We
treat the crystallization of proteins, such as lysozyme, as an association of charged
spheres in the presence of salts (1). The model is in good agreement with lysozyme
crystal solubility data as functions of temperature, pH, and salt. A key conclusion is
that because the crystal is macroscopic, it must be electroneutral, so: (a) increasing
the protein’s charge has little effect on its crystal stability because counterions are
sequestered in proportion to the charge, and (b) the reason that crystals melt upon
heating is because it melts the counterion `glue’, not because it melts the proteinprotein interactions.
Amyloid aggregation. What are the forces of amyloid aggregation? We consider
3 states in equilibrium: monomeric amyloid peptide molecules, oligomers (micellelike loose clusters of a few peptide chains), and fibrils (ordered fibers of many
chains). We suppose the chains are driven by hydrophobic interactions into the
oligomeric state and by additional interactions due to steric zipping (packing plus
hydrogen bonding) into the fibrillar state (2). The model predicts two transitions,
monomer to oligomer, and oligomer to fibril, the latter of which, interestingly, is
predicted to be essentially independent of peptide concentration. The model predicts that if the oligomers are the toxic species, then the fibrils are ‘good guys’,
because they soak up oligomeric chains and buffer their concentration. The model
resolves an experimental puzzle from two research groups regarding denaturant
disruption. Slightly different peptide concentrations lead to either stable oligomeric
intermediates, or none. Model predictions are in good agreement with dependences
on salt and pH.
Proteome stability. We compute the stabilities of all the proteins in various proteomes, including Ecoli, worm and yeast based on a simple thermodynamic parameterization of the database of known protein stabilities (3). We find that proteomes
are marginally stable. That is, even though thousands of proteins have a stability
averaging around 6 kcal/mol, about 650 of Ecoli’s proteins are less stable than
4 kcal/mol. Stability distributions such as these are useful for computing the temperature at which the whole proteome denatures, which coincides closely with the
experimental temperatures of cell death.
References
1. 1. J. D. Schmit and K. A. Dill. J Phys Chem B 114, 4020-4027 (2010).
2. 2. J. Schmit, K. Ghosh, and K. A. Dill. Bio phys J 100, 450-458 (2011).
3. 3. K. Ghosh and K. A. Dill. Bio phys J 99, 3996-4002 (2010).
Ken A Dill1*
Kings Ghosh2
Jeremy Schmit3
1Stony
29
Brook University
2Denver
University
3University
of California at San Francisco
*[email protected]
1000
30
Sanjeev Kumar Singh*
Sunil Kumar Tripathi
Department of Bioinformatics,
Alagappa University,
Karaikudi, Tamil Nadu, 630003, India
*[email protected]
Virtual Screening and Theoretical Activity Prediction
of Idenopyrazole Derivatives of CDK2 Inhibitors:
A QPLD and MM-GBSA approach
Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery
that govern the transition between phases during cell cycle progression. Genes
involved in cell cycle are frequently mutated in human cancer and deregulated CDK
activity represents a characteristic of malignancy. Among them CDK2 is essential
in the mammalian cell cycle and is required to complete G1 and to activate the
S phase (1). Lately there have been successful implementation of computer-aided
drug design to develop new therapeutics (2-5), and we have employed these designs
to develop anticancer drugs targetting CDK2. In this study we have taken 119 compounds of idenopyrazole derivatives, with in vitro biological activity data for CDK2
inhibition (6). Here, we used Virtual screening protocol to obtain effective idenopyrazole derivatives of CDK2 via VS workflow of Schrodinger package, ADME and
Lipinski- filter options. 17 top-ranked molecules obtained from this screening were
subjected for Molecular docking with GLIDE module and Quantam Polarized Ligand
Docking (QPLD) using QPLD module of Schrodinger package (7, 8).
The top-ranked 17 compounds were post-scored using molecular mechanics and
continuum solvation (MM-GBSA) (9, 10). The validity of the virtual screening
protocol was supported by (i) Testing of the MM-GBSA procedure (ii) Agreement
between predicted and crystallographic binding poses (iii) Recovery and identification of top-scoring potent idenopyrazole derivatives. With these combined
approach of ADME, Docking, QPLD and MM-GBSA, we found that compound
13, 24, 25 and 46 having properties essential for effective drugs so biological activity testing can be carried out on selected leads, for getting effective and potent
anticancer drug.
Figure: Compound 48 showing interaction with Glu12, Asn132 and Asp145 of CDK2.
1001
References
1. M. Malumbres and M. Barbacid. Nat Rev Cancer 9(3), 153-166 (2009).
2. C. Y. Chen. J Biomol Struct Dyn 27, 627-640 (2010).
3. C. Y. Chen, Y. H. Chang, D. T. Bau, H. J. Huang, F. J. Tsai, C. H. Tsai, and C. Y. C. Chen.
J Biomol Struct Dyn 27, 171-178 (2009).
4. H. J. Huang, K. J. Lee, H. W. Yu, C. Y. Chen, C. H. Hsu, H. Y. Chen, F. J. Tsai, and
C. Y. C. Chen. J Biomol Struct Dyn 28, 23-37 (2010).
5. H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, and C. Y. C. Chen. J Biomol Struct
Dyn 28, 187-200 (2010).
6. S. K. Singh, N. Dessalew, P. V. Bharatam. Eur J Med Chem 41(11), 1310-1319 (2006).
7. R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T. Mainz,
M. P. Repasky, E. H. Knoll, M. Shelley, J. K. Perry, D. E. Shaw, P. Francis, P. S. Shenkin.
J Med Chem 47, 1739-1749 (2004).
8. A. E. Cho, V. Guallar, B. J. Berne, R. Friesner. J Comput Chem 26, 915-931 (2005).
9. P. D. Lyne, M. L. Lamb, J. C. Saeh. J Med Chem 49, 4805-4808 (2006).
10. G. Barreiro, C. R. Guimarães, I. Tubert-Brohman, T. M. Lyons, J. Tirado-Rives,
W. L. Jorgensen. J Chem Inf Model 47, 2416-2428 (2007).
Homologous and Heterologous Crystallin
Interactions in Cataract
Age-related cataract is the most common cause of blindness worldwide. Nearly
fifty percent of Americans above the age of 75 are diagnosed with this disease,
and surgical intervention is the sole method of treatment at present. In the developing world, even this treatment is not readily available. These are compelling
reasons to search for better treatments to delay, prevent or arrest cataract formation. Recent evidence suggests that age-related cataracts also have a genetic
component (1). Therefore, determining the mechanisms underlying genetic cataracts with a known association to a protein-mutation is one important strategy
towards understanding the molecular basis for cataract formation. This approach
has the added advantage of addressing mechanisms of congenital and childhood
cataracts which are difficult to treat because surgical intervention frequently
leads to serious consequences (2). For these reasons, we have been determining
the molecular mechanisms underlying a number of genetic cataracts by studying
the mutant proteins associated with them.
The most common human cataract-associated mutations occur in γD-crystallin
(HGD). In the singly-substituted HGD mutants we have studied so far, we find
that the protein structure and stability do not change, despite a significant lowering
in protein solubility in most cases, which leads to the formation of a condensed
phase and consequent light scattering and opacity. One example is the Pro23 to
Thr (P23T) mutation in HGD in which the mutant protein shows a dramatically
lowered solubility, which is about 1/100th of that of the normal protein. Moreover,
the solubility profile of P23T is “retrograde”  i.e. it increases as the temperature
decreases. For this mutant we have shown that hydrophobic surface patches emerge
as a result of the mutation, which are largely responsible for its retrograde solubility and aggregation (3). Recently, we have identified the residues, Tyr16, His22,
Asp21, and Tyr50, in the P23T mutant that give rise to novel hydrophobic surface,
as well as several residues where backbone fluctuations in different time-scales
are restricted, providing a comprehensive understanding of how lens opacity could
result from this mutation. For P23T and several other mutants, we have shown
that changes in the homologous protein-protein interactions (or self-association)
are responsible for the formation of the distinct condensed phase, which in turn is
likely to lead to light scattering in the cataractous lens.
Priya R. Banerjee
Jayanti Pande*
University at Albany,
Albany, NY 12222
[email protected]
*[email protected]
31
1002
In an interesting recent development, we found that changes in homologous interactions leading to protein condensation and light scattering may not be the only
mechanism leading to cataract (4). In our study of the Glu107 to Ala (E107A) mutant,
we found that not only is the mutant protein very similar in structure and stability to
the normal protein  as in all the previous cases we studied  but it is also as soluble
as the normal protein (5). In fact, the E107A mutant does not form a condensed protein phase that could account for light scattering and cataract. However, mixtures of
the mutant with another lens crystallin, namely α−crystallin, show increased light
scattering compared to normal α−γ−crystallin mixtures. There is also a striking difference in the liquid-liquid phase separation behaviors: The two coexisting phases
in the E107A−α mixtures differ much more in protein density than those that occur
in HGD−α mixtures. In HGD−α mixtures, the de-mixing of phases occurs primarily by protein type while in E107A−α mixtures it is increasingly governed by
protein density. Therefore, here it is clearly the heterologous attractive interactions
between two different crystallins in the lens that lead to increased light scattering.
It has been known for some time that the attractive interactions between various
crystallins are optimized for stability, and any change  attractive or repulsive 
is likely to lead to instability (5). Our data (5) support these theoretical predictions and emphasize the importance of examining detailed molecular mechanisms
to build a comprehensive understanding of a complex disease. A brief commentary
on our work is presented in (6).
References
1. C. J. Hammond, H. Snieder, T. D. Spector, and C. E. Gilbert. N Engl J Med 342, 1786-1790
(2000).
2. C. Zetterstrom, A. Lundvall, and M. Kugelberg. J Cataract Refract Surg 31, 824-840
(2005).
3. A. Pande, K. S. Ghosh, P. R. Banerjee, and J. Pande. Biochemistry 49, 6122-6129 (2010).
4. P. R. Banerjee, A. Pande, J. Patrosz, G. M. Thurston, and J. Pande. Proc Natl Acad Sci USA
108, 574-579 (2010).
5. A. Stradner, G. Foffi, N. Dorsaz, G. Thurston, and P. Schurtenberger. Phys Rev Lett 99,
198103 (2007).
6. N. Asherie. Proc Natl Acad Sci USA 108, 437-438 (2010).
32
Adeleh Divsalar1*
Sajedeh Ebrahim-Damavandi2
Ali Akbar Saboury2
Hassan Mansouri- Torshizi3
1Department
of Biological Sciences,
Tarbiat Moallem University, Tehran, Iran
2Institute
of Biochemistry and
Biophysics, University of Tehran,
Tehran, Iran
3Department
Sistan & Baluchestan, Zahedan, Iran
*[email protected]
Improving in Digestion and Antioxidant Activity of
b-lactoglobulin Using Newly Designed Copper
Complexes as Artificial Proteases
Beta-lactoglobulin (BLG) is a major protein of whey; it is a major carrier protein
and is known to interact with Pd(II) anti-tumor compounds (1). It has wide application as a food ingredient (2-3). Stable structure of bovine whey proteins, especially BLG, restricts their hydrolysis by proteases and it leads to allergy reactions
(4). Now-a-days, the design of synthetic metallo-proteases that cleave proteins at
a specific site has elicited much interest (5). Hence, in the present investigation,
we have decided to design and synthesize a new class of cooper(II) complexes
({Cu(bpy)Cl2}, {Cu(bpy)2}Cl2, {Cu(dien)OH2}(NO3)2 and {Cu(trien)}(NO3)2) as
artificial protease in order to hydrolyze resistant BLG. We also examine the antioxidant activity of fragmented BLG using SDS-PAGE and different spectrophotometric methods (Fluorescence and UV-Visible). Incidentally copper complexes
are very important in biological systems; they have been used to locate nucleosome
positioning (6), they occur widely in oxidative proteins such as laccases (7), and
participate in many many cellular processes and are implicated in pathogenesis of
many diseases (8).
1003
SDS -PAGE of BLG incubated with different Cu(II) complexes for 30 h represented fragmentation of the protein. Also, increasing fluorescamine intensity measurements prove hydrolysis or fragmentation of protein in the presence of different
Cu(II) complexes. SDS-PAGE and fluorescamin studies show that complex 3
has higher protease activity against BLG. The antioxidant activities of native and
hydrolyzed BLG were determined by an ABTS°+ radical cation assay. The degree
of decolorization of ABTS radical induced by native and hydrolyzed BLG resulting from different Cu(II) complexes were compared to that induced by Trolox.
Results have represented that hydrolyzed BLG using complexes 2 and 3 exhibit
signiﬁcantly greater antioxidant activity than the other hydrolyzed or native BLG,
probably signifying the greater number of solvent-exposed amino acids available
for scavenging of the free radicals.
From above results, it can be concluded that our new designed Cu(II) complexes
have artificial protease activities against model protein of BLG. Also, hydrolysing
of BLG can enhance its fragmentation by proteases and increase its antioxidant
activity.
References
1. A. Divsalar, A. A. Saboury, H. Mansoori-Torshizi, M. I. Moghaddam, F. Ahmad,
G. H. Hakimelahi. J Biomol Struct Dyn 26, 587-597 (2009).
2. S. C. Cheison, M. Schmitt, L. Elena, T. Letzel, and U. Kulozik. Food Chemistry 121, 457467 (2010).
3. L. Tavel, C. Moreau, S. Bouhalla, and E. Li-Chan. Food Chemistry 119, 1550-1556 (2010).
4. M. Besler, P. Eigenmann, and R. H. Schwartz. Food Allergens 4, 199-106 (2002).
5. M. S. Kim and J. Suh. Bull Korean Chem Soc 26, 1911-1919 (2005).
713-724 (2010).
7. M. T. Cambria, D. Di Marino, M. Falconi, S. Garavaglia, and A. Cambria. J Biomol Struct
Dyn 27, 501-509 (2010).
8. H. F. Ji and H. Y. Zhang. J Biomol Struct Dyn 26, 197-201 (2008).
SANJEEVINI-A Lead Molecule Design Software
The ability of macromolecules to bind to their substrates in a highly specific manner
is an important feature in many biological processes. The challenge for computer
aided drug discovery is to achieve this specificity - with small molecule inhibitors in binding to their biomolecular targets, at reduced cost and time while ensuring
synthesizability, novelty of the scaffolds and proper ADMET profiles. Sanjeevini
is a drug design software suite (1-7) developed to provide a computational pathway
for automating lead molecule design. A sample of the various current approaches
to drug design can be found in many of the recent articles published in this Journal, for example references 8-11. Our methodology treats macromolecular target
and the lead compound at the atomic level and solvent as a dielectric continuum.
It comprises several modules for diverse functionalities such as automated identification of potential binding sites (active sites) for the ligands (5), a rapid screening for identifying good candidates for any target protein from a million molecule
database (6), optimization of their geometries and determination of partial atomic
charges using quantum chemical / in-house methods (7), docking the candidates
in the active site of target via Monte Carlo methods (4-5), estimating binding free
energies through empirical scoring functions (1-2), followed by rigorous analyses
of the structure and energetics of binding for further lead optimization. Each module is individually validated on a large data set of protein-ligand and DNA-ligand
complexes with known structures and binding affinities. The Sanjeevini software is
freely accessible at http://www.scfbio-iitd.res.in/sanjeevini/sanjeevini.jsp.
Goutam Mukherjee
Tanya Singh
B. Jayaram*
33
Department of Chemistry and
Supercomputing Facility for
Bioinformatics and Computational
Biology, Indian Institute of Technology,
*[email protected]
1004
References
1. T. Jain and B. Jayaram. FEBS Letters 579, 6659-6666 (2005).
2. S. A. Shaikh and B. Jayaram. J Med Chem 50, 2240-2244 (2007).
3. S. A. Shaikh, T. Jain, G. Sandhu, N. Latha, and B. Jayaram. Current Pharmaceutical Design
13, 3454-3470 (2007).
4. A. Gupta, A. Gandhimathi, P. Sharma, and B. Jayaram. Protein and Peptide Letters 14,
632-646 (2007).
5. T. Singh, D. Biswas, and B. Jayaram. “An Automated Active Site Identifier”, Manuscript
in preparation.
6. G. Mukherjee and B. Jayaram. “A Rapid Identification of Hit Molecules for Target Proteins
via Physico-Chemical Descriptors”, Manuscript in preparation.
7. G. Mukherjee, N. Patra, P. Barua, and B. Jayaram. Journal of Computational Chemistry,
32, 893-907 (2011).
8. T. T. Chang, H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, M. F. Sun, and
9. A. K. Kahlon, S. Roy, and A. Sharma. J Biomol Struct Dyn 28, 201-210 (2010).
10. T. C. Ramalho, M. V. J. Rocha, E. F. F. da Cunha, L. C. A. Oliveira, and K. T. G. Carvalho.
11. L. I. D. S. Hage-Melim, C. H. T. D. P. Da Silva, E. P. Semighini, C. A. Taft, and S. V. Sampaio.
1005
Inferred Biomolecular Interaction Server–A Method
and a Server to Analyze and Predict Protein
Interacting Partners and Binding Sites
Recently we have developed a new database and method called “IBIS” (Inferred
Biomolecular Interaction Server, http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.
cgi) which enables to conveniently study biomolecular interactions that have been
observed in protein structures. Moreover, through inference by homology IBIS
allows to formulate predictions/hypotheses for biomolecular interactions, even if
the data for specific biomolecules is not available. Similar binding sites are clustered together based on their sequence and structure conservation. To emphasize
biologically relevant binding sites, several algorithms are used for verification in
terms of evolutionary conservation, biological importance of binding partners, size
and stability of interfaces, as well as evidence from the published literature. IBIS
organizes, analyzes and predicts interaction partners and locations of binding sites
in proteins for five different types of binding partners (protein, chemical, nucleic
acid, peptides and ions), and facilitates the mapping of a comprehensive biomolecular interaction network for a given protein query. The method was validated by
comparison to other binding site prediction methods and to a collection of manually curated annotations. It has been shown that it achieves a sensitivity of about
72-75% at predicting biologically relevant sites for protein-small molecule and
protein-protein interactions.
34
Benjamin A. Shoemaker
Dachuan Zhang
Ratna R. Thangudu
Manoj Tyagi
Jessica H. Fong
Aron Marchler-Bauer
Stephen H. Bryant
Thomas Madej
Anna R. Panchenko*
National Center for Biotechnology
Information, National Institutes of
Health, Bethesda, MD 20894, USA
*[email protected]
1006
This research has been supported by the National Institutes of Health/DHHS (Intramural Research program of the National Library of Medicine).
References
1. Shoemaker, et al. Nucleic Acids Res Jan;38:D518-24 (2010).
2. Thangudu, et al. BMC Bioinformatics Jul 1;11:365 (2010).
35
Integrating CADD Methodologies for the Design of
Novel COMT Inhibitors
Nidhi Jatana
Aditya Sharma
N. Latha*
Catechol-O-methyltransferase (COMT) catalyzes the methylation of catecholamines, including neurotransmitters like dopamine, epinephrine and norepinephrine, leading to their degradation. COMT has been a subject of study as it
has implications in numerous neurological disorders like Parkinson’s disease (PD),
depression, schizophrenia and several mood disorders (1).
Bioinformatics Infrastructure Facility,
Availability of crystal structure of human soluble-COMT (S-COMT) (2) has helped
us in modeling of membrane-bound COMT (MB-COMT) and thus paved the way
for the discovery of a novel lead molecule using integrated computer aided drug
design (CADD) methodologies (3-8). Combination of both structure-based (molecular docking, de novo ligand design) and ligand-based (QSAR, pharmacophore
modeling) approaches that models separate facets of the natural system, will allow
us to use all available information to screen a chemical database in a more objective
and meaningful way.
Sri Venkateswara College (University of
Delhi), Benito Juarez Road,
Dhaula Kuan, New Delhi 110021, India
*[email protected]
In this study, we examine the structure of MB-COMT refined by molecular dynamics simulations. We have also conducted detailed computational modeling studies to understand the molecular mechanisms for the catalytic behavior of human
MB-COMT with respect to AdoMet (the methyl donor) and various substrates.
We have also carried out docking studies with known COMT inhibitors to (5, 9)
understand physico-chemical interactions in the protein-ligand complex in order to
identify the optimal binding geometry. The virtual screening procedure has been
implemented in a docking pipeline that performs a step-by-step, target specific,
filtering approach for data reduction to discover inhibitors with novel scaffolds. We
hope that the combination of energy terms from structure-based docking studies
with the chemical knowledge of a ligand-based pharmacophore search will leverage the strengths of both approaches to produce a good diversity of active molecules. Results of these studies will be discussed and presented.
References
1. P. T. Mannisto and S. Kaakkola. Pharmacol Rev 51, 593-628 (1999).
2. K. Rutherford, I. L. Trong, R. E. Stenkamp, and W. W. Parson. J Mol Biol 380, 120-130
(2008).
3. P. G. Mezey. J Mol Model 6, 150-157 (2000).
4. S. Y. Yang. Drug Discov Today 15, 444-50 (2010).
5. H. C. Guldberg and C. A. Marsden. Pharmacol Rev 27, 135-206 (1975).
6. A. Kaur Kahlon, S. Roy, and A. Sharma. J Biomol Struct Dyn 28, 201-210 (2010).
7. H. J. Huang, K. J. Lee, H. W. Yu, C. Y. Chen, C. H. Hsu, H. Y. Chen, F. J. Tsai, and
9. M. J. Bonifacio, P. N. Palma, L. Almeida, and P. S. da Silva. CNS Drug Rev 13, 352-379
(2007).
1007
Liquid State NMR Spectroscopy as a Tool to Obtain
Structural and Binding Information of Drug
Molecules Metabolized by P450BM3 Enzymes
Cytochrome P450 is a heme protein family ubiquitous in all domains of life and
involved in many biological processes, including the degradation and activation of
drugs by catalyzing the monooxygenation of a ligand (1). Cytochrome P450BM3
from Bacillus megaterium is one of the most promising monoxygenases for biotechnological applications because it is the most active P450 so far identified.
Mutated forms of P450BM3 are highly efficient biocatalysts to metabolize steroids
and other drugs in-vitro in order to produce potential new lead compounds (2). In
this contribution, liquid state NMR spectroscopy is shown to be a valuable tool to
provide a structural basis for drug metabolism by P450BM3 enzymes. First, the
regioselective hydroxylation of steroids by P450BM3 enzymes will be discussed.
Secondly, a new methodology for acquiring homonuclear decoupled proton NMR
spectra of drug metabolites will be discussed.
We show that by a single mutation (A82W) in the substrate binding pocket of
P450BM3 mutants, the regioselective hydroxylation of steroids at position 16-ß is
largely increased. Moreover, this enhanced regioselective hydroxylation is further
investigated by means of binding affinity, as studied by UV-VIS spectroscopy, and
the orientation of the steroid testosterone in the heme-active sites of these mutants,
as studied by T1 paramagnetic relaxation NMR. It is shown that testosterone has
an increased binding affinity and preferred orientation in the A82W mutant when
compared to its wild-type counterpart.
NMR spectroscopy is not only a very powerful tool to provide information on
ligand binding, but its main power is to provide structural information on large molecules, like RNA’s, DNA’s and enzymes, and small molecules. The determination
of structures of small molecules, i.e. metabolites which are formed by P450BM3
enzymes, by liquid state NMR is a standardized but complicated procedure (2).
Small molecules often have a dense network of J-coupled protons, which hampers
the assignment of NMR spectra, due to broad multiplets and subsequent spectral
overlap. We will show a novel methodology to acquire homonuclear decoupled
proton NMR spectra with a high resolution (~1 Hz) and higher sensitivity than currently reported for these types of NMR experiments.
36
A. J. Kolkman1*
V. Rea2
J. Draaisma1
M. Tessari1
E. V. Vottero2
K. A. M. Ampt1
J. N. M. Commandeur2
H. Irth3
N. P. E. Vermeulen2
M. Honing4
S. S. Wijmenga1
1Department
of Biophysical Chemistry,
Radboud University Nijmegen
2LACDR
division of Molecular
Toxicology, VU University Amsterdam
3LACDR
division of Biomolecular
Analysis, VU University Amsterdam
4MSD
Research Institute,
Medicinal Chemistry Oss
*[email protected]
References
1. A. W. Munro and D. G. Leys, et al. Trends Biochem Sci 27, 250-257 (2002).
2. J. S. B. de Vlieger and A. J. Kolkman, et al. J Chromatogr B 878, 667-674 (2010).
Structure-function Investigation of Metalloproteinases
Provides Novel Insights into Drug Design
Matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs)
are endopeptidases central to the degradation and remodeling of the extracellular
matrix (ECM). These proteases also exhibit regulatory activity in cell signaling pathways and thus tissue homeostasis under normal conditions and in many diseases (1).
Consequently, individual members of the MMP and ADAM protein families were
identified as important therapeutic targets. However, designing effective inhibitors
in vivo for this class of enzymes appears to be extremely challenging. This is attributed
to the broad structural similarity of their active sites and apparently to the dynamic
functional interconnectivity of MMPs with other proteases, their inhibitors, and substrates (the so-called degradome) in healthy and disease tissues, demonstrated to be
detrimental to the therapeutic rationale (2-3). We present recent advancements in
Netta Sela-Passwell
Alla Trahtenhercts
Irit Sagi*
37
Department of Biological Regulation,
The Weizmann Institute of Science,
Rehovot, 76100, Israel
*[email protected]
1008
our understanding of MMPs structures, dynamics and their function as master regulators (4-7). We highlight the use of structural-kinetic experimental approach for
protein-based drug design strategies, e.g. antibodies and protein scaffolds, targeting
extracatalytic domains, which are central to proteolytic and non-proteolytic enzyme
functions (3). Such rationally designed function blocking inhibitors may create new
opportunities in disease management and in emerging therapies that require control
of dysregulated MMP activity without causing severe side-effects.
References
1. N. Sela-Passwell, G. Rosenblum, T. Shoham, and I. Sagi. Biochim Biophys Acta 1803, 29-38
(2010).
2. I. Sagi and M. Milla. Anal Biochem 372, 1-10 (2008).
3. N. Sela-Passwell, A. Trahtenhercts, A. Krüger, and I. Sagi. Expert opinion on Drug Discovery
(2011) In Press.
4. A. Solomon, B. Akabayov, M. Milla, and I. Sagi. PNAS U S A 104, 4931-4936 (2007).
5. G. Rosenblum, P. Van den Steen, S. R. Cohen, G. J. Grossmann, A. Frenkel, R. Sertchook,
N. Slack, R. W. Strange, G. Opdenakker, and I. Sagi. Structure 10, 1227-36 (2007).
6. G. Rosenblum, P. Van den Steen, S. Cohen, A. Bitler, A. D. D. Brand, G. Opdenakker, and
I. Sagi. PLos One 5, 9-15 (2010).
7. M. Grossman, D. Tworowski, M. Lee, Y. Levy, G. Murphy, and I. Sagi. Biochemistry 49,
6184-6192 (2010).
38
Hridoy R Bairagya*
Bishnu P Mukhopadhyay**
Payel Mallik
Archana K. Srivastava1
Department of Chemistry, National
Institute of Technology–Durgapur,
W.B., 713 209, India
*[email protected]
**[email protected]
Conserved Water Mediated H-bonding Dynamics of
Carboxamide group in NAD to Catalytic Asp 274 and
His 93 in Human IMPDH
The Inosine Monophosphate Dehydrogenase (hIMPDH)-II of human is led to
new interest as an excellent target for the design of isoform specific anti leukemic agent (1, 2). The stereodynamics and recognition of mono and di nucleotide
binding pockets (3) through one conserved water molecular center (4) and the participation of catalytic Arg 322 through a conserved water molecular triad (5) seem
to be important concerning to water mimic drug design. Conserved water molecules are also played a significant role in conformational transition or flexibility of
“loop” and “flap” regions in unliganded form of enzyme, and was also used for anti
leukemic drug design (6). Involvement of Asp–Gly–Gly–Ile–Gln (S1: 364–368)
and Gly–Ser–Leu (S2: 387–389) sequences in the recognition of inhibitor CPR
(6-Chloropurine Riboside 50-Monophosphate) through conserved water molecules
have also indicated the rationality for modified CPR ligand (7). Attempt has been
employed to investigate the role of water molecules in the recognition of NAD or
its structural analogs to conserved catalytic D 274 of hIMPDH, which may be useful for IMPDH inhibitor design.
Extensive MD-Simulation studies of solvated x-ray structures of hIMPDH (1B3O,
1NFB, 1NF7 and 1JCN) and their complexes with NAD or its analogs have revealed
the presence of conserved water mediated interaction of those ligand to carboxylate
center of catalytic Asp 274 in the different isoforms of enzyme (Figure 1). In the
unliganded IMPDH, one conserved water molecule (W1) forms H- bond to D 274
(OD2) and H 93. However, during the complexation of NAD or its analogs with
IMPDH, that W1 retains its position and two other water molecules (W2 and WC)
are also observed to form H-bond with the carboxyl group of Asp 274. Position of
these three water molecules (WC, W1 and W2) are also be conserved during MDsimulation. All the conserved water molecules have stabilized through H-bonds
and occupied the three corners of a distorted trigonal pyramid with carboxyl oxygen (OD2) at apex (Figure 2). The water molecule WC interacts to carboxamide
1009
Figure 1: Presence of conserved water molecules ( W1, W2,WC ) and water mediated recognition of NAD to D 274 and H93 in the MD-simulated structure of
1B3O, 1NFB, 1NF7 and 1JCN. *MYD( NAD analog inhibitor ): C2 mycophenolic adenine dinucleotide
group of nucleotide or its analogs. The recognition geometry of carboxamide NN7
of NAD or analogs to carboxyl group of Asp 274 through conserved water (WC)
mediated interaction and their stereochemical features may be used for IMPDH
inhibitor design. Possibly, the chemical signals of cofactor binding region (NAD
or its structural analogs) could be transmitted toremote part of protein (Figure
3) through Base (NAD NN7) ----- Water (WC) ----- Acid (D 274 OD2) ----- Base
(H93 NE2) H-bonding interaction.
1010
Figure 2: The transition of unliganded IMPDH to NAD –bound IMPDH structure in human.*DN= Di-nucleotide Ligand (NAD/structural
analogs)
Figure 3: Conserved water mediated H-bonding interaction and the chemical signaling (Base --Water --- Acid ---) of the NAD binding region in hIMPDH
References
1. V. Nair and Q. Shu. Antivir Chem Chemother 18 , 245-258 (2007).
2. T. D. Colby, K.Vanderveen, M. D. Stricker, G. D. Markham, and B. M. Goldstein. Proc Nat
Acad Sci - Biochemistry 96, 3531-3536 (1999).
3. C. Branden and J. Tooze. Introduction to Protein Structure, Garland Publishing, New York
and London (1991).
4. H. R. Bairagya, B. P. Mukhopadhyay, and K. Sekar. J Biomol Struct Dyn 26, 497-508
(2009).
5. H. R. Bairagya, B. P. Mukhopadhyay, and K. Sekar. J Biomol Struct Dyn 27, 149-158
(2009).
6. H. R. Bairagya, B. P. Mukhopadhyay, and Asim K Bera. Journal of Molecular Recognition
24, 35-44 (2011).
7. H. R. Bairagya, B. P. Mukhopadhyay, and S. Bhattacharjee. Journal of Molecular Structure:
THEOCHEM 908, 31-39 (2009).
1011
Structure and Topology of Phospholamban Monomer
and Pentamer by a Hybrid Solution and Solid-State
NMR Method
Phospholamban (PLN) is an integral membrane protein that regulates calcium
homeostasis by inhibiting sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)
in cardiac muscle. PLN exists in two primary oligomeric forms: (1) a monomer
that directly binds to and inhibits SERCA (1, 2) and (2) a pentamer that indirectly
influences SERCA activity by regulating the pentamer-monomer equilibrium
(1, 2). To completely describe the fold space and ultimately the biological function
of PLN and other membrane proteins, it is necessary to determine the membrane
protein’s structure and the specific interactions with the lipid bilayer (i.e., topology). In this work, we describe a new hybrid method to calculate the structures of
the PLN monomer and pentamer using a combination of solution and solid-state
NMR restraints in detergent micelles and lipid bilayers, respectively (3, 4). These
high-resolution images of the PLN not only describe the structures, but also the
topologies of all the protein domains with respect to the lipid bilayer.
To minimize the structure/topology of the monomer and pentamer, we implemented
our hybrid objective function into XPLOR-NIH software (5) that utilizes geometrical (Echem) and solution (Esol-NMR) and solid-state NMR (EssNMR) restraints:
39
Nathaniel J. Traaseth1,2
Raffaello Verardi1
Lei Shi1
Gianluigi Veglia1
1Departments
of Chemistry and
Biochemistry, Molecular Biology &
Biophysics,
University of Minnesota, Minneapolis,
MN 55445
2Department
of Chemistry, New York
University, New York, NY 10003
Etotal 5 Echem 1 Esol–NMR 1 EssNMR
Figure 1: Structure of PLN monomer
from Traaseth et al. (Ref. 3).
1012
We obtained short-range distance and angular restraints from solution NMR of PLN
reconstituted into DPC detergent micelles and orientation restraints (anisotropic
chemical shifts and dipolar couplings) from 2D separated local field experiments
such as PISEMA (6, 7) in mechanically aligned lipid bilayers. The final stage of
refinement was to incorporate explicit lipids around the protein structure and carry
out minimization to reveal the interactions between the lipid and protein domains.
In our structures, we find that the N-terminal helical domain Ia (residues 1-16) of
monomer and pentamer rests on the surface of the lipid bilayer with the hydrophobic face of domain Ia embedded in the bilayer interior. The helix comprised
of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with tilt angles of ~11° (pentamer) and ~24° (monomer; structure in Figure 1). Hybrid methods such as the one presented in this work will be
necessary to tackle challenging biophysical problems such as membrane protein
structure determination.
References
1. Y. Kimura, K. Kurzydlowski, M. Tada, and D. H. Maclennan. J Biol Chem 272,
15061-15064 (1997).
2. L. G. Reddy, L. R. Jones, and D. D. Thomas. Biochem 38, 3954-3962 (1999).
3. N. J. Traaseth, L. Shi, R. Verardi, D. M. Mullen, G. Barany, and G. Veglia. Proc Natl Acad
Sci 106, 10165-10170 (2009).
4. L. Shi, N. J. Traaseth, R. Verardi, A. Cembran, J. Gao, and G. Veglia. J Biomolec NMR 44,
195-205 (2009).
5. C. D. Schwieters, J. J. Kuszewski, N. Tjandra, and G. M. Clore. J Magn Reson 160,
65-73 (2003).
6. C. H. Wu, A. Ramamoorthy, and S. J. Opella. J Magn Reson 109, 270-272 (1994).
7. T. Gopinath and G. Veglia. J Am Chem Soc 131, 5754-5756 (2009).
40
Loren E. Hough1
Kaushik Dutta2
Jaclyn Tetenbaum-Novatt1
Michael Rout1
David Cowburn3*
1Laboratory
of Cellular and Structural
Biology, Rockefeller University
2New
York Structural Biology Center
3Dept.
of Biochemistry
Albert Einstein College of Medicine
of Yeshiva University
Bronx, NY 10461
*[email protected]
The Solution and Binding Behavior of the Intrinsically
Disordered FG Nups Determined by in cell NMR
The nuclear pore complex (NPC) mediates all transport between the nucleus and
cytoplasm. The channel of the NPC is lined with “FG Nups”, a family of intrinsically disordered proteins characterized by phenylalanine-glycine repeat motifs.
FG nups form the exquisitely selective filter of the NPC; nonbinding proteins are
excluded while the binding of transport factors to the FG nups facilitates their passage through the NPC. Like other intrinsically disordered proteins, the FG nups
appear to be very sensitive to their environment, showing vastly different behavior in different experimental conditions; in vitro, the observed behavior of the FG
varies from rigid gels to flexible random coil polymers. We used stint-NMR to
protein behavior of a model FG not within the living cellular environment. In a
stint-NMR experiment, NMR observations are directed performed on bacteria,
in vivo co expressing an isotopically labeled protein and an unlabeled binding partner. We found that the solution state of the FG nups within living cells is disordered,
while NMR spectra significantly change in vitro buffers, presumably from numerous intra- or inter-molecular contacts. Moreover, the binding interface between
transport factors in the FG nups differs considerably between solution and cellular
conditions. Thus a key determinant to FG nups behavior is the local environment.
1013
These results indicate that the proper behavior of the FG naps is dependent on the
normal cellular milieu, and is not necessarily represented in vitro; our findings
have important implications for the various current models regarding the molecular
mechanisms of nuclear cytoplasmic transport and for behavior are weak cellular
interactions generally.
Supported by A Charles Revson postdoctoral fellowship, NIH grans GM067854,
GM071329 (MR), GM66354 (DC).
A Database/Webserver for Size-Independent
Quantification of Ligand Binding Site Burial Depth in
Receptor Proteins: Implicationson Protein Dynamics
We have developed a database/webserver that implements the two complementary methods to quantify the degree of burial of ligand and/or ligand binding
site (LBS) in a protein-ligand complex, namely, the ‘secant plane’ (SP) and the
‘tangent sphere’ (TS) methods, which we reported earlier (1). To recapitulate the SP
and TSmethods: the protein molecular centroid (global centroid, GC), and the LBS
centroid (local centroid, LC) are first determined. The SP is defined as the plane
passing through the LBS centroid (LC) and normal to the line passing through the
LC and the protein molecular centroid (GC). The “exterior side” of the SP is the
side opposite GC. The TS is defined as the sphere with center at GC and tangent to
the SP at LC. The percentage of protein atoms (a.) inside the TS (a.k.a. ‘TS index’)
and (b.) on the exterior side of the SP (a.k.a. ‘SP index’), are two complementary
measures of ligand or LBS burial depth since the latter is directly proportional to
(b.) and inversely proportional to (a.). We tested the SP and TS methods using a test
set of 67 well characterized protein-ligand structures (2), as well as the theoretical
case of an artificial protein in the form of a cubic lattice grid of points in the overall
shape of a sphere and in which LBS of any depth can be specified. Results from
both the SP and TS methods agree very well with reported data (2), and results
from the theoretical case further confirm that both methods are suitable measures
of ligand or LBS burial. There are two modes by which one can utilize our database/ webserver. In the first mode we term the ‘ligand mode’, the user inputs the
PDB structure coordinates of the protein as well as those of its ligand(one ligand
at a time if more than one). The second mode - the ‘LBS mode’-is the same as the
first except that the ligand coordinates are assumed to be unavailable, hence the
user inputs what s/he believes to be the LBS amino acid residues’ coordinates. In
both cases, the webserver outputs the SP and TS indices. LBS burial depth is an
important parameter as it is usually directly related to the amount of conformational
change a protein undergoes upon ligand binding, and ability to quantify it could
allow meaningful comparison of protein flexibility and dynamics. The URL of our
database/webserver will be http://tortellini.bioinformatics.rit.edu/sxc6274/thesis1.
phpand will be made freely available to the community very soon.
References
1. V. M. Reyes. J Biomol Struct & Dyn 26, 875-875 (2009).
2. R. A. Laskowski, N. M. Luscombe, M. B. Swindells, and J. M. Thornton. Protein Sci 5,
2438-2452 (1996).
Srujana Cheguri
Vicente M. Reyes*
41
Biological Sciences Dept., Sch. of
Medical & Biological Sciences,
College of Science, Rochester Institute
of Technology,
Rochester, NY 14623-5603
*[email protected]
1014
42
Srujana Cheguri
Vicente M. Reyes*
Medical & Biological Sciences,
of Technology,
*[email protected]
Using Cylindrical Coordinates to Represent
Rod-Shaped and Other Fibrous Protein 3D
Structures: Potential Advantages and Applications
Based on overall 3D structure, proteins may be grouped into two broad, general
categories, namely, globular proteins or ‘spheroproteins’, and elongated or ‘fibrous
proteins’, and the former comprises the significant majority. Our research group
is trying to use alternative representations for proteins structures, and has made
progress on representing spheroproteins using spherical coordinates (ρ,φ,θ) (1).
This work concerns the second general category of protein structures, namely, the
fibrous or rod-shaped class of proteins. Unlike a spheroprotein, a rod-shaped protein (RSP) possesses a visibly conspicuous axis along its longest dimension. To take
advantage of this potential symmetry element, we decided to represent RSPs using
cylindrical coordinates, (ρ,θ,z), with the z-axis as the main axis and one ‘tip’ of the
protein at the origin, with ‘tip’ being defined as one of two points lying along the
protein axis and defining its longest dimension. To do this, we first visually identify the two tips T1 and T2 of the protein using appropriate graphics software, then
determine their Cartesian coordinates, (h,k,l) and (m,n,o), respectively. Arbitrarily
selecting T1 as the tip to coincide with the origin, we translate the protein by subtracting (h,k,l) from all structural coordinates. We then find the angle α (in degrees)
between vectors T1T2 and the positive z-axis by computing the scalar product of
vectors T1T2 and OP where P is an arbitrary point along the positive z-axis, which is
typically (0,0,p), where p is the approximate length of the rod-shaped protein under
investigation. Then we compute the cross product of the two vectors to determine
the axis about which we should rotate vector T1T2 so it coincides with the positive z-axis.We then use a matrix form of Rodrigues’ formula to perform the actual
rotation. Finally we apply the Cartesian to cylindrical coordinate transformation
equations to the system. Thus far, we have applied the above transformation to 15
rod-shaped proteins, prominent among which are 1DXX, 2KOL,2KZG, 3LHP and
3MQC. We have also created a database/webserver that can take in the PDB coordinate file of a rod-shaped protein and output its cylindrical coordinates based on
the transformation steps described above. We shall implement this process in both
all-atom and reduced protein representations (2). The URL will be http://tortellini.
bioinformatics.rit.edu/sxc6274/thesis2.php and it will be made freely available to
the community very soon.
References
1. V. M. Reyes. Interdiscipl Sci: Comp Life Sci (2011, in press).
2. V. M. Reyes and V. N. Sheth. In: Handbook of Research in Computational and Systems
Biology: Interdisciplinary Approaches, L. A. Liu, D. Wei, and Y. Qing (Eds.), Chap. 26
(2011, in press).
1015
Towards a Spherical Coordinate System Metric for
Quantitative Comparison of Protein 3D Structures
Although observed protein structures generally represent energetically favorable
conformations that may or may not be “functional”, it is also generally agreed that
protein structure is closely related to protein function. Given a collection of proteins
sharing a common global structure, variations in their local structures at specific,
critical locations may result in different biological functions. Structural relationships among proteins are important in the study of the evolution of proteins as well
as in drug design and development.
Analysis of geometrical 3D protein structure has been shown to be effective with
respect to classifying proteins. Prior work has shown that the double-centroid
reduced representation (DCRR) model (1) is a useful geometric representation for
protein structure with respect to visual models, reducing the quantity of modeled
information for each amino acid, yet retaining the most important geometrical and
chemical features of each: the centroids of the backbone and of the side-chain.
Thus far, DCRR has not yet been applied in the calculation of geometric structural
similarity.
Meanwhile, multi-dimensional indexing (MDI) of protein structure combines protein structural analysis with distance metrics to facilitate structural similarity queries and is also used for clustering protein structures into related groups. In this
respect, the combination of geometric models with MDI has been shown to be
effective.
Prior work, notably Distance and Density-based Protein Indexing (DDPIn) (2),
applies MDI to protein models based on the geometry of the C backbone. DDPIn’s
distance metrics are based on radial and density functions that incorporate spherical-based metrics, and the indices are built from metric tree (M-tree; 3) structures.
This work combines DCRR with DDPIn for the development of new DCRR
centroid-based metrics: spherical binning (4) distance and inter-centroid spherical distance. The use of DCRR models will provide additional significant structural information via the inclusion of side-chain centroids. Additionally, the newly
developed distance metric functions combined with DCRR and M-tree indexing
should improve upon the performance of prior work (DDPIn), given the same data
set (5), with respect to both individual k-nearest neighbor search queries as well as
clustering all proteins in the index.
References
Biology: Interdisciplinary Approaches, L. A. Liu, D. Wei and Y. Qing (Eds.), Chap. 26
(2011, in press).
2. D. Hoksza. Proc 6th Ann IEEE Conf Comp Intel Bioinf Comp Biol CIBCB’09, 263-270 (2009).
3. P. Ciaccia, M. Patella, and P. Zezula. Proc 23rd Intl Conf Very Large Data Bases VLDB’97,
426-435 (1997).
5. O. Çamoglu, T. Kahveci, and A. K. Singh. Proc IEEE Comp Soc Conf Bioinf CSB’03,
148-158 (2003).
James DeFelice
Vicente M. Reyes*
43
Medical & Biological Sciences
of Technology
Rochester, NY 14613
*[email protected]
1016
44
PreetyPriya
Vicente M. Reyes*
Biological Sciences Dept.,
Sch. of Medical & Biological Sciences,
College of Science,
Rochester Institute of Technology,
*[email protected]
Cancer Meets the “Omics”: A Comprehensive Cancer
Biotherapy Database with Links to Multiple
Bioinformatics Websites/WebServers – Facilitating
the Search for Anticancer Biological Agents
‘Cancer biotherapy’ – as opposed to ‘cancer chemotherapy’ - is the use of macromolecular biological agents instead of small organic chemicals or ‘drugs’ to treat
cancer (1, 2). Although there are several important differences between cancer biotherapy and cancer chemotherapy, it suffices to saythat due to the much higher
selectivity of biological agents than chemical agents for cancer cells over normal
cells, there is much less toxic side effects in biotherapy as compared to chemotherapy, and as a result, patient survival is usually dramatically enhanced.We have built
the foundations of acomprehensive cancer biotherapy database for use as a lifesaving resource by cancer patients, and as a sounding board for scientific ideas by
cancer researchers. The database/webserver will have information about 12 main
families of cancer biotherapy regimens to date, namely, 1.) Protein Kinase Inhibitors, 2.) Ras Pathway Inhibitors, 3.) Cell-Cycle Active Agents, 4.) MAbs (monoclonal antibodies), 5.) ADEPT (Antibody-Directed Enzyme Pro-Drug Therapy), 6.)
Cytokines (interferons, interleukins, TNF, etc.), 7.) Anti-Angiogeneis Agents, 8.)
Cancer Vaccines (peptides, proteins,DNA), 9.) Cell-based Immunotherapeutics,
10.) Gene Therapy, 11.) Hematopoietic Growth Factors, and 12.) Retinoids. For
each biotherapy regimen, we will extract the following attributes in populating
the database: (a.) cancer type, (b.) gene/s and gene product/s involved, (c.) gene
sequence (GenBank ID), (d.) organ/s affected, (e.) available chemo treatment, (f.)
reference papers, (g.) clinical phase/stage, (h.) survival rate (chemo. vs. biother.),
(i.) clinical test center locations, (j.) cost, (k.) patient blog, (l.) researcher blog, etc.
The most salient feature of our database/webserver, besides its focus on biological
agents, is its multiple links to most, if not all, publicly available databases and webservers, including structural proteomics, metabolomics, glycomics, and lipidomics
webservers. It is hoped that these links can provide the researcher with up-to-date
knowledge about the structure and function of the specific biomolecules involved
with the type of cancer they are studying. Knowledge of these “cancer signatures”
or biomarkers is expected to facilitate the work of cancer researchers. As a public
resource, on the other hand, the database will include a description attribute that
will explain in simple, layman’s language the 12 biotherapy regimens well as other
technical items included to ensure public accessibility.The database attributes will
be regularly updated for novel attributes as discoveries are made.
References
1. A. Young, L. Rowett, and D. Kerr (Eds.). Cancer Biotherapy: An Introductory Guide,
Oxford Univ. Press, Oxford, U.K. (2006).
2. P. T. Rieger. Biotherapy: A Comprehensive Overview, 2nd ed., Jones & Barlett Publ.,
Sudbury, MA (2001).
1017
Using Cartographic Techniques to Project
Protein 3D Surfaces onto the 2D Plane: Potential
Applications and Implications
Study of protein surfaces is quite important in protein sciencesince the biological properties of proteins are largely (but not solely) determinedby their surface
properties. Meanwhile, it is estimated that a significant majority of all proteins
fold into compact globular structures (‘spheroproteins’), and as such may be likened to the earth. For centuries, the surface of the earth has been represented and
analyzed using cartographic spherical projection methods, such as the Mercator,
Eckert, Lambert, Werner and Aitoff projections. We have recently developed a
program that transforms protein 3D structure coordinates from Cartesian (x,y,z) to
spherical (ρ,φ,θ) coordinates, whose origin is the geometric centroid of the protein
(1). In this system, rho (ρ) plays the role of the earth’s radius from its center to an
entity in the protein structure, phi (φ), the latitudes, and theta (θ), the longitudes. 2D
projection of the surface of this sphere, with elevations fromthe “sea level” (ESLs),
may be achieved using the transformation equations for the particular projection
method. The ESLs, on the other hand, can be determined by choosing areference
“sea level” ρ0 (e.g., distance from center of sphere to the smallest nonzero ρvalue)
and using the “heights” of surface points above ρ0 as the z-coordinates (elevations
along the z-axis). Established methods in geography/cartography may then be used
to analyze such projections with elevations. Plots of these 2D raise-relief maps (a.k.a.
terrain models) may be easily rendered using MATLAB. These 2D projection methods present a number of benefits for protein surface analysis over 3D-based methods,
among which are (a) simplicity of presentation (i.e., 2D vs. 3D), (b) entire protein surface may be viewed all at once, (c) simpler and more direct protein surface comparisons (e.g., between similar but non-identical proteins, or between identical proteins in
different conformations or liganded states), and (d) applicability of the myriad wellestablished cartographic techniques for analysis and comparison of protein surfaces.
Thus far, we have written/implemented FORTRAN 77/90 programs for the following
map projections: 1.) Mercator, 2.) Miller, 3.) Mollweide, 4.) Robinson and 5.) GallPeters. We are currently expanding this list and comparing the methods with each other
in both all-atom and reduced protein representations (2) to determine which best represents the proteins’ surface properties by correlation with the proteins’ bioactivity.
Vicente M. Reyes
45
Medical & Biological Sciences
of Technology
[email protected]
References
Biology: Interdisciplinary Approaches, L. A. Liu, D. Wei and Y. Qing (Eds.), Chap. 26
(2011, in press).
Self Assembly Study of the Human GPCR Protein
b2-Adrenergic Receptor Using Coarse Grained
Molecular Dynamics Technique
Integral membrane proteins roughly constitutes of 25 to 30% to the human genome,
of which the G-Protein Coupled Receptors (GPCRs) encode for nearly 3-4% of
all the genes, regulating various physiological processes through signal transduction. As a consequence of this, these receptors have become the targets of several
modern day drugs. Most of the studies aimed at designing new drugs for targeting GPCRs have assumed that these receptors function in monomeric form. However, this assumption has recently been changed by the description of a number of
GPCRs that can be found in oligomeric state within the cellular environment (1).
Although many unsolved problems still remain, the idea that GPCRs directly interact to form oligomers, both homomers as well as heteromers, has been gradually
accepted. The mechanism of GPCR dimer or oligomer formation, and its effect on
receptor function, is not currently well understood.
46
Anirban Ghosh
Uddhavesh B. Sonavane
Rajendra Joshi*
Bioinformatics Group,
Computing,
Pune University Campus,
*[email protected]
1018
In the present study, coarse grained molecular dynamics (CGMD) approach was
adopted for studying the self-assembly process of the human amine GPCR protein β2-adrenergic receptor (β2-AR), for which several experimental evidences
of oligomerization process and its effect on its function are available (2, 3). PDB
entry 2RH1 was taken as the starting structure. Since 2RH1 lacks ICL3 (residue
231-262), initially the missing loop was modeled in SYBYL and simulated for 10
ns using restrained MD in order to get a stable conformation. The final structure
was then used for further studies. To mimic a cellular environment, 16 copies of
β2-AR were inserted into DSPC bilayer at a protein to lipid ratio of 1:104 and then
solvated with water. The entire system was represented using the MARTINI CG
convention (4), resulting in a total system size of 57296 CG beads. The system was
then simulated for 3 µs using the GROMACS package with MARTINI force filed
parameters. An increased time-step of 30 femto-second was used which resulted in
stable integration. At the end of the simulation period, proper dimers and tetramers
of β2-AR were found to be formed through the self-assembly mechanism which
were further validated through various analysis methods. The gradual decrease in
SASA values calculated with a probe radius of 0.52 nm confirmed that the monomers were indeed coming together to form aggregates. The lipid bilayer analysis
also helped to quantify the assembly mechanism. In order to identify the exact
residues or domains which are responsible for this oligomerization, a conversion of
the CG system back to an all-atom model and simulated annealing simulations are
being presently carried out.
References
1. S. R. George, B. F. O’Dowd, and S. P. Lee. Nat Rev Drug Discov 1, 808-20 (2002).
2. A. Salahpour, S. Angers, J. F. Mercier, M. Lagacé, S. Marullo, and M. Bouvier. J Bio Chem
279, 33390-7 (2004).
3. T. E. Hebert, S. Moffett, J. P. Morello, T. P. Loisel, D.G. Bichet, C. Barret, and M. Bouvier.
J Biol Chem 271, 16384-93 (1996).
4. Luca Monticelli, Senthil K. Kandasamy, Xavier Periole, Ronald G. Larson, D. Peter
Tieleman, and Siewert-Jan Marrink. J Chem Theory Comput 4, 819-34 (2008).
47
Amit Saxena1
Anirban Ghosh1
George A. Komatsoulis2
Hemant Darbari1
Anil Srivastava2
Ravi Madduri3
P.K. Sinha1
Rajendra Joshi1*
1Centre
for Development of Advanced
Computing, Pune–411007, India
2Center
for Biomedical Informatics and
Information Technology, NCI, NIH, USA
3University
of Chicago, USA
*[email protected]
Protein Structure Modelling on the Indo-US
Cancer Research Grid
The importance of protein structures can be understood easily from the fact that the
function of any protein is directly correlated to its structure (1). The three dimensional structure of a protein directs its function within a cellular environment. Any
mutation in the protein sequence leads to changes in its structure which in turn may
render the protein non-functional or even attribute some adverse functions (2-6)
leading to diseases like cancer. Over the decades cancer has become one of the
most prevalent diseases with an estimate of reaching over 12 million deaths in 2030
according to World Health Organization. Proteins from almost 1% of the human
genome have been identified to be involved in oncogenesis (7). In the absence of
resolved structural data (RCSB database has 65847 resolved protein structures as
opposed to 525207 sequence entries in UniProtKB) one has to resort to computational techniques to get the 3D structures of proteins in order to properly understand
their functions.
The Bioinformatics Group at the Centre for Development of Advanced Computing
(C-DAC) in collaboration with cancer Biomedical Informatics Grid (caBIG®) has
developed a grid-enabled web-based automated pipeline (Figure 1) for ab initio
prediction of protein structures with an emphasis on cancer related proteins. The
pipeline has been deployed on the Bioinformatics Resources & Applications
Facility (BRAF) hosted at C-DAC, Pune India. The upstream component of the
pipeline retrieves a protein sequence (according to user input) from the gridPIR
1019
Figure 1: Schematic representation of the protein prediction pipeline.
service of caBIG that provides a data resource of high quality annotated information on all protein sequences supported by UniProtKB. The retrieved sequence in
a FASTA format is then fed to the prediction pipeline. At its core the pipeline
uses the ROSETTA prediction algorithm (8) for determining the 3D structures. The
graphical user interface of the pipeline enables the user to choose various control
parameters like which secondary structure prediction algorithms to use, number of
iterations, number of output structures, uploading NMR constraint files etc. Once
submitted, the jobs get distributed over multiple processors in the form of multiple
threads on Biogene supercomputing system at BRAF, which highly reduces the prediction time. The resultant output comes in the form of predicted structures in PDB
format and parsed energy log files which can be downloaded by the user. All the file
transfers are secured over the network by SFTP. JMol has been integrated within
the pipeline to provide a visual inspection of the predicted models. Test cases have
been run using the pipeline with a few cancer related proteins, whose results will be
discussed. This pipeline provides a hassle-free high throughput structure prediction
platform. Java has been used for coding the entire pipeline with Struts, AJAX and
Hibernate framework. The upstream gridPIR searching module parses XML results
using SAX parser while the GUI has been built using JSP.
References
1.
2.
3.
4.
5.
6.
7.
8.
H. Hegyi and M. Gerstein. J Mol Biol 288, 147-64 (1999).
M. Soskine and D. S. Tawfik. Nat Rev Genet 11, 572-82 (2010)
L. Zhong. J Biomol Struct Dyn 28, 355-361 (2010).
Y. Yu, Y. Wang, J. He, Y. Liu, H. Li, H. Zhang, and Y. Song. J Biomol Struct Dyn 27, 641-649
(2010).
M. S. Achary and H. A. Nagarajaram. J Biomol Struct Dyn 26, 609-623 (2009).
A. A. Moosavi-Movahedi, S. J. Mousavy, A. Divsalar, A. Babaahmadi, K. Karimian,
A. Shafiee, M. Kamarie, N. Poursasan, B. Farzami, G. H. Riazi, G. H. Hakimelahi,
F. Y. Tsai, F. Ahmad, M. Amani, and A. A. Saboury. J Biomol Struct Dyn 27, 319-329
(2009).
P. A. Futreal, L. Coin, M. Marshall, T. Down, T. Hubbard, R. Wooster, N. Rahman, and
M. R. Stratton. Nat Rev Cancer 4, 177-83 (2004).
C. A. Rohl, C. E. Strauss, K. M. Misura, and D. Baker. Methods Enzymol 383, 66-93
(2004).
1020
48
Sam Tonddast-Navaei*
George Stan
*[email protected]
The Role of Central Pore Residues of p97/VCP on
Substrate Unfolding and Translocation: A
Computational Model
The p97/VCP nanomachine, a double ring member of the AAA1 superfamily, is
involved in substrate protein unfolding within the proteasomal degradation pathway
(1). Currently, it is unclear how p97/VCP interacts with its substrate. Revealing the
underlying mechanism of p97 could lead to better understanding of its bacterial
homologues ClpA and ClpB.
P97/VCP has a homo-hexameric structure that encloses a central pore. Within each
subunit, there are two nucleotide binding domains, D1 and D2, and the N domain,
which is connected to D1 and is known to interact with p97/VCP’s adaptors. ATP
hydrolysis leads to large scale conformational changes in D2 domain, which affects
the topology of its pore (2, 3).
Conserved loops at the entrance of D2 pore are suggested to enable substrate propagation through the pore via ATP-driven paddling motion of Trp551 and Phe552
residues. Two other essential residues inside the D2 pore, Arg586 and Arg599, contribute to the p97 function (4). We propose that the substrate, which enters through
the D1 pore, binds to the Arg599 sites on the D2 cavity lining. Repetitive ATP-driven
cycles of p97 mediate the complete translocation of the substrate protein into the D2
pore via the paddling motion of the D2 loops (centered onto Trp551 and Phe552).
To test this hypothesis, we perform implicit solvent simulations of the SsrA-SsrA
peptide threading through p97/VCP. Our results confirm the role of Arg599 as
binding sites. These simulations reveal that these Arginines interact with the substrate primarily via hydrogen-bonds formed with the peptide backbone, indicating
a non-specific interaction type.
Using the results from implicit solvent simulations, we develop a coarse-grained
model that extends our simulations to biologically relevant timescales. We investigate the unfolding mechanism of a four helix bundle protein fused with the SsrA
peptide coupled to ATP-driven conformational changes in the D2 domain of p97
(figure 1). Our simulations show that complete unfolding and translocation is due to
the collaboration between Arginine residues and the critical residues at the paddling
D2 loop, such that substrate is held by the Arginine residues of adjacent subunits and
the force exerted by the D2 loops pull the substrate through the central pore of p97.
Figure 1: A snapshot of unfolding and translocation of the four helix bundle (purple) and the SsrA
peptide (orange) by the p97/VCP nanomachine (N-D1 yellow, D2 green). To show the location of the
D2 loops (blue) and the Arginines (brown), the two front subunits are not presented in the figure.
1021
This research has been supported by a grant from the American Heart Association
and by the National Science Foundation CAREER grant to G. S. and a University
Research Council fellowship at the University of Cincinnati to M.J.
References
1. A. Beskow, K. B. Grimberg, L.C. Bott, F. A. Salomons, N. P. Dantuma, and
P. Young. J Mol Biol 394, 732-746 (2009). 2. B. DeLaBarre, J. C. Christianson, R. R. Kopito, and A. T. Brunger. J Mol Cell Biol
22, 451-462 (2006). 3. Q. Wang, C. Song, X. Yang, and C. H. Li. J Biol Chem 278, 32784-32793 (2003). 4. J. M. Davies, A. T. Brunger, and W. I. Weis. Struct. 16, 715-726 (2008). 5. A. Kravats, M. Jayasinghe, and G. Stan. Proc Natl Acad Sci USA (in press).
49
Computer-Aided Pathway to Increasing the
Thermostability of Small Proteins
A major task of modern bioengineering is the development of molecules with designated properties (1) such as increased stability, including their thermostability.
The augmented thermostability of proteins allows to increase the speed of enzymatic catalysis, as well as the duration of their storage.
A few years ago a possible thermostabilization mechanism of small globular proteins (2) was developed in the Laboratory of Structure and Dynamics of Biomolecular Systems at the Institute of Cell Biophysics (Russian Academy of Sciences).
It is based on the alternative hydrogen bonding mechanism between side chains of
amino acid residues on protein surface (2). This hypothesis based on experimental
data has been essentially supplemented later by modelings of dynamics of for proteins from thermophilic and mesophilic organisms (3, 4).
Our work uses this theory for improving the thermostability of human Peroxiredoxin 6. This protein (5) is a promising antioxidant for burn treatment. The spatial
structure of human Peroxiredoxin 6 was reported previously (6) (Figure 1.) and its
Figure 1: Structure of human Peroxiredoxin 6 (pdb entry: 1PRX) and active site location.
Maxim Kondratyev*
Artem Kabanov**
Alexander Samchenko
Vladislav Komarov
Nikolay Khechinashvili
Institute of Cell Biophysics
Pushchino, Russia
*[email protected]
**[email protected]
1022
homologs from various organisms have been well characterized, including their
thermodynamic properties (7). We propose to predict what point mutaions in Peroxiredoxin 6 will increase its themostability using our knowledge about alternative
hydrogen bonding, as well as the known structure of human Peroxiredoxin 6 and
its known homologs.
Data of alignment of Peroxiredoxin 6 homologs (Figure 2) give us information
about the variable and stable parts of the amino acid sequence of this protein. The
most probable sites of mutations reside only in the evolutionary variable areas of
amino acid sequence because changes in stable regions can affect the functional
properties of the protein.
It should be noted that certain homologs of Peroxiredoxin 6 possess higher thermostability in native state in comparison to the human protein. It is important to
notice that human and rat Peroxiredoxins have the highest homology (91.5%, i.e.
19 residues) (Figure 2.). At the same time, rat protein possesses the greatest thermostability (7) among the homologs studied. Thus, by comparing rat and human
Peroxiredoxin 6 amino acid sequences, we can get additional information about
the preferred locations of mutations to increase the thermostability of human
Peroxiredoxin 6.
The structures of native human Peroxiredoxin 6 protein and it homologs have been
studied by molecular dynamics (MD) at various temperatures on GPU NVIDIA
(8). The MD provides a powerway to follow formation and destruction of hydrogen bonds in all biological macromolecules (9-12). In the case of Peroxiredoxin 6
protein we tested the amount of hydrogen bonds on the surface of protein globules
in each frame of an MD-trajectory on the pairs of studied proteins. Solvent was
considered both in explicit and implicit models.
From the data of sequence alignment and our MD calculations, we predict four
amino acid substitutions, which we believe will lead to increased thermostability
of human Peroxiredoxin 6 protein without violating the spatial structure and functional properties.
The problem of substrate specificity of Peroxiredoxin 6 will also be discussed.
Figure 2: Alignment of Peroxiredoxins 6 from various organisms ([7]). (Note, that active site of all Peroxiredoxin’s 6 (CYS47) is located in stable region of
amino acids sequence.)
1023
References
1. Donald Lee Wise. Encyclopedic handbook of biomaterials and bioengineering: Applications, vol. 2, New York: Marcel Dekker, 1995.
2. N. N. Khechinashvili, M. V. Fedorov, A. V. Kabanov, S. Monti, C. Ghio, and K. Soda.
3. A. V. Kabanov and N. N. Khechinashvili. J Biomol Struct Dyn 24, 756-756 (2007).
4. N. N. Khechinashvili, S. A. Volchkov, A. V. Kabanov, and G.Barone. Biochim Biophys Acta
Proteins & Proteomics, 1784 (11), P.1830 92084).
5. I. V. Peshenko, V. I. Novoselo, V. A. Evdokimov, Y. V. Nikolaev, T. M. Shuvaeva,
V. M. Lipkin, and E. E. Fesenko. FEBS Lett 381, 14-19 (1996).
6. H. J. Choi, S. W. Kang , C. H. Yang, S. G. Rhee, and S. E. Ryu. Nat Struct Biol 5, 400-406
(1998).
7. M. G. Sharapov, V. I. Novoselov, and V. K. Ravin. Mol Biol (Mosk) 43, 505-11 (2009).
8. http://www.nvidia.com/object/cuda_home_new.html
9. Z. Gong, Y. Zhao, and Y. Xiao. J Biomol Struct Dyn 28, 431-441 (2010).
10. J. Wiesner, Z. Kriz, K. Kuca, D. Jun, and J. Koca. J Biomol Struct Dyn 28, 393-403 (2010).
12. F. Mehrnejad and M. Zarei. J Biomol Struct Dyn 27, 551-559 (2010).
Generating Conformational Ensembles for Flexible
Protein-Ligand Docking by Elastic Network Model
Guided Molecular Dynamics Simulations: Application
to Beta 2 Adrenergic Receptor
Studying the entire motion spectrum of a protein is necessary for a complete understanding of its function(s). However, this is not trivial since protein motions are
complex ranging from femtosecond scale local atomic vibrations to millisecond
scale large motions. We previously developed Anisotropic Network Model (ANM)
restrained Molecular dynamics (MD) that takes advantage of these two complementing methods to sample long time scale biologically relevant global motions
of a biomolecular system with realistic deformations favored by a detailed atomic
force field in the presence of the explicit environment (1). Here, we use ANMrestrained-MD method to generate conformational ensembles of a pharmacologically relevant G-protein Coupled Receptor (GCPR), Beta 2 Adrenergic Receptor
(β2AR). It has been shown that the binding of GPCRs to structurally diverse ligands
and their activation is a complex process that requires these receptors passing through
multiple conformationally distinct states (2, 3). Along with the existing crystal structures, the conformational ensembles are utilized for understanding the dynamics and
binding modes of β2AR to its known agonists by performing docking against them.
We observe that the residues Ser203, Ser204, and Ser207 on H5 become accessible
at the ligand binding site by the rotation of H5. Additional to Serines on H5, Val114
and Thr118 on H3 and “the rotamer toggle switch”, Phe290, on H6 stabilize the
aromatic rings and the catecholamine moieties of the agonists of β2AR by forming
hydrogen bonds and phi-stacking interactions. Furthermore, our study also shows
that the long acting agonist drug that is currently prescribed for asthma, salmeterol,
folds uniquely at the ligand binding pocket of β2AR and stabilizes its extracellular
region of forming a “beta sheet-like” structure with the extracellular loop 2.
Authors would like to thank Drs Ivet Bahar, Klaus Schulten and Emad Tajhkorshid
for their valuable contributions during the development of ANM-guided-MD algorithm.
References
1. B. Isin, K. Schulten, E. Tajkhorshid, and I. Bahar. Biophys J 95, 789-803 (2008).
2. G. Liapakis, W. C. Chan, M. Papadokosktaki, and J. M. Javitch. Mol Pharmacol 65, 11811190 (2004).
3. B. K. Kobilka and X. Deupi. Trends Pharmacol Sci 28, 397-406 (2007).
Basak Isin1*
Guillermina Estiu2
Olaf Wiest2
Zoltan N. Oltvai1
1Department
50
of Pathology, and
Computational & Systems Biology,
Pittsburgh, PA, 15261
2Department
of Chemistry and
Biochemistry,
University of Notre Dame,
Notre Dame, IN, 46556
*[email protected], [email protected]
1024
51
Valery V. Petrov
Institute of Biochemistry and
Physiology of Microorganisms,
RAS, 142290 Pushchino, Russia
[email protected]
Role of M5-M6 Loop in the Biogenesis and Function
of the Yeast Pma1 H+-ATPase
Yeast Pma1 H+-ATPase belongs to P2-type ATPases which couple ATP hydrolysis to transport of cations across biomembranes where they are embedded by 10
membrane segments. The determinants of cation specificity and stoichiometry lie
in M4, M5, M6, and M8 segments; point mutations in these segments affect normal
functioning and biogenesis of the enzyme and change both stoichiometry (1-3) and
specificity (4). During the reaction cycle P2-ATPases undergo significant conformational changes: M1-M6 segments bend, unwind partially and even shift normal
to the membrane (5). Ala substitutions of the residues in the M6 N-terminal half
interfered markedly with the Pma1 functioning and biogenesis, or both (3). The
results described here extend a systematic study of the yeast H+-ATPase by focusing on the extracytoplasmic loop between M5 and M6 segments of this enzyme,
searching for residues that may play a role in H+ transport or any other aspect of the
enzyme functioning and biogenesis. To explore role of this loop in the structurefunction relationship of the S. cerevisiae Pma1 ATPase, Ala-scanning mutagenesis
was used. The loop consists of 7 residues: 714-DNSLDID. L717 is the most conservative among them. Accordingly, only L717A led to a complete block in membrane trafficking that prevented the ATPase from reaching secretory vesicles (SV)
which points to a severe defect in protein folding, causing the abnormal ATPase
to be retained in the endoplasmic reticulum (ER). Mutation D714A was expressed
poorly in SV (Figure 1), displaying very low ATPase activity with no detectable
H+ pumping. The remaining 5 mutations were expressed at 34 to 94% with ATPase
activities of 35 to 101% of the WT level. Given the known contribution of M5 and
M6 segments to the transport pathway of P2-ATPases and their mobility during
reaction cycle, it was of particular interest to ask whether any of the mutations
Figure 1: Effect of Ala substitutions of the residues in the M5-M6 loop of the Pma1 ATPase on the enzyme
expression (left columns) and activity (right columns), %.
1025
in the M5-M6 loop affected H+ pumping. For most of the mutants, the coupling
ratio was close to that of the WT (1.00). Only I719A mutant gave the ratio significantly lower (0.29), pointing to a partial uncoupling between ATP hydrolysis
and H+ transport. Thus, 3 of 7 residues in M5-M6 loop seem to be important for
the proper function and biogenesis of the yeast Pma1 H+-ATPase. L717 is located
in the middle of the loop; since the enzyme reaction cycle is accompanied by significant conformational changes, substitution of this residue with a smaller Ala
may strongly affect the mobility of M5 and M6 segments causing misfolding and
retaining of impaired enzyme in ER. D714 is also important for structure and, especially, functioning of the enzyme; it probably plays a role similar to D739 in M6
which neutralizes neighboring positive charge and, thus, stabilizing the protein (3).
Finally, I719 replacement with Ala caused significant uncoupling between ATP
hydrolysis and H+ transport.
Acknowledgements
Author is grateful to scientific adviser of this project Prof. C. W. Slayman (Yale
School of Medicine).
References
1. V. V. Petrov, K. P. Padmanabha, R. K. Nakamoto, K. E. Allen, and C. W. Slayman.
J Biol Chem 275, 15709-15716 (2000).
2. G. Guerra, V. V. Petrov, K. E. Allen, M. Miranda, J. P. Pardo, and C. W. Slayman. Biochim
Biophys Acta 1768, 2383-2392 (2007).
3. M. Miranda, J. P. Pardo, and V. V. Petrov. Biochim Biophys Acta (epublished Dec.
13, 2010).
4. D. Mandal, T. B. Woolf, and R. Rao. J Biol Chem 275, 23933-23938 (2000).
5. C. Toyosima and H. Nomura. Nature 418, 605-611 (2002).
Point Mutation in M9-M10 Loop of the Yeast Pma1
H+-ATPase Affects Both ATPase Functioning and
Polyphosphate (PolyP) Distribution
Both ATP and linear polymers of inorganic phosphate (Pi) PolyP are involved in
cell energetics and metabolism. Role of ATP and ATPases is well established,
while less is known about PolyP. However, it is clear that PolyP are involved in
storage of Pi, membrane channel formation, cation binding, regulation of enzyme
activities, gene expression (1). There are very little data on the interaction between
ATP and PolyP metabolism. Yeast plasma membrane (Pma1) H+-ATPase generates electrochemical H+ gradient providing energy for operating the secondary solute transport systems. The enzyme is embedded in the membrane by 10 segments
(M1-M10) with most of the molecule located in cytosole or in the membrane; only
5% of the molecule face extracellular space. Yeast Pma1 H+-ATPase is regulated
by glucose: during glucose consumption ATP is produced, this triggers activation of Pma1 functioning manifested in 3-10-fold increase of Vmax, and decrease
of Km and Ki. ATPase activation is structurally accompanied by the enzyme multiple phosphorylation (2); two tandemly positioned sites are located in the enzyme
C-terminal tail (3).
Most of the plausible residues for the ATPase phosphorylation (Ser, Thr, Asp, and
Glu) are located in the inner parts of the enzyme; however, there are several phosphorylable residues located in the Pma1 outer parts: D714, S716, D718, and D720
in the M5-M6 loop and S846, E847, T850, and D851 in the M9-M10 loop which
is close to the enzyme regulatory C-tail. It seems reasonable that multiple phosphorylation of Pma1 goes subsequently, and first of such sites could be located in
52
Alexander A. Tomashevsky
Valery V. Petrov*
Institute of Biochemistry and
Physiology of Microorganisms,
RAS, 142290 Pushchino, Russia
*[email protected]
1026
Figure 1: Left: Pma1 ATPase activity in the WT and T850A mutant strains under carbon-starved (CS)
and glucose-metabolizing (GM) conditions (%). Right: PolyP3 content in the WT and T850A strains in
the logarithmic (log) and stationary (stat) phases (µmol P/g wet weight).
the extracellular part of the enzyme. The M5-M6 loop residues, except D714, were
found not to be important for the enzyme structure-function relationship; the D714A
mutant activity was unessential to be studied further (4). Therefore, we choose to
replace with Ala one of the residues in the M9-M10 loop of the enzyme which
could be phosphorylated – T850. When T850 is replaced with Ala, the mutated
enzyme activity dropped significantly; at the same time the ability of the mutated
enzyme to be activated by glucose was strongly impaired (Figure 1, left). In parallel
with ATPase activity assay, distribution of PolyP fractions (PolyP1-PolyP5) was
analyzed. No significant changes were found between most PolyP fractions in logarithmic and stationary phases of the wild type (WT) and T850A mutant. However,
PolyP3 fraction stood out of the rest displaying almost twofold increase of PolyP
amount in the mutant during stationary phase compare with only a quarter in the
WT (Figure 1, right). Since ATPase is more active during logarithmic phase (similar to GM and CS conditions in Figure 1, left), it points to a connection between
ATP and Poly metabolisms. Significant increase of PolyP3 amount in the T850A
mutant in stationary phase may point to the lack of one of the phosphorylation sites.
Further study of this and similar mutants, although methodologically challenging,
seems certain to yield useful insights into the fundamental mechanisms of ATP and
PolyP interactive metabolisms.
References
1. I. S. Kulaev, V. M. Vagabov, and T. V. Kulakovskaya. High-molecular inorganic polyphosphates: biochemistry, cell biology and biotechnology. Moscow, Scientific World (2005).
2. A. Chang and C. W. Slayman. J Cell Biol 115, 289-295 (1991).
3. S. Lecchi, C. J. Nelson, K. E. Allen, D. L. Swaney, K. L. Thompson, J. J. Coon,
M. R. Sussman, and C. W. Slayman. J Biol Chem 282, 35471-35481 (2007).
4. V. V. Petrov. J Biomol Struct Dyn http://www.jbsdonline.com/product-p18051.html
(2011).
1027
Role of Conserved Water Molecules in Binding
of Thyroxin and Analogs Inhibitors
to Human Transthyretin: A Study
on Water-Mimic Inhibitor Design
Transthyretin (TTR) is a very important protein associated with the transportation
of thyroxin hormone and vitamin-A in the serum as well as in ceribro-spinal-fluid
of human (1). The protein is also responsible to cause amyloid diseases like FAP
(Familial Amyloidotic Poly-neuropathy) and SSA (Senile systematic Amyloidosis) due to formation of amyloid fibrils (2, 3) by the protein and their deposition
in extra cellular matrixes and tissues in human body (4). Simulation of different
X-ray structures of TTR [available as dimer in Protein Data Bank, Resolution
1.3-2.0Å] and their water dynamics have revealed the presence of six conserved
water molecules in the buried core of A-chain, 4- in B-chain and 6- in the interface
of the dimer. Among these conserved water molecules, one in each monomer seems
to play an important role in the thyroxin binding with the protein. Comparative
analysis of unliganded TTR and TTR-Thyroxin complex structures (5) and their
53
Avik Banerjee
Hridoy R. Bairagya
Bishnu P. Mukhopadhyay*
Tapas K. Nandi
National Institute of
Technology- Durgapur,
West Bengal,
Durgapur –713209, India
* [email protected]
Figure 1: Interaction of conserved water
molecule (W) with Ser 117 and Thr 119 in
unliganded form of Protein. Transition of TTRthyroxin complex to Unliganded TTR.
1028
water molecular dynamics reveal that in the unliganded structures the conserved
water molecule forms H-bond with the side-chains of two important residues,
Ser-117 and Thr-119, of the thyroxin binding pocket (6). The Ser-Thr bound conserved water molecule seems to migrate when the thyroxin molecule enters in the
pocket and forms complex with the protein. Again, during migration of that water
molecule, side chains of the respective Ser-117 and Thr-119 adopt a trans like
conformation which stereo-chemically assist the thyroxin molecule to occupy the
specific binding pocket of TTR [Figure1]. The positional invariance of that water
molecule with the 5’-iodine atom of thyroxin molecule seems to be interesting.
These results may provide further insight and complementary information into thyroxin/inhibitor (thyroxin analog) binding chemistry, which may be used as a new
strategy in search of a new effective TTR-inhibitor design.
References
1. P. A. Peterson. J Biol Chem 246, 44-49 (1971).
2. L. K. Chang, J. H. Zhao, H. L. Liu, J. W. Wu, C. K. Chuang, K. T. Liu, J. T. Chen,
W. B. Tsai, and Y. Ho. J Biomol Struct Dyn 28, 39-50 (2010).
3. L. -K. Chang, J. -H. Zhao, H. -L. Liu, K. -T. Liu, J. -T. Chen, W. -B. Tsai, and Y. Ho. J
Biomol Struct Dyn 26, 731-740 (2009).
4. X. Hou, M. I. Aguilar, D. H. Small. FEBS J 274, 1637-1650 (2007).
5. A. Wojtczak, V. Cody, J. R. Luft, and W. Pangborn. Acta Crystallogr Sect D 52, 758-765 (1996).
6. A. Banerjee, H. R. Bairagya, B. P. Mukhopadhyay, T. K. Nandi, and A. K. Bera. IJBB 47,
197-202 (2010).
54
Matthew A. Young*
Douglas M. Jacobsen
Zhao-Qin Bao
Department of Biological Chemistry
and Bioinformatics Program
University of Michigan
Ann Arbor, 48109
*[email protected]
Structures and Dynamics of the Complete Protein
Kinase Catalytic Cycle of CDK2/CyclinA
Like many protein kinases, CDK2 is known to be a rather flexible enzyme and conformational transitions and protein dynamics are believed to play important roles
in both the catalytic mechanism and the regulation of catalytic activity. We have
determined high-resolution crystal structures of multiple steps along the complete
reaction cycle of CDK2, including a transition-state complex consisting of CDK2/
CyclinA bound to ADP, a substrate peptide and MgF3–, a structural mimic for the
gamma-phosphate of ATP in the transition-state. Compared to structures of active
CDK2 bound to its substrates or its products, the catalytic subunit of the kinase in
the transition-state adopts a more closed conformation of the active site and, for the
first time, a second catalytic Mg ion is observed in the active site. Coupled with a
strong [Mg] effect on in vitro kinase activity, this structure suggests that the transient
binding of a second Mg ion is necessary to achieve maximum rate-enhancement
of the chemical reaction and Mg concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the
simultaneous binding of substrate peptide, ATP and two Mg2+ ions is able to stabilize the closed and also more rigid organization of the active site that functions to
orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated gamma-phosphate from solvent. Once the phosphoryl-transfer
step is complete, the second Mg is released and the active site returns to the more
open conformation to release the products.
1029
Protein Flexibility Methods to Compare Protein
Structure Predictions
Geometric measurements such as RMSD or GDT score are routinely used as a
preliminary tool to assess the quality of a model to a certain reference structure
(usually high resolution X-ray or NMR data). Two problems with these measures
are: (1) at a given temperature there is not one single structure, but an ensemble
of them; (2) geometric analysis gives global information, is highly degenerate and
does not take the protein’s topology into consideration. We investigating the utility
of using an energetic measure based on a protein’s intrinsic flexibility pattern to
complement geometry measurements in model quality evaluation.
To account for flexibility we use the elastic network model (ENM) theory [1-3],
which is computationally inexpensive and gives an accurate approximation to protein flexibility. In ENM the protein is represented as a series of nodes (Calphas). A
simple Hamiltonian is derived by: (1) finding all pairs of nodes closer than some
cutoff distance; and (2) connecting them via springs. Calculation of the hessian
and its diagonalization yields a set of eigenvector-eigenvalue pairs representing the
directions of maximum deformability in the protein system. This information can
be used to calculate deformation energies needed to deform the reference structure
into the model one. This energy will rapidly increase when deforming regions of
the protein that are not intrinsically flexible. Thus, two structures with the same
RMSD (or same GDT score) might have very different energies. Conversely, two
structures that have the same energy value might be far apart in RMSD (see figure
below). Having an energetic measure is helpful in identifying structures within
thermal noise of each other (e.g. within the native ensemble), meaning that the
model is of good enough quality. It might be useful in events such as CASP [4] to
quantify how hard refinement of a given template is going to be.
AP acknowledges support from EMBO long-term fellowship.
Figure: Top is a reference crystal structure in CASP9 (TR569). Below are two submitted models. The
one on the left would receive a higher score in geometric measurements, but the main difference is in a
very flexible area of the protein, resulting in similar deformation energies for both models.
References
1.
2.
3.
4.
A. R. Atilgan, et al. Biophys J 80:505-515 (2001).
M. Tirion, Phys Rev Lett 77, 1905-1908 (1996).
K. Hinsen, Proteins 33, 417-429 (1998).
Proteins: Structure, Function, and Genetics 23(3), 295-460 (1995).
Alberto Perez*
Justin L. MacCallum
Yang Zhang
Ivet Bahar
Ken A. Dill
55
Laufer Center for Physical and
Quantitative Biology, Stony Brook
University, Department of Pharmaceutical
Chemistry, University of California,
San Francisco, and University of
Pittsburgh
*[email protected]
1030
56
Ahmet Bakan
Ivet Bahar*
Department of Computational and
Systems Biology, School of Medicine,
University of Pittsburgh, 3501 Fifth Ave,
Suite 3064 BST3, Pittsburgh, PA 15260
*[email protected]
Computational Generation of Inhibitor-bound
Conformers of p38 MAP Kinase and Comparison
with Experiments
We developed an extensible framework, ProDy, for structure-based analysis of
protein dynamics (http://www.csb.pitt.edu/ProDy/). ProDy allows for quantitative analysis of heterogeneous experimental structural datasets and comparison
with theoretically predicted conformational dynamics (1). Datasets include structural ensembles composed of a given family or subfamily members, mutants and
sequence homologues, in the presence/absence of their substrates, ligands or inhibitors. We demonstrate the utility of ProDy by way of application to exploring the
dynamics of p38 MAP kinases, a family of enzymes which play a critical role in
regulating stress-activated pathways, and serve as molecular targets for controlling
inflammatory diseases. Computer-aided efforts for developing p38 inhibitors have
been hampered by the necessity to include the enzyme conformational flexibility in
ligand docking simulations. A useful strategy in such complicated cases is to perform
ensemble-docking provided that a representative set of conformers is available for
the target protein either from computations or experiments. Using ProDy, we explore
the abilities of two computational approaches, molecular dynamics (MD) simulations and anisotropic network model (ANM) normal mode analysis, for generating
potential ligand-bound conformers starting from the apo state of p38, and benchmark
them against the space of conformers inferred from the principal component analysis
of 134 experimentally resolved p38 kinase structures (2). The ANM-generated conformations are found to provide a significantly better coverage of the inhibitor-bound
conformational space observed in experiments, compared to the conformers generated by MD simulations performed in explicit water. The results suggest that ANMbased sampling of conformations can be advantageously employed for generating
structural models to be used as input in docking simulations.
Support from NIH grants 1R01GM086238-01, 5R01GM086238-02 and
5R01LM007994-06 is gratefully acknowledged by I. Bahar.
References
1. A. Bakan and I. Bahar. Proc Natl Acad Sci USA 106, 14349-14354 (2009).
2. A. Bakan and I. Bahar. Pacific Symposium on Biocomputing 16, 181-192 (2011).
1031
Dynamics of AMPA-subtype Glutamate Receptor
Using Elastic Network Models
Ionotropic glutamate receptors (iGLURs) are known to mediate several excitatory
neurotransmission in the central nervous system. These receptors are ligand gated
ion channels that couple agonist binding to a ligand binding core, to open and
desensitize the ion channel. Dysfunction of iGLURs due to injury or other stimuli,
results in acute neurological disorders attracting the attention of pharmaceutical
industry to iGLURs as potential drug discovery targets. The recent structure of the
intact α-amino-3-hydroxy-5-methyl-4-isozazole propionic acid (AMPA) receptor
(1) paved the way for a structural analysis of the dynamics of the receptor. Global,
large-scale, co-operative motions of the structure are obtained using the well know
Gaussian Network Model (GNM) and Anistropic Network Model (ANM) (2, 3).
The slowmodes within this model, enabled identification of hinge residues at the
N-terminal domain (NTD), Ligand Binding domain (LBD) and at the Transmembrane Domain (TMD). The Markovian Stochastic Model (MSM) (4), enabled the
identification of several key residues with low-commute times. The residues identified as hot-spots by MSM and the hinge residues identifed by GNM and ANM
although scattered in the different domains, are located at structurally ‘strategic’
places such as NTD/LBD interface, LBD/TMD interface or even the linker region.
Several of these residues are also highly conserved among the iGLUR family,
and are thus anticipated to play a role in the signal transduction machinery of the
AMPA-receptor.
References
1. A. I. Sobolvesky, M. P. Rosconi, and E. Gouaux. Nature 462, 745-756 (2009).
2. A. R. Atilgan, S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar.
Biophys J 80, 505-515 (2001).
3. I. Bahar, T. R. Lezon, A. Bakan, and I. H. Shrivastava. Chem Rev 110, 1463-1497 (2010).
4. C. Chennubhotla and I. Bahar. PloS Comp Biol 3, e172 (2007).
Indira Shrivastava*
Anindita Dutta
Ivet Bahar
57
University of Pittsburgh, Department of
Computational and Systems Biology,
Fifth Avenue, 3501, Biomedical Science
Tower-3, Pittsburgh, PA 15213
*[email protected]
1032
58
Elia Zomot
Ivet Bahar*
Department of Computational & Systems
Biology, School of Medicine, University
of Pittsburgh, 3064 BST3, 3501 Fifth
Avenue, Pittsburgh, PA 15213
*[email protected]
Protonation of Glutamate-208 Induces the Release
of Agmatine in an Outward-Facing Conformation
of Arginine/Agmatine Antiporter
Virulent enteric pathogens have developed several systems that maintain intracellular pH in order to survive extreme acidic conditions. One such mechanism is
the exchange of arginine (Arg+) from the extracellular region with its intracellular
decarboxylated form, agmatine (Agm2+). The net result of this process is the export
of a virtual proton from the cytoplasm per antiport cycle (1). Crystal structures of
the arginine/agmatine antiporter from E. coli, AdiC, have been recently resolved in
both the apo and Arg+-bound outward-facing conformations (2, 3), which permit us
to assess for the first time the time-resolved mechanisms of interactions that enable
the specific antiporter functionality of AdiC. Using data from approximately 1µs
of molecular dynamics simulations, we show that the protonation of E208 selectively causes the dissociation and release of Agm2+, but not Arg+, to cell exterior.
The impact of E208 protonation is transmitted to the substrate binding pocket via
the reorientation of I205 carbonyl group at the irregular portion of transmembrane
(TM) helix 6. This effect, which takes place only in the subunits where Agm2+ is
released, invites attention to the functional role of the unwound portion of TM helices (TM6 W202-E208 in AdiC) in facilitating substrate translocation, reminiscent
of the behavior observed in structurally similar Na+-coupled transporters.
This research was supported by NIH grants 1R01GM086238-01 and
1U54GM087519-01A1, and by the NSF through TeraGrid resources provided by Kraken (NICS) and Ranger (TACC) under grant number
TG-MCB100108.
References
1. R. Iyer, C. Williams, and C. Miller. J Bacteriol 185, 6556-6561 (2003).
2. X. Gao, F. Lu, L. Zhou, S. Dang, L. Sun, X. Li, J. Wang, and Y. Shi. Science 324,
1565-1568 (2009).
3. X. Gao, L. Zhou, X. Jiao, F. Lu, C. Yan, X. Zeng, J. Wang, and Y. Shi. Nature 463, 828-832
(2010).
59
Mert Gur
Ivet Bahar
University of Pittsburgh, 3501 Fifth Ave,
Suite 3064 BST3, Pittsburgh, PA 15260
[email protected]
[email protected]
Transition Pathways of Enzymes Explored by
Combining the Anisotropic Network Model,
Molecular Dynamics Simulations and a Monte Carlo
Sampling of Conformational Space
The conformational transition between the open and closed forms of Escherichia
coli adenylate kinase (AK) is explored using a molecular dynamics (MD) simulation protocol which is guided by the normal modes derived from the coarse
grained anisotropic network model (ANM). The methodology applies to the cases
where the passage from one substate to another (e.g., the open and closed forms
of an enzyme) within a global energy minimum (native state) involves relatively
low energy barriers, based on the assumption that low energy barriers may be surmounted/overlooked by adopting a coarse-grained description of the structure and
energetics, which smoothes out the energy landscape. The basic approach is to
deform the structure along ANM modes, similar to the adaptive ANM (aANM)
procedure adopted in our previous work, (1) but with the major improvement that
1033
the intermediate structures are selected by a Monte Carlo scheme and energy minimized by short MD runs. While the detailed energy landscape may usually comprise multiple microstates and multiple barriers/pathways, the coarse-graining of
the transition path between the open and closed forms of AK highlights three substates. In agreement with previous work, the conformational change is undergone
in two steps: Closing of the LID (shown in red in Figure1) region succeeded by that
of the nucleotide binding domain (colored orange). Some residues are observed to
experience high internal energies during the simulated transition, this highlighting the critical interactions that play a dominant role in destabilizing or stabilizing
particular substates.
Support from NIH grants 1R01GM086238-01is gratefully acknowledged by
I. Bahar.
Reference
1. Zheng Yang , Peter Májek, and Ivet Bahar. PLoS Comput Biol 5(4): e1000360 (2009).
Intrinsic Dynamics and Allostery: Learning from
Theory, Computations and Experiments
The significance of protein dynamics in achieving molecular functions in the cell
is widely recognized. The old view, ‘a unique structure for each protein’, is now
replaced by ‘an ensemble of conformers’ accessible near native state conditions.
Many studies suggest that fluctuations between the conformers, or transitions
between their representative substates, underlie, if not enable, functional events.
Elastic network models (ENMs) and spectral graph theoretical analysis methods
are broadly used for exploring the collective dynamics intrinsically accessible to
biomolecular systems to elucidate structure-encoded dynamics and function. In
parallel with the multiplicity of conformers accessible under physiological conditions, the Protein Data Bank contains multiple structures of the same protein in different forms (e.g., orthologs, mutants, substates visited during an allosteric cycle,
or various complexes, multimers or assemblies). These datasets usually convey
valuable information on functional changes in structure. Furthermore, they can help
benchmark and improve theoretical models, computational methods and software.
Our recent work suggests that the information inferred from these experimental
datasets and those predicted by theory and computations can be advantageously
combined to gain insights into the allosteric mechanisms of activation or inhibition
of target proteins. Recent applications will be presented, along with a discussion
Ivet Bahar
60
[email protected]
1034
of the limits of applicability of ENM-based approaches and molecular dynamics
simulations, and future directions to overcome these limitations (1).
Support from NIH 5R01GM086238-02 is gratefully acknowledged.
Reference
1.
61
Z. N. Gerek
A. Bolia
S. B. Ozkan*
Center for Biological Physics,
Arizona State University,
Tempe, AZ 85042
*[email protected]
I. Bahar, T. R. Lezon, L. -W. Yang, and E. Eyal. Annu Rev Biophys 39, 23-42 (2010).
Perturbation Response Scanning Method
for Identifying Allosteric Transitions and
Utilizing in Flexible Docking
We have recently developed coarse-grained method; perturbation response scanning (PRS) that couples elastic network models (1) with linear response theory
(LRT). It computes the response of the protein structure (i.e. displacement vector)
upon exerting directed random forces on selected residues. The method has proven
successful in reproducing residue displacements for a set of 25 proteins that display
a variety of conformational motions upon ligand binding (2). Using PRS we analyzed two PDZ domain proteins (PSD95 PDZ3 domain and hPTP1E PDZ2 domain)
whose allosteric behavior play a key role in signaling. By PRS, we first identified the residues that give the highest response upon perturbing the binding sites.
Strikingly, we observe that the residues that give the highest response agree with
experimentally determined residues involved in allosteric pathways. Second, we
constructed the allosteric pathways by clustering the residues giving same type of
response upon perturbation of the binding sites. Interestingly our analysis provided
molecular understanding of experimentally observed hidden allostery of PSD95.
We have shown that removing the distal alpha helix from the binding site alters the
allosteric pathway and decreases the binding affinity. Overall, these results indicate
that (i) PRS is successful in capturing the conformational changes upon binding
(2), (ii) it can identify key residues that mediate long-range communication in PDZ
Figure 1: Allosteric pathways of wild type PSD95 and truncated (distal α-helix) PSD 95. Allosteric pathway upon truncation the distal a-helix changes and this also leads a change in binding affinity and confirmed with our flexible
docking and experimental analysis.
1035
domain proteins (3), (iii) we can construct the allosteric pathways and show how
the allosteric pathway changes upon minor alteration in the fold with PRS (3).
Utilizing these exceptional features of PRS, we have recently developed a flexible
docking scheme which predicts the peptide-protein interactions accurately.
References
1. E. D. Akten, S. Cansu, and P. Doruker. J Biomol Struct Dyn 27, 13-25 (2009).
2. C. Atilgan, Z. N. Gerek, S. B Ozkan, and A. R. Atilgan. Biophys J 99, 933-943 (2010).
3. Z. N. Gerek and S. B Ozkan. PLoS Comp Biol, submitted (2010).
Linking Allostery in Chaperonins to Protein Folding
Chaperonins consist of two back-to-back stacked oligomeric rings with a cavity at
each end in which protein folding can take place under confining conditions (1).
They are molecular machines that assist protein folding by undergoing large-scale
ATP-driven allosteric transitions between protein substrate binding and release
states (1, 2). The intra-ring conformational changes in chaperonins were found to
be concerted in the case of the homo-oligomeric prokaryotic chaperonin GroEL
and sequential in the case of the hetero-oligomeric eukaryotic chaperonin CCT
(2). Previously (3), we hypothesized that a sequential allosteric mechanism might
be more beneficial for eukaryotic proteins that tend to be larger and multi-domain
as it may enable one domain to detach from the chaperonin and start folding while
the other domain(s) is still bound, thereby mimicking co-translational folding. By
contrast, we reasoned that a concerted mechanism is likely to be more beneficial for
prokaryotic proteins that tend to be smaller and single-domain and, thus, may need
to be released in an all-or-none fashion in order to fold efficiently.
Support for this hypothesis was first obtained from lattice model simulations of single- and double-domain protein folding in chaperonin cages that undergo concerted
or sequential allosteric transitions (4). We then took advantage of a GroEL mutant
(D155A) that undergoes sequential intra-ring allosteric transitions (5) to test our
hypothesis experimentally. In one test, we used a chimeric fluorescent protein substrate, CyPet-Ypet, for which it was possible to determine the folding yield of each
domain from its intrinsic fluorescence and that of the entire chimera by measuring
FRET between the two domains. Hence, it was possible to determine whether release
(and thus also folding) of one domain is accompanied by release of the other domain
(concerted mechanism) or if their release is not coupled. Our results showed that
the chimera’s release tends to be concerted when its folding is assisted by wild-type
GroEL but not when it is assisted by the D155A mutant that undergoes a sequential
allosteric switch (6). In a second test, we used chimeras in which a substrate whose
release requires the co-chaperonin GroES is fused to a GroES-independent substrate
(7). In the case of the D155A mutant, release and folding of the GroES-dependent substrate was found to take place in a step-wise fashion upon addition of ATP whereas, in
the case of wild-type GroEL, substrate release was found to have a sigmoidal dependence on ATP concentration. Our results, therefore, demonstrate that changes in the
allosteric mechanisms of chaperonins can impact their folding function.
References
1. A. L. Horwich, W. A. Fenton, E. Chapman, and G. W. Farr. Annu Rev Cell Dev Biol 23,
115-145 (2007).
2. A. Horovitz and K. R. Willison. Curr Opin Struct Biol 15, 646-651 (2005).
3. D. Rivenzon-Segal, S. G. Wolf, L. Shimon, K. R. Willison, and A. Horovitz. Nat Struct Mol
Biol 12, 233-237 (2005).
4. E. Jacob, A. Horovitz, and R. Unger. Bioinformatics 23, i240-i248 (2007).
5. O. Danziger, D. Rivenzon-Segal, S. G. Wolf, and A. Horovitz. Proc Natl Acad Sci USA 100,
13797-13802 (2003).
6. N. Papo, Y. Kipnis, G. Haran, and A. Horovitz. J Mol Biol 380, 717-725 (2008).
7. Y. Kipnis, N. Papo, G. Haran, and A. Horovitz. Proc Natl Acad Sci USA 104, 3119-3126 (2007).
Amnon Horovitz
62
Department of Structural Biology,
Weizmann Institute,
Rehovot 76100, Israel
[email protected]
1036
63
George Stan1*
Andrea Kravats1
Sam Tonddast-Navaei1
Manori Jayasinghe2
1Department
of Cincinnati, Cincinnati, OH 45221
2Department
Computational Modeling of Allostery-Driven
Unfolding and Translocation of Substrate Proteins
Molecular chaperones employ diverse ATP-dependent mechanisms to effect
protein folding and degradation. Chaperonin nanomachines assist protein folding
through concerted allostery in bacteria (1, 2) and sequential allostery in eukaryotic and archaeal organisms (3, 4). Clp ATPases, which are hexameric ring-shaped
AAA+ nanomachines that perform substrate protein (SP) unfolding and translocation for protein degradation, are suggested to undergo sequential allostery(5). We
use coarse-grained simulations to study the protein remodeling actions of ClpY,
which contains a single ATP-binding domain per subunit, onto a tagged four-helix
bundle SP (Figure 1). Our results indicate that unfolding is initiated at the tagged
C-terminus via an obligatory intermediate (6). Translocation proceeds on a different timescale than unfolding and involves sharp stepped transitions. We find
that an ordered sequential mechanism is more effective than random or concerted
of Chemistry,
Northern Kentucky University,
Highland Heights, KY 41099
*[email protected]
Figure 1: Unfolding and translocation of the fusion protein formed by a four-helix bundle protein
(purple) and the SsrA peptide (yellow) by the ClpY ATPases (green). For clarity, two of the six ClpY
subunits are not shown.
allostery. In the absence of allosteric motions, mechanical unfolding of the SP in
atomic force microscopy experiments proceeds via multiple unfolding pathways. SP
threading through a non-allosteric ClpY nanopore involves simultaneous unfolding
and translocation effected by strong pulling forces.
The p97 nanomachine is a homologue of ClpA and ClpB, which contain two ATPbinding domains per subunit. We use coarse-grained and atomistic simulations to
investigate the unfolding mechanism of the four-helix bundle protein coupled with
1037
ATP-driven conformational changes in the D2 domain of p97. Our simulations
suggest that SP unfolding and translocation takes place as a result of the collaboration between strongly conserved sites, Arg586 and Arg599 residues and the
D2 central pore loop. The mechanism of SP translocation involves a mechanical
force exerted by the D2 central pore loop combined with substrate binding at the
Arginine sites of adjacent subunits. Unlike ClpY-assisted action, SP unfolding and
translocation actions effected by p97 are simultaneous. We find that accumulation
of the SP chain within the central cavity of the D2 does not result in significant SP
refolding.
This research has been supported by a grant from the American Heart Association
and by the National Science Foundation CAREER grant to G. S. and an University
Research Council fellowship at the University of Cincinnati to M.J.
References
1. D. Thirumalai and G. H. Lorimer. Annu Rev Biophys Biomol Struct 30, 245-269 (2001).
2. G. Stan, G. H. Lorimer, D. Thirumalai, and B. R. Brooks. Proc Natl Acad Sci USA 104,
8803-8808 (2007).
3. A. Horovitz and K. Willison. Curr Op Struct Biol 15, 646-651 (2005).
4. M. Jayasinghe, C. Tewmey, and G. Stan. Proteins: Structure, Function, and Bioinformatics
78, 1254-1265 (2010).
5. A. Martin, T. A. Baker, and R. T. Sauer. Nature 437, 1115-1120 (2005).
6. A. Kravats, M. Jayasinghe, and G. Stan. Proc Natl Acad Sci USA (in press).
64
Energetically Favourable Communication Pathways
in Pyrrolysyl-tRNA Synthetase
Aminoacyl-tRNA synthetases (aaRS) play a pivotal role in the protein biosynthetic
machinery, ensuring the correct translation of the genetic code. The response to
cognate tRNA binding is elicited in the release of the activated amino acid from
the pre-transfer complex to the 3’ end of the tRNA. Such efficient communication across distant sites (1) underlies allostery, making the aaRS an excellent
model for studying this phenomenon. Several studies at atomistic detail (2, 3),
including investigations on aaRS (such as MetRS and TrpRS) from our own lab
(4, 5), have elucidated allosteric communications. Here we have chosen an atypical aaRS, pyrrolysyl-tRNA synthetase (from D.hafniense [DhPylRS]), in its three
different states of ligation (Sys1: native DhPylRS, Sys2: DhPylRS+2YLY, Sys3:
DhPylRS+2YLY+2tRNA) for our study. Interestingly, in contrast to canonical
aaRS, DhPylRS exhibits a diffused recognition of tRNA bases (not residing in the
triplet codon). Recent crystal structure of DhPylRS [dimer] bound to tRNAPyl has
given insights into the unique protein-tRNA interactions accounting for the orthogonality of this aaRS-tRNA pair (6).
In this study, we investigate the interaction energy weighted (7) protein-tRNA
network and its dynamical properties to gain insight into the functioning of this
non-canonical tRNA-synthetase and understand the mechanism of long-range communications. The concept of pre-existing paths of communication (8) and their
manifestations at different liganded forms are probed in this study. Specifically, the
ensemble derived interaction energy between residues is a parameter that regulates
the transmission of perturbation upon ligand binding between distant functional
sites in DhPylRS. Interaction energy based long-range residue coupling from the
MD ensembles is found to contribute to global signal transfer. Our analysis reveals
Moitrayee Bhattacharyya
Saraswathi Vishveshwara*
Molecular Biophysics Unit, Indian
Institute of Science, Bangalore, India
*[email protected]
1038
the importance of side-chain interactions while backbone conformational changes
are not significant for Sys1-3. The ligand induced changes in conformation and
communication pathways are efficiently captured by protein-tRNA energy networks for the three systems. We also probe different weighted network parameters
(e.g., betweenness and funneling) to obtain the key residues for signal propagation
across distant sites. The cost of communication between distant functional sites
and pre-existence of the optimal and sub-optimal pathways in the MD ensemble is
also investigated. Furthermore, asymmetry in terms of communication efficiency
between the two subunits is clearly evident from all our studies for Sys1-3 to various extents, with Sys3 being maximally asymmetric. Interestingly, the concept of
half-sites reactivity discussed in literature agrees well to this asymmetry between
the two subunits. Additionally we find that the transfer of activated amino acid to
the 3’ end of tRNA is co-ordinated by alternation of global rigidity/flexibility. Our
results exhibit good correlation with mutagenesis experiments for DhPylRS. Based
on these observations, a general mechanistic insight for allosteric communication
and the relevance of asymmetry in the dimeric protein is presented.
Abbreviations: YLY: adenylated pyrrolysine; Pyl: pyrrolysine.
References
1. T. Zhu, B. Wu, B. Wang, and C. Zhu. J Biomol Struct Dyn 27, 573-579 (2009).
2. C. Chennubhotla and I. Bahar. PLoS Comput Biol 3, e172 (2007).
3. A. Sethi, J. Eargle, A. A. Black, and Z. Luthey-Schulten. ProcNatl Acad Sci USA 106, 6620
(2009).
4. A. Ghosh and S. Vishveshwara. Proc Natl Acad Sci USA 104, 15711 (2007).
5. M. Bhattacharyya, A. Ghosh, P. Hansia, and S. Vishveshwara. Proteins: Struct, Funct, and
Bioinfo 78, 506 (2010).
6. K. Nozawa, P. O/’Donoghue, S. Gundllapalli, Y. Araiso, R. Ishitani, T. Umehara, D. Soll,
and O. Nureki. Nature 457, 1163 (2009).
7. M. Vijayabaskar and S. Vishveshwara. Biophys J (accepted), (2010).
8. A. del Sol, C. -J. Tsai, B. Ma, and R. Nussinov. Structure 17, 1042 (2009).
1039
Modeling Three Dimensional Structures of
Complete PKS Modules for Understanding
Inter-domain Interactions
Modular polyketide synthases (PKS) utilize multiple copies of distinct sets of
catalytic domains, called modules for catalyzing biosynthesis of a variety of pharmaceutically important natural products (1). Even though bioinformatics analysis
has played a major role in discovery of novel secondary metabolites and rational
design of natural product analogs, most of these computational methods have used
sequence information alone. However, recently available crystal structures of mammalian FAS and large polypeptide stretches from modular PKS indicate that, the
polyketide biosynthesis is brought about by a tightly coupled network of catalytic
and structural domains (2). Therefore, it is necessary to model three-dimensional
structures of complete PKS modules for understanding role of inter domain interactions in substrate channeling.
Structure based sequence analysis on a data set of 662 KS, 541 AT, 308 DH, 99 ER,
450 KR and 562 ACP domains from 55 modular PKS clusters indicate that, except
for the structural sub-domain of KR, all other domains show significant sequence
similarity with available structural templates. Despite the high sequence divergence,
the structural sub-domain of KR can be modeled using threading approach. The
dimeric structure of complete PKS module could also be modeled based on the relative orientation of different domains in mechanistically analogous mammalian FAS
structure (3). Modeling of a bi-modular PKS protein using this approach has provided valuable clues for recent discovery of a novel modularly iterative mechanism
of mycoketide biosynthesis in Mycobacterium tuberculosis (4). Since the mammalian FAS structure lacks ACP domain, we have tried to predict its orientation
with respect to other catalytic domains using protein-protein docking and molecular
dynamics methods (5, 6). Long molecular dynamics simulations on the KS-AT
di-domain structure indicate that, the extent of inter domain movement within a
module is not large enough to bring them in proximity for acyl transfer. Thus,
intrinsic flexibility of the linker regions preceding ACP might facilitate interaction
of ACP with other catalytic domains. These results on inter domain interactions
within PKS modules have interesting implications for design of domain swapping
experiments for obtaining natural product analogs by biosynthetic engineering.
References
1. B. Shen. Curr Opin Chem Biol 7, 285-95 (2003).
2. R. S. Gokhale, R. Sankaranarayanan, and D. Mohanty. Curr Opin Struct Biol 17, 736-43
(2007).
3. S. Anand, M. V. Prasad, G. Yadav, N. Kumar, J. Shehara, M. Z. Ansari, and D. Mohanty.
Nucleic Acids Res 38, W487-96.
4. T. Chopra, S. Banerjee, S. Gupta, G. Yadav, S. Anand, A. Surolia, R. P. Roy, D. Mohanty,
and R. S. Gokhale. PLoS Biol 6, e163 (2008).
5. P. Sklenovsky and M. Otyepka. J Biomol Struct Dyn 27, 521-539 (2010).
(2010).
Swadha Anand*
Debasisa Mohanty
65
National Institute of Immunology, Aruna
Asaf Ali Marg, New Delhi-110067, India
*[email protected]
1040
66
Andrea Kravats1*
Manori Jayasinghe2
George Stan1
1Department
of Cincinnati, Cincinnati, OH 45221
2Department
of Chemistry, Northern
Kentucky University, Highland Heights,
KY 41099
*[email protected]
Molecular Dynamics Simulations of
Protein Unfolding and Translocation Resulting
from Allosteric Motions of ClpY
Clp ATPases are macromolecular machines which use the energy released from
ATP hydrolysis to unfold, translocate and degrade misfolded proteins. ClpY, a bacterial unfoldase within this family, assembles into a homohexameric ring structure
with a narrow central pore. Flexible diaphragm forming loops within this channel
undergo large scale conformational changes driven by ATP binding and hydrolysis. The result is unfolding and translocation of a tagged substrate protein. We
employ coarse grained molecular dynamics simulations to probe coupling between
the allosteric motions of the central pore loops and the unfolding and translocation
of a four helix bundle protein (Figure 1). We determine that minimal unfolding of
the SP into an obligatory non-native intermediate, a three helix bundle, is required
for translocation. The pathway for unfolding is unraveling from the C-terminus,
which is in agreement with experiments (1). Multiple translocation pathways are
observed following the initial unfolding event. Weak mechanical forces exerted by
the pore loops accompanied by transient SP binding to the I domain effect translocation (2). We also investigate the ordering of allosteric transitions within individual subunits of the ClpY ring. Experiments suggest non-concerted allostery (3);
however, the preference between sequential and random allostery remains unclear.
To determine the efficacy of the possible allosteric mechanisms, we perform simulations of concerted, random, and sequential (clockwise and counterclockwise,
viewed proximal to the I domain) mechanisms. The concerted simulations do not
result in translocation, in accord with experiments (1). Our results indicate that
sequential clockwise simulations are the most efficient in the handling and translocation of the substrate protein.
Figure 1: Unfolding and translocation of a substrate protein formed from the four helix bundle protein
(magenta) and ssrA degradation tag (yellow). Two subunits of ClpY (green) have been removed.
1041
References
1. C. Lee, M. Schwartz, S. Prakash, M. Iwakura, and A. Matouschek. Mol Cell 7, 627-637
(2001).
2. A. Kravats, M. Jayasinghe, and G. Stan. Proc Natl Acad Sci USA 108, 2234-2239 (2011).
3. A. Martin, T. A. Baker, and R. T. Sauer. Nature 437, 1115-1120 (2005).
67
Evolution of Structure and Dynamics for a Family
of Intrinsically Disordered Proteins
Intrinsically disordered proteins (IDPs) perform essential functions in organisms
from all phyla. IDPs do not form tertiary structures and contain varying amounts of
secondary structure. In order to develop general relationships between the structure
and function of IDPs, we are investigating the structure and dynamics of protein
families that are intrinsically disordered. The work is being placed in an evolutionary context to permit the identification of important structural features by virtue
of their conservation and constitutes the first attempt to quantify the relationship
between sequence identity and structural similarity for IDPs. The intrinsically
disordered transactivation domain of the tumor suppressor, p53 (p53TAD) is one
model system chosen for study. Significant differences are observed in the secondary structure and dynamics of mammalian homologues of p53TAD. These differences are primarily localized to the binding sites for the ubiquitin ligase, MDM2
and the 70 KDa subunit of replication protein A (RPA70) and appear to influence
the kinetics and thermodynamics of binding. We are also investigating the role of
prolines in controlling the structure and dynamics of IDPs. Mutating the conserved
prolines that flank the MDM2 binding site has a striking effect on the structure and
dynamics of this region. An analysis of other IDPs that fold when they bind to their
protein partners shows that proline residues are enriched in the regions flanking
the binding sites, suggesting the structural and dynamical effects observed for the
p53TAD homologues are general.
This work is supported by the American Cancer Society (RSG-07-289-01-GMC)
and the National Science Foundation (MCB-0939014).
Gary W. Daughdrill1*
Wade Borcherds1
Hongwei Wu1
Bin Xue2
Vladimir Uversky2
1Department
of Cell Biology,
Microbiology, and Molecular Biology
and Center for Drug Discovery and
Innovation
2Department
of Molecular Medicine,
University of South Florida,
Tampa, Fl 33612
*[email protected]
1042
68
David Eliezer
Department of Biochemistry,
Weill Cornell Medical College,
New York, NY 10065
[email protected]
69
Akihiko Takashima
Laboratory for Alzheimer’s disease,
Brain Science Institute, RIKEN,
2-1 Hirosawa, Wako-shi,
Saitama 350-0198
Japan
[email protected]
Disordered Proteins in Parkinson’s and Alzheimer’s
Disease: Linking Structural Transformations to
Function, Aggregation and Toxicity
The proteins tau and alpha-synuclein are linked to neurodegenerative disease both
through their appearance within protein-rich deposits in diseased brain and through
the identification of mutations in their associated genes which cause hereditary
forms of disease. Both protein share the property of being highly disordered when
isolated in solution, but both proteins can undergo disorder-to-order transitions
either upon interactions with appropriate binding partners or upon aggregating into
fibrillar amyloid-like aggregates similar to those found in brain deposits. Structural
studies of these proteins provide the basis for new hypothesis regarding how their
structural transformations may mediate both the normal function of these proteins,
as well as they pathological aggregation.
Tau Change is a Key for Understanding Brain
Aging and Alzheimer Disease
Neurofibrilarlly tangles (NFTs), which consists fibrillar aggregate of hyperphosphorylated tau, are commonly seen in aging and Alzheimer’s disease brain. Based
on Braak staging of NFTs, NFT first observed in entorhinal cortex. Then, NFTs
spread from entorhinal cortex to limbic and neocortex. NFTs formation in entorhinal cortex may be correlating with memory loss in brain aging, because entorhinal
cortex is involved in memory formation, and NFTs in limbic and neocortex may
cause dementia in AD, because limbic and neocortex serve higher order brain functions. These suggest that regional development of NFTs is correlated with decline
of brain functions in aging and AD. Recent reports sugge sted that the process of
NFT formation, but not NFT itself is involved in neuronal dysfunction. Normally
tau binds to microtubules and stabilize them. Once tau receives hyperphosphorylation by activating tau kinases, tau dislodge from microtubules, and starts tautau interaction in cytoplasm, forming tau oligomers. When tau oligomers possess
b-sheet structure, tau oligomer forms insoluble granular tau aggregate. Granular
tau aggregate sticks together, and form NFT. From analysis of tau Tg mouse, we
found that hyperphosphorylated tau is involved in synapse loss, and granular tau
aggregate is involved in neuronal loss. Thus, during NFT formation, different tau
aggregation induces synapse loss, and neuronal loss, leading to brain dysfunction
in brain aging and AD. A process of tau aggregation was analyzed by ThT fluorescence and AFM observation. Role of different tau aggregates was determined by
analysis of tau Tg mouse. Before tau fibril formation, tau formed soluble oligomer,
insoluble granular tau aggregate. Soluble phosphorylated and oligomer tau may be
involved in synapse loss, and insoluble granular tau aggregates may play a role in
neuronal death. Inhibition of phosphorylated tau, and granular tau aggregation is
expected to block a progression of AD symptom by preventing synapse loss and
neuronal loss.
On the next page is illustrated a diagram showing the connection between each tau
aggregates on synapse loss and neuron loss.
1043
Connection between each tau aggregates on synapse loss and neuron loss
70
Understanding The Molecular Basis of Pathogenicity
or Lack Thereof of Serum Amyloid a Isoforms
Acute-phase protein Serum Amyloid A (SAA) is an important biomarker of inflammation and the precursor protein responsible for amyloid A (AA) amyloidosis.
Under normal circumstance, SAA is found associated with high-density lipoproteins
(HDL), but during infection or injury, SAA levels can increase as high as 1000-fold
in ~24hrs. Under chronic inflammatory conditions, persistence of high levels of SAA
leads to amyloid deposits composing of whole length and fragments of SAA leading
to the systemic AA amyloidosis disease. In mouse models, SAA1.1 predominates in
amyloid deposits both as full length and fragmented forms. However, the CE/J type
mouse, which expresses a single isoform (i.e. SAA2.2), was resistant to amyloidosis.
SAA1.1 and SAA2.2 differ only by six amino acids, suggesting that factors such as
fibrillation kinetics, oligomeric states and ligand interactions might play a critical role
in determining their pathogenicity. The present study attempts to understand the oligomeric and aggregation mechanism of the pathological and non-pathological forms
of SAA, SAA1.1 and SAA2.2 respectively. Our data show that both SAA isoforms
exhibit marked differences in oligomeric propensities, fibrillation kinetics and thermal stabilities suggesting the importance of location of specific amino acid residues.
SAA1.1 was found to be more intrinsically disordered and exhibited low thermal stability when compared to SAA2.2. Furthermore, SAA1.1 showed a longer lag phase
(3-4 days) prior to fibrillation, when compared to SAA2.2 (3-6 hrs). To obtain insights
on the molecular basis of these observed differences, point mutations were performed
on SAA2.2 to make it resemble SAA1.1, one amino acid at a time. Overall, comparison of the stability, oligomeric structure and aggregation properties of SAA2.2 and
SAA1.1 provides insight that explains their difference in pathogenicity. Furthermore,
the intrinsically disordered structure of SAA2.2 and SAA1.1 and their ability to form
a diversity of self-assembled structures at 37°C suggests that the structure of SAA
might be modulated in vivo to form different biologically relevant species.
Sai Praveen Srinivasan1,2
Yun Wang1,2
Zhuqiu Ye1,2
Marimar Lopez2
Wilfredo Colón1,2*
1Department
of Chemistry and Chemical
Biology
2Center
for Biotechnology and
Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy,
New York, 12180
*[email protected]
1044
71
Nadrian C. Seeman
New York University,
New York, NY 10003, USA
[email protected]
DNA: Not Merely the Secret of Life
Structural DNA nanotechnology is based on using stable branched DNA motifs,
like the 4-arm Holliday junction, or related structures, such as double crossover
(DX), triple crossover (TX), and paranemic crossover (PX) motifs. The sequence
design of stable branched molecules is based on the notion of minimized sequence
symmetry. We have been working since the early 1980’s to combine these DNA
motifs to produce target species. From branched junctions, we have used ligation to
construct DNA stick-polyhedra and topological targets, such as Borromean rings.
Branched junctions with up to 12 arms have been produced. We have also built
DNA nanotubes with lateral interactions.
Nanorobotics is a key area of application. PX DNA has been used to produce a
robust 2-state sequence-dependent device that changes states by varied hybridization topology. We have used this device to make a translational machine that prototypes the simplest features of the ribosome. Two protein-activated devices have
been developed that can measure the ability of the protein to do work, and bipedal
walkers, both clocked and autonomous have been built. We have also built a robust
3-state device that includes a state corresponding to a contraction.
One of the long-sought goals of nanotechnology has been the construction of
molecular assembly lines. We have combined a DNA origami layer with three
PX-based devices, so that there are eight different states represented by the arrangements of these 2-state devices; we have programmed a novel DNA walking device
to pass these three stations. As a consequence of proximity, the devices add a cargo
molecule to the walker. We have demonstrated that all eight products (including the
null product) can be built from this system. More extensive origami systems could
be used to make even more diverse and complex products. Most recently, we have
used DNA origami in a diagnostic tool.
A central goal of DNA nanotechnology is the self-assembly of periodic matter. We
have constructed 2-dimensional DNA arrays from many different motifs. We can
produce specific designed patterns visible in the AFM. We can change the patterns
by changing the components, and by modification after assembly. Recently, we
have self-assembled a 3D crystalline array and have solved its crystal structure to
4 Å resolution, using traditional unbiased crystallographic methods. Nine other
crystals have been designed following the same principles of sticky-ended cohesion. We can use crystals with two molecules in the crystallographic repeat to control the color of the crystals. Thus, structural DNA nanotechnology has fulfilled its
initial goal of controlling the structure of matter in three dimensions. A new era in
nanoscale control is beginning.
This research has been supported by grants GM-29554 from the National Institute
of General Medical Sciences, CTS-0608889 and CCF-0726378 from the National
Science Foundation, 48681-EL and W911NF-07-1-0439 from the Army Research
Office, N000140910181 and N000140911118 from the Office of Naval Research
and a grant from the W.M. Keck Foundation.
1045
The Label-Free Unambiguous Detection and
Symbolic Display of Single Nucleotide
Polymorphisms on DNA Origami
Single Nucleotide Polymorphisms (SNPs) are the most common genetic variation
in the human genome. Kinetic methods based on branch migration have proved
successful for detecting SNPs because a mispair will inhibit the progress of branch
migration in the direction of the mispair. Biased single-stranded branch migration
is used prominently for changing the shapes of DNA nanomachines, because it
involves the isothermal removal of strands from a DNA machine frame, enabling
a change in topology. Here, we have combined the effectiveness of this approach
with atomic force microscopy (AFM) of DNA origami patterns to produce a direct
visual readout of the target nucleotide contained in the probe sequence. The origami contains graphical representations of the four possible nucleotide alphabetic
characters, A, T, G and C. Each of the components of the letters contains a nucleotide at the test site that is the complement of one of the nucleotides in the probe.
Consequently, the symbol containing the test nucleotide identity vanishes in the
presence of the probe. Computer processing of a statistically significant group of
images produces a direct symbolic readout that directly identifies the nucleotide
carried by the probe.
The figure above shows the schematic diagram of DNA Origami tile used (on the
left) and the averaged AFM image of the origami tile (on right), showing the character readouts (the scale bar shows a distance of 50 nm). This method works not
only with a single SNP, but also with two different probes, as would be found in the
case of a heterozygous diploid organism.
This research has been supported by NIGMS, NSF, ARO, ONR and the
W. M. Keck Foundation.
72
Hari K. K. Subramanian
Banani Chakraborty
Ruojie Sha
Nadrian C. Seeman
Department of Chemistry, New York
University, New York, NY 10003, USA
[email protected]
[email protected]
[email protected]
[email protected]
1046
Topological Bonding of DNA Nanostructures
73
Yoel Ohayon1
Ruojie Sha1
Ortho Flint2
Nadrian C. Seeman1*
1Department
2Department
of Mathematics, University
of Western Ontario, London,
ON N6A 5B7, Canada
*[email protected]
The properties of DNA that allow it to act as the storage medium of genetic
information also make it an outstanding molecule for use in nanotechnology.
This fact has led to the development of DNA nanotechnology which is based on
Watson-Crick base pairing. The backbone structures involved these constructs are
complex species, not simple linear duplex molecules. These motifs and various
programmable structures in one, two and three dimensions are constructed by using
sticky-ends which are short-linear extensions that hold complementary structures
together. The PX (Paranemic Crossover) motif (1) has also been reported as another
form of cohesion for large DNA structures. It may be useful in overcoming some
of the weaknesses of sticky-ended cohesion (2). Both sticky-ended and PX cohesion are based on hydrogen-bonded interactions that cannot withstand denaturing
conditions (3). In the work presented, we used two approaches to demonstrate that
the hydrogen-bonded cohesion of DNA nanostructures can be transformed into
a topological interaction. In the first case, we created a topological interaction
between DNA structures that cohere via both PX and sticky-ends. Covalent linkages were created between the functionalized 3’ and 5’ ends of the sticky-ends.
The torus-like structures were obtained via enzymatic ligation. In the second case,
we converted the PX interaction of DNA circles containing cohesive loops into
catenated structures via the use of Topo I enzyme by creating a linkage between
the loops. The two methods were used to construct topologically linked onedimensional DNA arrays assembled from different types of PX cohering tiles. The
construction of these poly-catenated scaffolds allowed for a new method to position nano-particles on linear structures. The program Knotilus (4, 5) was used to
determine and display the topology of the catenated DNA structures.
This work is supported by the grants from the National Institute of General
Medical Sciences, the National Science Foundation, the Army Research Office and
the Office of Naval Research.
References
1. Z. Shen, H. Yan, T. Wang, and N. C. Seeman. J Am Chem Soc 126, 1666-1674 (2004).
2. X. Zhang, H. Yan, Z. Shen, and N. C. Seeman. J Am Chem Soc 124, 12940-12941 (2002).
3. C. H. Spink, L. Ding, Q. Yang, R. D. Sheardy, and N. C. Seeman. Biophys J 97, 528-538
(2009).
4. O. Flint. “The Master Array, a complete invariant for prime alternating links” (Thesis,
2007).
5. http://knotilus.math.uwo.ca/
A Controlled DNA Biped Walker on a DX Track
1047
Molecular machines perform elegant work with high efficiency in biological
systems. They have inspired attempts to create artificial machines that mimic
the ability to produce controlled motion (1, 2). We describe the construction of
an extendable DNA walker-track system. A monomer DX track with three footholders is constructed, and the biped walker moves along the cylinder side of the
track. Therefore the terminus of the monomer track could be used to create sticky
ends for track extension and the total steps the walker could take increases as
more monomer tracks are added. A dimer DX track with six foot-holders demonstrates the feasibility of track extension. By means of sequential addition of
DNA set/unset strands (3, 4), the walker is programmed to move forward and
then backward along the track. PAGE analysis demonstrates that the walker-track
complex forms well. Psoralen cross-link monitoring is performed with an aliquot
of material at each step. The PAGE analysis results further establish the step-wise
formation of the expected products.
74
The schematic above shows the attachment of walker to the monomer DX track via
a set strand (A), and the attachment of walker to a dimer DX track (B).
References
1. M. Fennimore, T. D. Yuzvinsky, Wei-Qiang Han, M. S. Fuhrer, J. Cumings, and A. Zettl.
Nature 424, 408-410 (2003).
2. N. C. Seeman. Nature 421, 427-431 (2003).
3. W. B. Sherman and N. C. Seeman. Nano Lett 4, 1203-1207 (2004).
4. J. Shin and N. A. Pierce. J Am Chem Soc 126, 10834-10835 (2004).
Dadong Li
Ruojie Sha
James Canary
Nadrian C. Seeman*
*[email protected]
1048
75
Tong Wang1
Ruojie Sha1
Jens J. Birktoft1
Jianping Zheng1
Chengde Mao2
Nadrian C. Seeman1*
1Department
2Department
of Chemistry,
Purdue University, West Lafayette,
IN 47907, USA
A DNA Crystal Designed to Contain Two Molecules
per Asymmetric Unit with Fluorescent Dyes
We have reported the X-ray crystal structure of a designed macroscopic selfassembled 3D crystal based on a robust motif (1). That crystal contained a single
molecular species, a tensegrity triangle (2), the molecule’s self-assembly into a
crystal was programmed through the sticky ends at the end of each DNA double
helix. One of the strengths of using the chemical information contained in sticky
ends to program self-assembly is that one ought to be able to control the number
of species contained within the unit cell. This capability has been demonstrated in
very small (∼1-10 µm) two-dimensional crystals (3), but macroscopic 3D crystals
provide the most interesting case. Here, we describe the self-assembly of a DNA
crystal that contains two tensegrity triangle molecules per asymmetric unit. We
have used X-ray crystallography to determine its crystal structure. In addition, we
have demonstrated control over the colors of the crystals by attaching either Cy3
dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crystals of corresponding colors. By doing those, we demonstrate that we could use
more than one component to self-assemble DNA crystal, put the covalently bonded
foreign molecules into the DNA crystal while not affecting its self-assembly and
grow the DNA crystal in the less strain condition such as low salt concentration and
room temperature.
*[email protected]
This work is supported by the National Institute of General Medical Sciences, the
National Science Foundation, the Army Research Office and the Office of Naval
Research.
References
1. J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R. Sha, P. E. Constantinou, S. L. Ginell, C. Mao,
and N. C. Seeman. Nature 461, 74-77 (2009).
2. D. Liu, W. Wang, Z. Deng, R Walulu, and C. Mao. J Am Chem Soc 126, 2324-2325
(2004).
3. E. Winfree, F. Liu, L. A. Wenzler, and N. C. Seeman. Nature 394, 539-544 (1998).
1049
A Toolkit for Site-Specific DNA
Interstrand Crosslinks
We report the development of technology that allows interstrand coupling between
various positions across minor or major grooves within one turn of DNA duplex.
2’-Modified nucleotides were synthesized as protected phosphoramidites and
incorporated into DNA structures (1, 2). The modified nucleotides contained fiveatom linkers, with either amine or carboxylic acid functional groups at their termini. Chemical coupling of amine and carboxylic acid groups in designed strands
resulted in the formation of an amide bond linking two complementary strands.
Interstrand crosslinks were formed specifically between nucleotides placed at preselected positions rather than selective formation between various positions as
observed in previous studies. The trajectory lengths of linkers determined if the
termini could reach each other to enable reactions to occur. Modeling (3) and calculated crosslink trajectory distances were in agreement our conclusions from experimental data. This technology enables approaches that can control regiospecific and
distance dependent linkage formation, offering tools to use site-specific cross-links
to probe biological phenomena (4). In addition, this approach can also be applied to
DNA nanotechnology to help build nanoscale structures along DNA templates. Miao Ye
Johan Guillaume
Yu Liu
Ruojie Sha
Risheng Wang
Nadrian C. Seeman
James W. Canary*
New York, NY 10003
*[email protected]
Acknowledgements
This research has been supported by the following grants to JWC and NCS:
National Science Foundation (CTS-0608889) and the Office of Naval Research
(N000140911118). This work has also been supported by grants to NCS from the
National Institute of General Medical Sciences (GM-29544) the National Science
Foundation (CCF-0726378), the Army Research Office (48681-EL and W911NF07-1-0439), and a grant from the W. M. Keck Foundation. This work was supported partially by the MRSEC Program of the National Science Foundation under
Award Number DMR-0820341.
References
1. Y. Liu, A. Kuzuya, R. Sha, J. Guillaume, R. Wang, J. W.Canary, and N. C. Seeman. J Am
Chem Soc 130, 10882-10883 (2008).
2. L. Zhu, P. S. Lukeman, J. W. Canary, and N. C. Seeman. J Am Chem Soc 125, 10178-10179
(2003).
3. S. Arnott, D. W. L. Hukins, and S. D. Dover. Biochem Biophys Res Comm 6, 1392 (1972).
4. D. M. Noll, T. M. Mason, and P. S. Miller. Chem Rev 106, 277-301 (2006).
76
1050
Amyloid Fibrils Captured inside
Twenty-Helix DNA Nanotubes
77
Anuttara Udomprasert1
Marie Bongiovanni2,3
Ruojie Sha1
William B. Sherman4
Monica Menzenski1
Paramjit Arora1
James W. Canary1
Sally L. Gras2,3
Nadrian C. Seeman1*
1Department
2Department
of Chemical and
Biomolecular Engineering,
The University of Melbourne, Parkville,
VIC 3010, Australia
3The
Bio21 Molecular Science
and Biotechnology Institute,
The University of Melbourne, Parkville,
VIC 3010, Australia
4Brookhaven
National Laboratory,
Upton, NY 11973, USA
*[email protected]
Amyloid fibrils are ordered and insoluble protein aggregates that were originally
found associated with neurodegenerative diseases such as Alzheimer’s disease.
They share a common core structure, an elongated stack of b-strands, perpendicular to the fibril axis (1). These molecules have rod-like structures with a very
high persistence length and also exhibit high thermal and chemical stability (2).
Short synthetic non-disease-related peptides can induce fibril in vitro (3). There
are increasing observations on how these fibrils form providing more opportunities to design novel functionalized motifs (2, 4). They offer great potential as selfassembling materials for nanotechnology and bionanotechnology (5). However,
they require the development of new methods to manipulate fibrils into organized
arrangements.
DNA nanotubes with a cylindrical structure can be used as sheaths around rodlike molecules in biological systems and nanotechnology (6). In this work, we
take advantage of the powerful features of DNA to form nanotubes to capture
amyloid fibrils formed from a short peptide fragment of protein transthyretin
(TTR105-115). The scaffolded DNA origami method is used to form DNA nanotubes,
using M13mp18 as a scaffold strand with more than 170 staple strands, designed to
have enough space for capturing the fibrils inside. We expect to be able to organize
these bio-inspired materials onto predefined surfaces via DNA-DNA interactions.
We report the results of sheathing experiments that are evaluated by atomic force
microscopy.
References
1. J. F. Smith, T. P. Knowles, C. M. Dobson, C. E. MacPhee, and M. E. Welland. Proc Natl
Acad Sci 103, 15806-15811 (2006).
2. C. E. MacPhee and C. M. Dobson. J Am Chem Soc 122, 12707-12713 (2000).
3. C. M. Dobson. Trends Biochem Sci 24, 329-332 (1999).
4. S. L. Gras, A. K. Tickler, A. M. Squires, G. L. Devlin, M. A. Horton, C. M. Dobson, and
C. E. MacPhee. Biomaterials 29, 1553-1562 (2008).
5. S. L. Gras. Aust J Chem 60, 333-342 (2007).
6. A. Kuzuya, R. Wang, R. Sha, and N. C. Seeman. Nano Lett 7, 1757-1763 (2007).
Crystalline Two-Dimensional DNA-Origami Arrays
1051
Nanotechnology aims to organize matter with the highest possible accuracy and
control. Such control will lead to nanoelectronics, nanorobotics, programmable
chemical synthesis, scaffolded crystals, and nanoscale systems responsive to their
environments. Structural DNA nanotechnology (1) is one of the most powerful
routes to this goal. It combines robust branched DNA species with the control of
affinity and structure (2) inherent in the programmability of sticky ends. The successes of structural DNA nanotechnology include the formation of objects (3), 2D
crystals (4), 3D crystals (5), nanomechanical devices (6), and various combinations
of these species (7). DNA origami (8) is arguably the most effective way of producing a large addressable area on a 2D DNA surface. This method entails the combination of a long single strand (typically the single-stranded form of the filamentous
bacteriophage M13, 7249 nucleotides) with about 250 staple strands to define the
shape and patterning of the structure. With a pixelation estimated at about 6 nm (8),
it is possible to build patterns with about 100 addressable points within a definable
shape in an area of about 10000 nm2. Many investigators have sought unsuccessfully to increase the useful size of 2D origami units by forming crystals of individual origami tiles (9). Herein, we report a double-layer DNA-origami tile with two
orthogonal domains underwent self-assembly into well-ordered two-dimensional
DNA arrays with edge dimensions of 2–3 μm (see schematic representation and
AFM image). This size is likely to be large enough to connect bottom-up methods
of patterning with top-down approaches.
78
Two-dimensional crystals from DNA origami tiles
Acknowledgements
This research has been supported by the following grants to NCS: GM-29544
from the National Institute of General Medical Sciences, CTS-0608889 and CCF0726378 from the National Science Foundation, 48681-EL and W911NF-07-10439 from the Army Research Office, N000140910181 and N000140911118 from
the Office of Naval Research and a grant from the W.M. Keck Foundation.
References
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N. C. Seeman. J Theor Biol 99, 237-247 (1982).
H. Qiu, J. C. Dewan, and N. C. Seeman. J Mol Biol 267, 881-898 (1997).
J. Chen and N. C. Seeman. Nature 350, 631-633 (1991).
E. Winfree, F. Liu, L. A. Wenzler, and N. C. Seeman. Nature 394, 539-544 (1998).
J. Zheng, J. J. Birktoft, Y. Chen, T. Wang, R. Sha, P. E.Constantinou, S. L. Ginell, C. Mao,
and N. C. Seeman. Nature 461, 74-77 (2009).
H. Yan, X. Zhang, Z. Shen, and N. C. Seeman. Nature 415, 62-65 (2002).
B. Ding and N. C. Seeman. Science 314, 1583-1585 (2006).
P. W. K. Rothemund. Nature 440, 297-302 (2006).
Z. Li, M. Liu, L. Wang, J. Nangreave, H. Yan, and Y. Liu. J Am Chem Soc 132, 1354513552 (2010).
Wenyan Liu
Hong Zhong
Risheng Wang
Nadrian C. Seeman*
*[email protected]
1052
Biosensor Design Using DNA Tile Lattices
79
Lauren Hakker1
Kimberly A. Harris2,3
Thom H. LaBean4
Paul F. Agris2
1Department
of Chemistry, University at
Albany-SUNY, Albany, NY 12222, USA
2The
RNA Institute, Department of
Biological Sciences, University at
Albany-SUNY, Albany, NY 12222, USA
3Department
of Molecular & Structural
Biochemistry, North Carolina State
Biosensors are of great scientific importance in many fields because of their ability to detect a physiological change or presence of various chemical or biological
materials. DNA has become known as an extremely useful building material in
nanotechnology, especially in biosensors (1, 2). DNA has the ability to stabilize
structure while allowing enough flexibility to achieve the desired shapes (4). Base
pairing interactions between designed DNA strands are used to construct tiles,
from which lattice structures are assembled (Figure 1). These lattices are useful for
diverse molecular scale nanofabrication tasks because of their high thermal stability and multiple attachment sites within and between tiles (3). These lattice structures consist of a central loop, four shell strands, and four arms, totaling from nine
strands of DNA (2). The central loop contains 12 unpaired thymine bases. One
goal is to adhere a single-stranded DNA (ssDNA) to the available thymine bases in
the central loop, creating a “crown” on top of the lattices. The ssDNA strand will
ultimately be designed to contain a biomolecule that will serve as a detector in a
biosensor. Experiments were performed with a fluorescein labeled poly-adenosine
ssDNA binding to the lattice and was detected by polyacrylamide gel electrophoresis in combination with a fluorescence imager. Circular dichroism was also used
to detect secondary structures of the interaction. Once preliminary experiments are
completed successfully, other biomolecules will be attached to the ssDNA to create
a tile lattice that will have the ability to function as a biosensor.
University, Raleigh, NC 27695, USA
4
Department of Computer Science,
Chemistry and Biomedical Engineering,
Duke University, Durham, NC 27708,
USA
Figure 1: (A) DNA strand structure of tiles (B) schematic drawing of tiles and lattices (C) AFM images of tiles and lattices (Adapted from 2).
References
1.
2.
3.
4.
T. H. LaBean and H. Li. Nanotoday 2, 26-35 (2007).
S. H. Park, G. Finkelstein, and T. H. LaBean. J Am Chem Soc 130, 40-41 (2007).
K. V. Gothelf and T. H. LaBean. Org Biomol Chem 3, 4023-4037 (2005).
T. H. LaBean. Nature 459, 331-332 (2009).
Deoxyribozyme Sensors for Nucleic Acid Analysis
1053
Deoxyribozyme (DNAzyme) sensors are biocompatible, chemically stabile, and
simple in terms of structural prediction and modification. In addition, such sensors have the potential of an improved limit of detection (LOD) due to their ability
to catalytically amplify signals. Two types of deoxyribozyme sensors have been
developed by us in recent years. The sensor architecture allows for a unique combination of high selectivity and the convenience of fluorescent or visible (color
change) signal monitoring in homogeneous solution. The LOD of the sensor was
found to vary from 10 nM to 1 pM. Deoxyribozyme sensors are promising tools for
the detection of single nucleotide substitutions in DNA and RNA molecules.
80
References
1. D. M. Kolpashchikov. ChemBioChem 8, 2039-2042 (2007).
2. D. M. Kolpashchikov. J Am Chem Soc 130, 2934-2935 (2008).
3. Y. V. Gerasimova, E. Cornett, and D. M. Kolpashchikov. ChemBioChem 11, 811-817
(2010).
4. D. M. Kolpashchikov. Chem Rev 110, 4709-4723 (2010).
Dmitry M. Kolpashchikov*
Yulia V. Gerasimova
Evan Cornett
University of Central Florida,
P.O. Box 162366,
4000 Central Florida Blvd.,
Orlando, FL 32816-2366, USA
*[email protected]
Molecular Dynamics Simulations of DNA Holliday
Junctions: Conformational Stability and Transitions
81
Holliday Junctions (4W-junctions) are highly conserved four-way DNA homologous
Elizabeth G. Wheatley*
replication junctions capable of undergoing salt-dependent conformational transiSusan Pieniazek
tions between mobile open planar and immobile stacked-X forms (1). This study
investigates the effect of salt concentration on Holliday Junction conformation and
David L. Beveridge
dynamics. All-atom Molecular Dynamics (MD) simulations are reported on 17 basepair Holliday Junctions with “Junction 3” (J3) cores (2). We carried out the calculaDepartment of Chemistry,
tions in different ionic concentrations to facilitate the transition between open and
stacked-X conformers. The results were analyzed in terms of free energy landscapes
Wesleyan University,
obtained with Principal Component Analysis. In low salt conditions (electroneutralMiddletown, CT 06459
ity), the open planar form transitioned to the lesser-observed Iso I stacked-X form
*[email protected]
by passing through a tetrahedral intermediate species capable, in theory, of adopting
several stacked conformations (3). The higher salt condition (70 mM
KCl) revealed a transition from open to the experimentally observed Iso
II stacked form without the presence of an intermediate species. A free
energy map of the lower salt condition reveals a multistate model with
three distinct minima, two for the open/stacked-X conformations, and
a third accounting for the tetrahedral intermediate state. On the other
hand, a free energy map of the high salt simulation reveals a distinctly
two-state model with two minima and a narrow pathway (energy barrier) connecting the two. The findings imply that there is a unique stacking pathway along the free energy surface for the J3 4W-junction in a
high salt condition where phosphate charges are adequately shielded.
Above: Holliday Junction open and stacked forms
References
1. F. Hays, J. Watson, and P. Ho. J B C 278, 49663-49666 (2003).
2. D. R. Duckett, A. Murchie, S. Diekmann, E. Kitzing, B. Kemper, and D. Lilley. Cell 58,
79-89 (1988).
3. J. Yu, T. Ha, and K. Schulten. NAR 32, 6683-6695 (2004).
1054
82
Jonas K. Hannestad*
Bo Albinsson
Department of Chemical and Biological
Engineering/Physical Chemistry,
Chalmers University of Technology,
SE-41296 Gothenburg, Sweden
*[email protected]
Multi-Step Energy Transfer as a Communication
Tool in Nanoscale DNA Assemblies
Bioinspired nanoscale technology requires new means to transfer information and
driving forces for reaction of relevant distances. One possible solution can be to use
excitation energy transfer in multiple steps to bridge large distances with high precision. This resembles the light harvesting complexes of photosynthetic organisms
where absorbed energy is transported in multiple steps to the reaction center. We
have created photonic systems based on multistep energy transfer capable of transferring excitation energy over long ranges with high specificity. Here, we present
a self-assembled DNA-based photonic wire where excitation energy is transported
over more than 20 nm (1). The wire utilizes the interlcalator YO-PRO to mediate
end-to-end energy transfer in the photonic wire. We also introduce a photonic network with selectable outputs incorporated in a self-assembled DNA nano-construct.
Though the binding of YO-PRO it is possible to direct energy transfer from one
output dye to the other. Our work show how self-assembled structures of DNA and
fluorophores can function as a basis for communication on the nanometer scale.
Figure: Self-assembled DNA-based photonic wire. End-to-end
energy transfer is facilitated by the meditative effect of YO-PRO.
References
1. J. K. Hannestad, P. Sandin, and B. Albinsson. J Am Chem Soc 130, 15889 (2008).
1055
Rationalizing the Outcome of One-Pot
DNA Nano-Assemblies
Here we present a fully addressable four-ring DNA nanonetwork composed of tripodal oligonucleotide building blocks and show that with a set of unique oligonucleotide building blocks, based on orthogonal sequence design, it is possible to
assemble non-repetitive networks with high information density. Our DNA based
assembly system is focused on building fully addressable networks, using the smallest practical units of DNA, i.e. one turn of the double helix (1, 2). Having addressable structures is a prerequisite for controlled positioning of functional units, being
components for energy transfer or chemical reaction centers. Huge progress has
been made in the past decade using DNA as a building block for nanoscale fabrication (3). The nanoscale fabrication relies solely on self-assembly of carefully
designed DNA molecules with the free energy of hybridization as the underlying
driving force. With extra consideration taken to the design of base sequences, a
desired structure can be created in a one-pot, one-step assembly reaction. In our
study we address a fundamental problem with DNA nanofabrication based on a
one-step assembly process, i.e. the reaction yield. The total yield of DNA assemblies in one-pot reactions can be described in terms of the yield of one hybridization
reaction, raised to the power of total number of events. We therefore look for and
suggest alternative assembly strategies, one of which being a fixation strategy based
on click chemistry, creating robust units for a modular build-up approach (4).
83
Erik P. Lundberg1*
Calin Plesa1
L. Marcus Wilhelmsson1
Per Lincoln1
Tom Brown2
Bengt Nordén1
1Department
of Chemical and Biological
2School
Southampton, Highfield, Southampton
SO17 1BJ, U.K.
*[email protected]
References
1. J. Tumpane, R. Kumar, E. P. Lundberg, P. Sandin, N. Gale, I. S. Nandhakumar, B. Albinsson,
P. Lincoln, L. M. Wilhelmsson, T. Brown, and B. Nordén. Nano Lett 7, 3832-3839 (2007).
2. J. Tumpane, P. Sandin, R. Kumar, V. E. C. Powers, E. P. Lundberg, N. Gale, P. Baglioni,
J. M. Lehn, B. Albinsson, P. Lincoln, L. M. Wilhelmsson, T. Brown, and B. Nordén. Chem
Phys Lett 440, 125-129 (2007).
3. N. C. Seeman. Nano Lett 10, 1971-1978 (2010).
4. E. P. Lundberg, A. H. El-Sagheer, P. Kocalka, L. M. Wilhelmsson, T. Brown, and B. Norden.
Chem Commun 46, 3714-3716 (2010).
1056
84
Graham D. Hamblin
Hanadi F. Sleiman*
Chemistry Department, McGill
University, 801 Sherbrooke St West,
Montreal, QC H3A2K6
*[email protected]
Programming Curvature in DNA Nanotubes
Organizing materials at the nanoscale with high precision is of great interest for
many applications, ranging from electronics to optics and biophysics (1). As one
of the most programmable self-assembling materials, DNA is an excellent building
block to template this fine organization. We have recently reported the construction
of DNA nanotubes with the ability to readily program geometry, length, single- or
double-stranded character, and the ability to encapsulate and selectively release
materials within these structures (2-4). Here, we report our progress towards higherorder control over these nanotubes. They are built in a modular fashion; by making
localized structural alterations to individual components, we examine how these
structural changes can be amplified into long-range motion throughout the final
tube. As an example, we present curved DNA nanotubes in which the degree and
occurrence of curvature is under external control: pH, light, and externally added
biomolecules are used as triggers to effect these changes. These assemblies could
find applications as biophysical tools, molecular machines, and dynamic templates
for nanocircuitry.
References
1. G. Cao. Synthesis, Properties and Applications; Imperial College: London, 110 (2004).
2. P. K. Lo, P. Karam, F. A. Aldaye, C. K. McLaughlin, G. D. Hamblin, G. Cosa, and
H. F. Sleiman. Nature Chemistry 2, 319-328 (2010).
3. P. K. Lo, F. Altvater, and H. F. Sleiman. J Am Chem Soc 132, 10212-10214 (2010).
4. F. A. Aldaye, P. K. Lo, P. Karam, C. K. McLaughlin, G. Cosa, and H. F. Sleiman. Nature
Nanotechnol 4, 349-352 (2009).
1057
DNA Nanoarchitechtures and Mechanical Devices
The idea behind our research is to use DNA as a programmable tool for directing
the self-assembly of molecules and materials. The unique specificity of DNA interactions, our ability to code specific DNA sequences and to chemically functionalize DNA, makes it the ideal material for controlling self-assembly of components
attached to DNA sequences. We have explored some new approaches in this area
such as the use of DNA for self-assembly of organic molecules and for electrochemical sensors.
In this presentation it is demonstrated how DNA origami (1) can be used to assemble organic molecules, study chemical reactions with single molecule resolution
(2), and position dendrimers and other materials. After initial 2D designs we made
a 3D DNA origami box with a lid that could be controlled and the lid motion was
monitored by FRET (3).
Kurt V. Gothelf
85
Centre for DNA Nanotechnology,
iNANO and Department of Chemistry,
Aarhus University,
8000 Aarhus C Denmark
[email protected]
Recently, we have designed a new type of DNA actuator that has a sliding type
of motion. It can be positioned in 11 discrete positions and be shifted between the
positions. The motion was followed by FRET and by performing chemical reactions that are only geometrically possible in certain states of the actuator (4).
References
1. P. K. W. Rothemund. Nature 440, 297 (2006).
2. N. V. Voigt, T. Torring, A. Rotaru, M. F. Jacobsen, J. B. Ravnsbaek, R. Subramani,
W. Mamdouh, J. Kjems, A. Mokhir, F. Besenbacher, and K. V. Gothelf. Nature Nanotech
5, 200-203 (2010).
3. E. S. Andersen, M. Dong, M. M. Nielsen, K. Jahn, R. Subramani, W. Mamdouh, M. M. Golas,
B. Sander, H. Stark, C. L. Oliveira, J. S. Pedersen, V. Birkedal, F. Besenbacher,
K. V. Gothelf, and J. Kjems. Nature 459, 73-76 (2010).
4. Z. Zhang, E. M. Olsen, M. Kryger, N. V. Voigt, T. Tørring, E. Gültekin, M. Nielsen,
R. M. Zadegan, E. Andersen, M. M. Nielsen, J. Kjems, V. Birkedal, and K. V. Gothelf.
Angew Chem Int Ed (2011), in press.
Nucleic Acid-based Molecular Devices
In recent years, the predictable interactions between complementary sequences of
DNA or RNA molecules have been utilized for the construction of a large variety of
synthetic biomolecular structures and devices. For instance, the recently developed
DNA origami technique enables the assembly of two- and even three-dimensional
molecular objects with almost arbitrary shape - and with nanoscale precision. These
structures can be used to arrange other molecular components, e.g. proteins, into
well-defined geometries. Researchers envision the utilization of such structures as
molecular assembly lines, or for the arrangement of artificial enzyme cascades.
In order to demonstrate the function of molecular-scale structures and devices, appropriate characterization tools are required. Typically, DNA nanostructures are studied
using scanning probe or electron microscopy techniques, but in many cases optical
characterization methods would be preferable. In the first part of the talk, it will be
discussed how modern super-resolution microscopy methods can be applied to study
DNA assemblies whose dimensions are well below the classical diffraction limit.
In addition to the realization of static molecular nanostructures one of the visions
of molecular nanotechnology is the generation of dynamic molecular assemblies
that resemble naturally occurring molecular machines. In fact, DNA and RNA molecules have already been utilized for the construction of a variety of molecular
devices that can be switched between several distinct conformational states, that
Friedrich C. Simmel
86
Physics Department, ZNN/WSI
TU München,
85748 Garching, Germany
[email protected]
1058
display nano-scale motion or that bind and release molecules on demand. Moreover, DNA recognition reactions have also been employed for the realization of
artificial regulatory circuits, which can be used to control the timing of molecular
assembly processes, or to direct the operation of nucleic acid-based nanodevices. In
the second part of the talk, an example for an artificial RNA-based reaction network
will be demonstrated that controls the motion of a DNA nanodevice.
References
1. S. Modi, D. Bhatia, F. C. Simmel, and Y. Krishnan. Journal of Physical Chemistry Letters
1, 1994-2005 (2010).
2. C. Steinhauer, R. Jungmann, T. Sobey, F. C. Simmel, and P. Tinnefeld. Angewandte Chemie
48, 8870-8873 (2009).
3. R. Jungmann, C. Steinhauer, M. Scheible, A. Kuzyk, P. Tinnefeld, F. C. Simmel. Nano
Letters 10, 4756-4761 (2010).
4. F. C. Simmel. Nanomedicine 2, 817-830 (2007).
87
C. Iulia Vitoc
Olga Buzovetsky
Jacob Litke
Yan Li
Ishita Mukerji*
Molecular Biology and
Biochemistry Department,
Molecular Biophysics Program,
Middletown, CT 06459
*[email protected]
Structure of DNA Four-Way Junctions: Effect
of Ions and Proteins
Holliday or DNA four-way junctions are important intermediates in recombination and repair processes (1-4). These structures can change conformation rapidly
in solution interconverting from an open, four-fold symmetrical structure, with
no central base stacking to one in which coaxial stacking of the helical arms has
been observed (5). The open structure, which is capable of branch migration, is
functionally relevant; however, many proteins have been observed to bind to and
stabilize the stacked form. Our investigations have focused on elucidating the conformational changes induced by the binding of ions and the architectural proteins,
HU and IHF, to improve understanding of the stacked form of the junction. Using
fluorescence spectroscopic methods, we have examined the ion-binding site and
have explicitly explored the coordination of ions to the central region of the junction. Förster resonance energy transfer (FRET) experiments have revealed that the
degree of stacking or interduplex angle (IDA) of the junction is modulated by ion
size, where larger ionic radii lead to larger IDAs. The proteins, HU and IHF, which
are known to bind and bend DNA, stabilize the junction in the stacked conformation and induce a greater degree of stacking upon binding (6). In contrast, the repair
protein, Msh2-Msh6, induces the junction to adopt an open conformation. All three
proteins recognize and bind to the junction structure with nanomolar affinity and
interestingly, Msh2-Msh6 binds the junction with higher affinity than a mismatch
site. A novel FRET-mapping approach has been employed to determine the location of these proteins on the junction and indicates that the proteins bind to the
central region of the junction.
This research has been supported by grants (MCB-0316625; MCB-0843656) from
the NSF awarded to I.M.
References
1. R. Holliday. Genet Res 5, 282-304 (1964).
2. Y. Liu and S. C. West. Nat Rev Mol Cell Biol 5, 937-944 (2004).
3. G. T. Marsischky, S. Lee, J. Griffith, and R. D. Kolodner. J Biol Chem 274, 7200-7206
(1999).
4. T. Snowden, S. Acharya, C. Butz, M. Berardini, and R. Fishel. Molecular Cell 15, 437-451
(2004).
5. S. A. McKinney, A. C. Declais, D. M. Lilley, and T. Ha. Nat Struct Biol 10, 93-97 (2003).
6. C. I. Vitoc and I. Mukerji. Biochemistry 50, 1432-1441 (2011).
1059
Towards the Design and Synthesis of an Artificial Cell
The complexity of modern biological life has long made it difficult to understand
how life could emerge spontaneously from the chemistry of the early earth. The key
to resolving this mystery lies in the simplicity of the earliest living cells. Through
our efforts to synthesize extremely simple artificial cells, we hope to discover plausible pathways for the transition from chemical evolution to Darwinian evolution.
We view the two key components of a primitive cell as a self-replicating nucleic acid
genome, and a self-replicating cell membrane. We have recently described a simple
and robust pathway for the coupled growth and division of a model primitive cell
membrane. Much of our current work is focused on the synthesis of self-replicating
nucleic acids, including a series of phosphoramidate nucleic acids. While the rate
of template copying by the polymerization of amino-sugar nucleotides can be quite
good in such model systems, the fidelity of chemical copying is low. This has led
us to explore various nucleobase analogs in search of simple ways to enhance the
fidelity of chemical replication. I will discuss recent progress towards the realization of an efficient and accurate system for the chemical replication of an informational polymer. I will also describe ways in which a chemically replicating nucleic
acid could lead to evolutionary changes in the membrane composition of a simple
protocell, as well as ways in which the evolving cell membrane could enhance
nucleic acid replication.
This work was supported by the Howard Hughes Medical Institute, the NSF
and NASA.
Jack W. Szostak
88
Department of Molecular Biology,
Massachusetts General Hospital,
Boston, MA 02114
[email protected]
1060
Towards Origin of Life and... What is Life, After All?
89
Czech Republic
Recent literature is full of sensations opening eyes on the origin of life problems,
such as creation of artificial bacteria (1), arsenic DNA (2), cyanobacteria in meteorites (3), not mentioning constantly advancing chemical systems aiming at the
life origin. Each of the overtures brings us closer to something apparently simple,
almost trivial, but elusive as well, since nobody knows what we are, actually, getting to. There is no established definition of life to be guided by. In perception of
many the phenomenon of life is beyond the perception limits. However, if in the
classical Cartesian body/mind dualism of life only material part is taken, one finds
himself at the brink of this something simple, almost trivial. Over hundred definitions are suggested by philosophers and scientists of many generations. The fact
that the list continues to grow only attests to actual lack of a convincing consensus.
The author humbly suggests one definition of life that appears to be a “principal
component” of all definitions. It is derived by a linguistic (word count) analysis
of the large corpus of the definitions. It turns out identical to the one introduced
earlier (4) on the basis of developing theory of early molecular evolution. And the
definition is:
[email protected]
Life is self-reproduction with variations.
Edward N. Trifonov1,2
1Genome
Diversity Center, Institute
of Evolution, University of Haifa,
Mount Carmel, Haifa 31905, Israel
2Department
of Functional Genomics and
Proteomics, Faculty of Science, Masaryk
University, Kotlarska 2, CZ-61137 Brno,
Irrespective of whether it is correct or any close to final, the formula allows one
to critically overview all known approaches to the problem of origin of life, and,
perhaps, enthuse researchers to further converge on the very point of origin, if only
its elusiveness is not due to yet another uncertainty principle.
References
1.
2.
3.
4.
90
Uwe J. Meierhenrich*
Jean-Jacques Filippi
Cornelia Meinert
Jan Hendrik Bredehöft
Jun-ichi Takahashi
Laurent Nahon
Nykola C. Jones
Soeren V. Hoffmann
University of Nice-Sophia Antipolis,
Nice, France
*[email protected]
D. G. Gibson, et al. Science 329, 52-56 (2010).
E. Pennisi and F. Wolfe-Simon. Science 330, 1734-1735 (2010).
R. B. Hoover. J Cosmology 13, in press.
E. N. Trifonov. Res Microbiol 160, 481-486 (2009).
Formation and Chiroptical Properties of Amino Acids
in Interstellar Ice Analogues
Comets are accretions of frozen volatiles and rocky debris left over from the formation of the outer Solar System (1). In order to better understand the chemical
and molecular composition of comets their formation was artificially and stepwise
reproduced in the laboratory. To this end, representative interstellar molecules containing C1 and N1 units such as 13CO and NH3 were condensed on a solid surface
at 12 K while being irradiated at Lyman‑α. The obtained interstellar ice analogues
were subjected to enantioselective gas chromatographic and mass spectrometric
analysis allowing us the identification of 16 different amino acids and diamino
acids (2). The molecular composition of these ices was found to be similar, but
not identical, to the amino acids and diamino acids identified in meteorites (3)
and will be discussed in the context of the molecular origin of life on Earth. Terrestrial life uses homochiral l‑amino acids for the expression of proteins (4). In
a follow-up study we investigated whether a chiral symmetry breaking in amino
acids is feasible under simulated interstellar conditions based upon an asymmetric
photochemical mechanism. In a UHV-chamber we deposited amorphous amino
acid films of defined thickness on MgF2 windows (see Figure) and subjected them
1061
to circularly polarized light. The differential absorption of circularly polarized light
by individual amino acid enantiomers, which determines speed and intensity of
enantioselective photolysis, was recorded in the vacuum-ultraviolet spectral range,
where massive circular dichroic transitions were observed (5). We report on the
formation of amino acids and diamino acids under interstellar conditions and on
chiroptical properties of amino acids in the solid state. These data are of importance
for the molecular understanding for both origin and evolution of life on Earth and
its molecular asymmetry.
This research has been supported by grants from the ANR and the European
Community’s Seventh Framework Program.
References
1.
2.
3.
4.
5.
A. Mann. Nature 467, 1013-1014 (2010).
G. M. Muñoz Caro et al. Nature 416, 403-406 (2002).
U. J. Meierhenrich et al. Proc Natl Acad Sci 101, 9182-9186 (2004).
U. J. Meierhenrich. Amino Acids and the Asymmetry of Life, Springer (2008).
U. J. Meierhenrich, et al. Angew Chem Int Ed 49, 7799-7802 (2010).
Spontaneous Generation of RNA in Water
Definition of life is an open problem. The largely accepted wording: “Life is a
self sustained chemical system capable of undergoing Darwinian evolution” (1)
attains a solid operative sense but it is more the description of a process than a
formal definition of a system. If we have difficulty in even formulating a rigorous
definition of life, certainly we do not know how it started. In recent years, progress
has been made in the search for the unitary chemical frame into which the first
reactions lighted up and started accumulating and evolving chemical information.
In collaboration with R. Saladino group (Università della Tuscia, Italy), we have
shown that formamide (HCONH2), one of the simplest molecules grouping the
four most common elements of the universe H, C, O and N, provides a chemical
frame potentially affording all the monomeric components necessary for the formation of nucleic polymers (lastly reviewed in Saladino 2). In the presence of the
appropriate catalysts and by moderate heating, formamide yields a complete set
of nucleic bases, acyclonucleosides and favours both their phosphorylation and
transphosphorylation.
Nucleotide phosphorylation and RNA oligomerization take place in water in nonenzymatic abiotic conditions. At moderate temperatures (40-90°C) RNA chains
up to 120 nucleotides long may form from 3’, 5’-cAMP and 3’, 5’-cGMP, in the
absence of enzymes or inorganic catalysts (3). Mechanisms of abiotic RNA chain
extension and ligation based on base-pairing and base-stacking interactions were
also observed (4, 5). The enzyme and the template-independent synthesis of long
oligomers in water, from prebiotically affordable precursors, approaches the concept of spontaneous generation and evolution of (pre)genetic information.
References
1. J. Joyce, in D. W. Deamer, and G. R. Fleischaker (eds.), the foreword of “Origins of life: the
central concepts”, Jones and Bartlett, Boston, 1994.
2. R. Saladino, C. Crestini, F. Ciciriello, F. Pino, G. Costanzo, and E. Di Mauro. Research in
Microbiology 160, 441-448 (2009).
3. G. Costanzo, S. Pino, F. Ciciriello, and E. Di Mauro. J Biol Chem 284, 33206-33215.
4. S. Pino, F. Ciciriello, G. Costanzo, and E. Di Mauro. J Biol Chem 283, 36494-36503
(2008).
5. S. Pino, G. Costanzo, A. Giorgi, and E. Di Mauro (2011). Biochemistry, in press.
Giovanna Costanzo1
Samanta Pino2
Fabiana Ciciriello2
Ernesto Di Mauro2
1Istituto
91
di Biologia e Patologia
Molecolari, CNR, Roma, Italy
2Dip.
Biologia e Biotecnologie
“Charles Darwin”, Università
“Sapienza”, Roma, Italy
*Ernesto. [email protected]
1062
RNA, a Precursor to the Origin of Life
92
James P. Ferris*
Prakash C. Joshi
Michael F. Aldersley
John W. Delano
Department of Chemistry and Chemical
Biology, Rensselaer Polytechnic Institute,
Troy, NY 12180
*[email protected]
The RNA world is believed to be the most likely precursor to the origins to life on
Earth (1). We observed initially that montmorillonite clay-catalyzed the formation of RNA oligomers from activated nucleotides to yield oligomers as long as
10 mers. The reactions were carried out at room temperature in water and High
Performance Liquid Chromatography (HPLC) separated the oligomers formed on
ion exchange and reverse phase HPLC columns. The next stage in the study was
the generation of oligomers by the elongation of decamers to form 30-50 mers. The
third stage in the study was when 1-methyladenine was used to activate the monomer in place of imidazole. The positive charge on the 1-methyladenine resulted in
the formation of 40-50 mers in one day rather than the in previous procedure where
it took 14 days to generate 40-50 mers. Current studies on our approach to the
RNA world are the investigation of the reactions of racemic activated monomers.
Mixtures of D, L-ImpA with D, L-ImpU were reacted on the premise that if RNA
oligomers were formed on the primitive Earth they would have been formed as
racemic mixtures. Since ImpA and ImpU are complementary we investigated the
reaction of D, L-ImpA with D, L-ImpU and then isolated the dimers, trimers and
tetramers. The linear dimers, cyclic dimers and trimers were separated by HPLC
using ion exchange first and then each fraction was collected and separated by
HPLC reverse phase chromatography. The homochirality of the linear dimers and
cyclic dimers did not yield noteworthy products but it was impressive that 75% of
the trimers were homochiral (2).
References
1. J. P. Ferris, P. C. Joshi, M. F. Aldersley, and J. W. Delano. J Am Chem Soc 13369-13374
(2009).
2. P. C. Joshi, M. F. Aldersley, J. W. Delano, and J. P. Ferris. Orig Life Evol Biosp 40, 000-000.
93
Understanding the Role of Passenger Mutations
in Cancer Progression
Christopher D. McFarland1
Shamil Sunyaev1,2
Leonid Mirny1,3,4
1Harvard
University Graduate Biophysics
Program, Cambridge, MA 02138
2Divison
of Genetics
Brigham and Women’s Hospital,
Harvard Medical School
3Departmetn
of Physics, Massachusetts
Institute of Technology
4Harvard-MIT
Division of Health
Sciences and Technology
The development of cancer can be considered an evolutionary process within an
organism: cells acquire mutations, compete for resources, and are subject to natural
selection. During this transformation, malignant tissues acquire tens of thousands of
somatic mutations, yet only a handful are believed to be responsible for the cancer
phenotype, termed driver mutations (1). The rest, occurring sporadically across cancer
genomes, are called passenger mutations. We hypothesize that many passenger mutations may be deleterious to cancer cells, yet occasionally fixate in the population.
We developed a stochastic, evolutionary model of cancer progression where cells
may acquire both driver mutations (advantageous to the cells) and passenger mutations (deleterious to the cells). We found that many passengers fixate in neoplastic populations, despite purifying selection against them, via a mechanism similar
to Muller’s Ratchet (2) and by hitchhiking with driver mutations. An analysis of
known somatic mutations in cancer found that many passenger mutations are predicted to be deleterious to human cells, based on their reduction in protein stability
and occurrence at conserved loci—corroborating our model’s findings. We also
found that biophysical properties of driver mutations may distinguish oncogenes
from tumor suppressors.
Through combined analytical and computational analysis, we identified two phases
of neoplastic behavior: one where driver mutations dominate dynamics and the population grows exponentially, and another where passenger mutations overwhelm
the cells causing prolonged dormancy or regression. We found that the conditions
1063
for dormancy and regression may be most exploitable in early metastases and are
currently testing our findings in a mouse model.
References
1. C. C. Maley, P. C. Galipeau, X. Liu, C. A. Sanchez, T. G. Paulson, and B. J. Reid. Cancer
Res 64, 3414-3427 (2004).
2. J. Felsenstein. Genetics 78, 737-756 (1974).
Intercalation-Mediated Assembly and the
Origin of Nucleic Acids
The continued increase of evidence for the central role of RNA in contemporary
life seems to provide ever-increasing support for the RNA world hypothesis (1).
However, it is difficult to imagine how RNA polymers would have appeared
de novo, as the abiotic formation of long nucleic acid polymers from mononucleotides or short oligonucleotides presents several formidable challenges in the
absence of highly evolved enzymes (2). For example, under solution conditions were
the backbone linkages formed between mononucleotides or oligonucleotides are
essentially irreversible very short cyclic products can be kinetically favored, which
severely limits polymer growth. We are investigating the hypothesis that reversible
non-covalent interactions between small planar molecules, similar to intercalating
dye molecules, originally organized and selected the base pairs of nucleic acids (3).
Recent experiments in our laboratory involving intercalation-mediated DNA and
RNA ligation have confirmed that intercalators, which stabilize and rigidify nucleic
acid duplexes, almost totally eliminate strand cyclization, allowing for chemical
ligation of tetranucleotides into duplex polymers of up to 100 base pairs in length
(4). In contrast, when these reactions are performed in the absence of intercalators, almost exclusively cyclic tetra- and octa- nucleotides are produced. Intercalator-free polymerization is not observed, even at tetranucleotide concentrations
10 000-fold greater than those at which intercalators enable polymerization. We
have also observed that intercalation-mediated polymerization is most favored if
the size of the intercalator matches that of the base pair; intercalators that bind
to Watson–Crick base pairs promote the polymerization of oligonucleotides that
form these base pairs. Additionally, intercalation-mediated polymerization is possible with an alternative, non-Watson–Crick-paired duplexes that selectively bind
complementary intercalators. These results support the hypothesis that intercalators
(acting as ‘molecular midwives’) could have facilitated the polymerization of the
first nucleic acids and possibly helped select the first base pairs, even if only trace
amounts of suitable oligomers were available. In another set of experiments, we
are exploring the utility of reversible backbone linkages to facilitate nucleic acid
polymer growth. Results from these experiments demonstrate how reversible linkages and intercalation can work together to promote the formation of extremely long
polymers by the assembly and “recycling” of previously cyclized oligonucleotides.
This research was supported by the NSF and the NASA Astrobiology Center
for Chemical Evolution (CHE-1004570) and the NASA Exobiology Program
(NNX08A014G).
References
1. R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds., The RNA World, Cold Spring Harbor Laboratory Press, 3rd Ed (2006).
2. A. E. Engelhart and N. V. Hud. Primitive genetic polymers, In Origins of Cellular Life,
D. Deamer and J. Szostak, Eds., Cold Spring Harbor Laboratory Press (2010).
3. N. V. Hud, S. S. Jain, X. Li, and D. G. Lynn. Chem Biodiver 4, 768-783 (2007).
4. E. D. Horowitz, A. E. Engelhart, M. C. Chen, K. A. Quarles, M. W. Smith, D. G. Lynn, and
N. V. Hud. Proc Natl Acad Sci USA 107, 5288-5293 (2010).
Aaron E. Engelhart
Ragan Buckley
Eric D. Horowitz
Nicholas V. Hud*
94
School of Chemistry and Biochemistry,
Center for Chemical Evolution,
Georgia Institute of Technology,
Atlanta, GA 30332
*[email protected]
1064
95
Irena Mamajanov*
Aaron E. Engelhart
Heather D. Bean
Nicholas V. Hud**
School of Chemistry and Biochemistry,
Parker H. Petit Institute for
Bioengineering and Bioscience,
Georgia Institute of Technology,
Atlanta, GA 30332-0400 (USA)
*[email protected]
**[email protected]
Characterization of DNA and RNA Secondary
Structures in Anhydrous Media
An aqueous environment has been long been postulated as being necessary for
the formation of nucleic acid secondary structures. While there has been considerable interest and debate regarding the actual number of water molecules required
to maintain a particular nucleic acid structure (1), there are obvious experimental
challenges to studying nucleic acid structures in the absence of water (e.g. low
nucleic acid solubility). Thus far, complementary DNA strands have been reported
to retain duplex structure only in anhydrous modified polyethylene glycol (2). However, DNA duplexes only exhibit marginal stability in other water-free organic solvents (3). An ever expanding set of new polar solvents, such as room temperature
ionic liquids (RTIL), provide new opportunities to study nucleic acids in waterfree environments. Here, we report that nucleic acids can form duplex, triplex and
G-quadruplex secondary structures that undergo reversible thermal denturation in a
water-free solvent (e.g. 0.25% water). This so-called deep eutectic solvent (DES)
is comprised of one part choline choride and two parts urea. The choline cholorideurea DES has a melting point of only 12˚C, whereas pure choline chloride melts
at 302˚C and urea at 133˚C (4). We have found that nucleic acid secondary structures exhibit different relative stabilities in the DES, compared to aqueous media
(5). Thermal transition midpoints of the duplex structures in the DES are lower
than those measured in water solution, whereas triplex and G-quadruplex structures can be more stable in the DES
compared to an aqueous solution
with the same ionic strength. Deep
eutectic solvents and ionic liquids
are currently of tremendous interest as nonvolatile media for a wide
range of chemical reactions and processes. Given previous reports that
these water-free solvents can support protein enzyme catalysis, it is
now feasible that catalytic nucleic
acids and protein enzyme-nucleic
acid complexes could be used in
these solvents.
References
1. T. V. Maltseva, P. Agback, and J. Chattopadhyaya. Nucleic Acids Res 21, 4246-4252
(1993).
2. A. M. Leone, S. C. Weatherly, M. E. Williams, H. H. Thorp, and R. W. Murray J Am Chem
Soc 123, 218-222 (2003).
3. G. Bonner and A. M. Klibanov. Biotechnol Bioeng 68, 339-344 (2003).
4. A. P. Abbott, G. Capper, D. L. Davies, R. K. Rasheed, and V. Tambyrajah. Chem Comm
70-71 (2003).
5. I. Mamajanov, A. E. Engelhart, H. D. Bean, N. V. Hud. Angew Chem Int Ed Eng 49,
6310–6314 (2010).
1065
Emergence and Evolutionary Connections between
Enzymatic Functions via Prototypes of Elementary
Functional Loops
Earlier studies of protein structure revealed closed loops with a characteristic size
25-30 residues and ring-like shape as a basic universal structural element of globular proteins (1, 2). Elementary functional loops (EFL) have specific signatures and
provide functional residues important for binding/activation and principal chemical transformation steps of the enzymatic reaction (3). We derive prototypes of
the elementary functional loops (EFLs) and use them for dissecting the enzymatic
function into its building blocks. As a result, previously uncharted evolutionary
connections between seemingly unrelated enzymes become apparent. We show
that proteins with different folds and biochemical reactions are built from limited
set of common elementary functions (3).
96
Alexander Goncearenco
Igor N. Berezovsky*
Computational Biology
Unit and Department of Informatics,
University of Bergen, Bergen, Norway
*[email protected]
We study relations between enzymatic functions in Archaeal superkingdom, using
elementary functional loops (EFLs). Connections between different superfamilies
and folds indicate events of EFLs’ recombination in the process of emergence of
new biochemical functions. We also consider methanogenesis, a metabolic pathway
typical for Archaea, as a case study of enzymes with specific biochemical functions
and unique cofactors. We show that some methanogenic enzymes reutilize already
existing folds with tuned original functions, while other methanogenic functions
are apparently built de novo from EFLs. Examples of common elementary functions and role of corresponding EFLs as basic units of enzymes is discussed.
References
1. I. N. Berezovsky, A. Y. Grosberg, and E. N. Trifonov FEBS Letters 466, 283-286 (2000).
2. E. N. Trifonov and I. N. Berezovsky. Curr Opin Struct Biol 13, 110-114 (2003).
3. A. Goncearenco and I. N. Berezovsky. Bioinformatics 26, i497-i503 (2010).
Mechanisms of Protein Oligomerization, the Critical
Role of Insertions and Deletions in Maintaining
Different Oligomeric States
Many proteins form homooligomeric complexes in a cell. Proteins that exist in different oligomeric states are responsible for the diversity and specificity of many
pathways, and transitions between different oligomeric states may regulate protein
activity. Despite the importance of homooligomers, the mechanisms of oligomerization are not very well understood and general principles have not been formulated.
In our study, we analyze sequence and structural features of homologous proteins in
different oligomeric states, and how they are involved in the interface formation (1).
We show that insertions and deletions which differentiate monomers and dimers
have a significant tendency to be located on the interaction interfaces. We also show
that about a quarter of all proteins and forty percent of enzymes in our dataset have
regions which mediate or disrupt the formation of homooligomers. These results
suggest that relatively small insertions or deletions may have a profound effect on
complex stability. Indeed, in many cases removal of enabling regions caused the
strong destabilization of the complexes. Moreover, we find that enabling regions
contain a larger fraction of hydrophilic residues, glycine and proline compared to
conventional interfaces and surfaces, resulting in a lower aggregation propensity.
Most likely, these regions may mediate specific interactions, prevent non-specific
dysfunctional aggregation and preclude undesired interactions between close paralogs therefore separating their functional pathways. When we closely examine the
Kosuke Hashimoto*
Anna R. Panchenko
97
National Center for Biotechnology
Information, National Library
of Medicine, National Institutes of
Health, Bethesda, MD 20894, USA
*[email protected]
1066
glycosyltransferase family, which consists of highly diverged members adopting different oligomeric states, we find that homooligomeric glycosyltransferases
appear as ancient as monomeric ones and go back in evolution to the last universal
common ancestor and many of them have enabling or disabling features on their
interfaces (2).
References
1. K. Hashimoto and A. R. Panchenko. Proc Natl Acad Sci USA 107, 20352-20357 (2010).
2. K. Hashimoto, T. Madej, S. H. Bryant, and A. R. Panchenko. J Mol Biol 28, 196-206
(2010).
98
William L. Duax*
Robert Huether
David Dziak
Courtney McEachon
University at Buffalo,
Buffalo, NY 14203
*[email protected]
An Evolutionary Tree Rooted in Actinobacteria
The three dimensional folds of each of the fifty ribosomal proteins (RibP) are fully
conserved in all bacterial species for which complete genomes have been reported
(2300 as of 3/1/11). Over 98% of all members of each bacterial RibP (bRibP) can
be accurately aligned with retention of a small number of fully conserved identities
commonly including Alanines (Ala), Prolines (Pro), Arginines (Arg), and immutable Glycines (Gly) and precisely located insertion or deletion sites (Indels) of
restricted size. Accurate full length alignment permits identification of sequence
positions where a single residue difference can separate Gram positive from Gram
negative bacteria. Co-evolution of different combinations of amino acids in other
positions in the aligned sequence separates bRibP sequences of different phylum,
classes, orders and genera from one another. Accurate alignment makes it possible
to follow sequence divergence through 3 billion years of evolution, reveals that the
entire sequences of the ribosomal proteins of individual genera have remained over
95% identical throughout their evolution, detects Gene bank errors in gene length
and annotation, and reveals that codon bias and use in ribosomal proteins is not
determined by tRNA population. We detect significant sequence homology between
all bRibPs and RibPs in the same two dozen chloroplasts (clRibP) and find that the
greatest sequence homology is between RibPs of cyanobacteria and these clRibPs.
On the basis of our analysis we find less sequence divergence, fewer Indels, and
more restricted codon use in RibPs of the small ribosomal subunit than those of
the large subunit. Conserved homology between the sequences of the bRibPs and
those of archaea, mitochondrial, and chloroplasts is significantly greater for the
proteins of the small subunit than the large. Of the RibPs analyzed thus far, the
sequence and three dimensional structure of ribosomal protein S19 from the small
subunit appears to be the most highly conserved in all species. When we align
3987 sequences of S19 (2353 bacteria, 1528 eukaryotes, and 106 archaea) only
two residues are fully conserved (a Gly and an Arg). One or more Gly residues in
each RibP family are immutable because the Gly conformations (phi and psi) are in
regions of the Ramachandran plot where other amino acids are rarely tolerated. Arg
conservation is associated with maintenance of charge balance and direct interaction with specific sites on rRNA and tRNA involved in ribosomal function. Sixteen
other sequence positions have 90% conservation of amino acid identities and one
sequence position, immediately adjacent to the fully conserved Gly, is occupied
by only two amino acids. An Asp in this position isolates 95% of all gram-positive
(G1) bacteria (866). An Asn in this position isolates 1487 bacteria, 1528 eukaryotes and 106 archaea. The 1487 bacteria include 95% of all gram-negative (G2)
bacteria. This [Asp,Asn] Gly sequence forms a tight turn with an internally hydrogen-bonded network and directly interacts with ribosomal RNA via five additional
hydrogen bonds [figure 1]. The amino acid composition of the S19 sequences is
found to diverge in the order G1 bacteria, to G2 bacteria, to eukaryotes. In G1
bacteria ten amino acids make up 76% of their total composition, amino acids of the
tRNA synthetase II family account for 61% of the composition. All G1 bacteria
have 46 residues conserved at 90% identity or greater of which 14 are G, A, R or P
(the amino acids encoded by the 8 codons composed of only guanine and cytosine).
Optimizing GARP content of the minimum universal fingerprint of S19 isolates
actinobacteria. Twelve species of Actinobacteria are found to have the highest
GARP composition. All of the RibPs of the large and small subunits in these species have full length sense/antisense open reading frames, a hallmark of the DNA
of genes of ancient species (1, 2). Examination of codon use in the proteins of the
30S subunits of six of these species reveals that 24 codons in which the third nucleotide is A or T are never or rarely found in their DNA. No tRNAs cognate to these
“unused” codons are found in the genomes of those six species. These findings
reveal that not all species use a 64 letter code, that the earliest peptides and proteins
had a bias in amino acid composition favoring amino acids of tRNA synthetase II
family, that immutable Gly residues are the key to aligning all members of major
protein families, that ribosomal protein S19 is a rosetta stone for accurate determination of a rooted evolutionary tree of all living species and that the last universal
common ancestor at the root of the tree is closely related to either K. radiotolerans
or C. flavogenans or both. We will trace the evolution of all 50 bRpros in an effort
to create a consistent evolutionary tree of all bacteria and unambiguously identify
the last universal common ancestor at its root.
Figure 1: The sequences of residues, N53G54K55, in a tight turn in S19 forms four hydrogen bonds
with the rRNA in the gram negative bacterium T. thermophilus. The immutably, 100% conserved, G54
has phi/psi values of 96.8o and -15.5o.
Support in part by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum
Fund, The East Hill Foundation and the generous help from a number of High
School students from the Buffalo NY area.
References
1. W. L. Duax, R. Huether, V. Z. Pletnev, D. Langs, A. Addlagatta A, et al. Proteins 61, 900906 (2005).
2. W. W. Carter and W. L. Duax. Mol Cell 10, 705-708 (2002).
3. W. L. Duax, R. Huether, V. Pletnev, and T. C. Umland. Int J Bioinform Res Appl 5, 280-294
(2009).
1067
1068
Recently Duplicated Human Genes: Basics of
Evolution
99
Alexander Y. Panchin2,3
Elena N. Shustrova3
Irena I. Artamonova1,2,3*
1Vavilov
Institute of General Genetics
RAS, Gubkina 3,
119991 Moscow, Russia
2Kharkevich
Institute for Information
Transmission Problems RAS,
Bolshoi Karetny pereulok 19,
127994 Moscow, Russia
3Lomonosov
Moscow State University,
Faculty of Bioengineering and
Bioinformatics, Vorobyevy Gory 1-73,
Moscow, Russia
*[email protected]
Gene duplications are one of the major sources of new protein functions. We studied the evolution of recently duplicated human genes. A genome-wide procedure
was used to find paralogous genes retaining detectable sequence similarity throughout most exons and introns. Similar genes were collected in families. Only families
containing three or more genes were analyzed.
A novel method was introduced for calculating the evolutionary rates of individual
genes from such families. It shows that negative selection, acting at a duplicated
gene and measured by the Kn/Ks test, is relatively weaker immediately after the
duplication event and then increases. Such changes of the negative selection pressure seem to be a major trend in the evolution of young human paralogs.
Another trend concerns the asymmetry of the evolution of two gene copies resulting from a recent duplication event. In about one fifth of recently duplicated gene
pairs from young paralogous gene families, the two gene copies accumulate amino
acid substitutions at significantly different rates. Differences in the gene expression
levels do not explain this asymmetry. The asymmetry in the rate of accumulation of
synonymous substitutions is much weaker and not significant. In asymmetric gene
pairs the number of deleterious mutations is higher in one copy, and lower in the
other copy as compared to genes comprising non-asymmetrically evolving pairs.
A possible explanation for this trend is the need for one of the two duplicated gene
copies to retain its initial function, as the other copy rapidly evolves to get a new
function.
We also compared the evolutionary rates for the original and derived copies of
recently duplicated paralogous genes. For primate-specific duplications it is possible to distinguish the original copy based on conservation of the gene neighborhood in rodents, although this can be done reliable only for a minor fraction of
genes produced by non-tandem duplications. The Kn values and the Kn/Ks ratios
were significantly lower for the original copies compared to the derived ones. Both
original and derived copies were evolving under negative selection.
This work is partially supported by the Russian Academy of Sciences (programs
“Molecular and Cellular Biology” and “Biodiversity”) and the state contract P1376.
100
T. J. Glembo
S. B. Ozkan*
Center for Biological Physics,
Arizona State University,
Tempe, AZ
*[email protected]
Structural and Functional Evolution of Proteins by
Conformational Diversity
Protein evolution has most commonly been studied either theoretically, by analyzing the sequence of the protein (1, 2), or experimentally, by resurrecting ancestral
proteins in the lab and performing ligand binding studies to determine function.
Thus far, structural and dynamic evolution have largely been left out of molecular
evolution studies. Here we incorporate both structure and dynamics to elucidate the
molecular principles behind the divergence in the evolutionary path of the steroid
receptor proteins. We begin by determining the likely structure of three evolutionary diverged, ancestral steroid receptor proteins (1) using the Zipping and Assembly Method with FRODA (ZAMF) (2). Our predictions are within 1.9Å RMSD
of the crystal structure of ancestral corticoid steroid receptor. Beyond comparing
static structure prediction, the main advantage of ZAMF is that it allows us to
observe protein dynamics. Therefore we can investigate differences in the diverged
proteins’ available dynamic space. Strikingly, our dynamics analysis of these
1069
predicted structures indicates that evolutionary diverged proteins do not share the
same dynamic subspace. Moreover, our dynamic analysis enables us to identify
critical mutations that most affect dynamics, therefore it shows the critical mutations leading to a divergence in function, which are verified experimentally.
References
1. P. Anbazhagan, M. Purushottam, H. B. K. Kumar, O. Mukherjee, S. Jain, and R. Sowdhamini.
2. Z. Liu, Y. Zu, L. Wu, and S. Zhang. J Biomol Struct Dyn 28, 97-106 (2010).
3. J. T. Bridgham, E. A. Ortlund, and J. W. Thornton. Nature 461, 515-519 (2009).
4. T. J. Glembo and S. B. Ozkan. Biophys J 98, 1046-1054 (2010).
101
Structure, Function and Evolutionary Relationship of
the Leptin Receptor (OB – Rb)
In view of the release of more and more genome sequences, the genome sequence
comparisons and phylogenetic tree constructions have become popular (1-3) and
it will be useful to use this methodology to understand body weight regulation
and energy homeostasis. Leptin, a 16 kDa adipocytic protein hormone is known to
play a central role in regulation of body weight and energy homeostasis; it is also
is also known to regulate various physiological functions such as reproduction,
hematopoiesis, inflammation etc. (4, 5). Leptin acts via its specific receptor (OB –
R) or LR. and till date six splice variants of OB – R have been identified and mapped
to the db gene locus on chromosome 1p in human. Of the six, the longest isoform
OB – Rb which is highly expressed in hypothalamus has the signaling capability
and the structure-functional relationship of this has been analysed in detail.
Alignment of the aminoacid sequences of leptin receptor (OB-Rb) from different
species such as human, mouse, pig, cattle, bat, turkey, frog, zebra and madaka fish
OB- Rb reveals that most of the regions including the N – terminal, transmembrane
(TM), disulphide bridges and C – terminal regions are highly conserved in mammalian species with subtle variations is madaka and zebra fish. Phylogenetic analysis is suggestive of evolutionary relationship between human, monkey, cattle etc.,
pig and sheep, rat and mouse, zebra fish and madaka fish with distinct variation in
elelephant, details of which will be discussed.
References
1. V. Sabbia, H. Romero, H. Musto, and H. Naya. J Biomol Struct Dyn 27, 361-369 (2009).
2. P. Anbazhagan, M. Purushottam, H. B. K. Kumar, O. Mukherjee, S. Jain, and R. Sowdhamini.
3. Z. Liu, Y. Xu, L. Wu, and S. Zhang. J Biomol Struct Dyn 28, 97-106 (2010).
4. Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, and J. M. Friedman. Nature 372,
425-432 (1994).
5. A. A. Steiner and A. A. Romanovsky. Prog Lipid Res 46(2), 89-107 (2007).
R. Malathi*
R. Raskin Erusan
Department of Genetics,
Dr. A.L.M. Post Graduate Institute of
Basic Medical Sciences,
University of Madras,
Chennai- 600 113, India
*[email protected]
1070
102
Ivana Besseova1*
Kamila Reblova1
Neocles B. Leontis2
Jiri Sponer1**
1Institute
of Biophysics, Academy of Sci-
ences of the Czech Republic,
Kralovopolska 135, 61265 Brno,
Czech Republic
2Department
of Chemistry, Bowling
Green State University, Bowling Green,
OH 43403, USA
*[email protected],
**[email protected]
Intrinsically Flexible Ribosomal RNA Segments.
Family C 3-Way Junctions and their Possible Role in
the Translation
We carried out extensive explicit solvent molecular dynamics analysis (1.4 µs) of
several RNA three-way junctions (3WJs) from the large ribosomal subunit. The
aim was to analyze the intrinsic flexibility of the 3WJs and to consider them in
the ribosomal context in available ribosomal crystal structures. The flexibility of
the RNA is inferred from stochastic thermal fluctuations sampled in unrestrained
simulations. The simulations identify the intrinsic low-energy deformation modes
of the molecules that can co-operate with the surrounding ribosomal elements to
achieve the functional dynamics.
All studied 3WJs possess significant anisotropic hinge-like flexibility between their
stacked stems and dynamics within the compact regions of their adjacent stems.
The most interesting is the GAC (GTPase associated center) 3WJ which may support large-scale dynamics of the L7/L12-stalk rRNA, i.e. rRNA Helices (H) 42-44
(1). Projection of the observed anisotropic movement into the ribosome shows that
H43/H44 rRNA is flexible in direction towards and away (closing-opening geometry path of the GAC 3WJ) of the large ribosomal subunit, see the Figure. When
the H42-H44 domain is in the overall “closed” conformation, the tip of the hairpin
loop of H89 can fit into the groove defined by the docking of the hairpin loops
of H43/44. However, such contact is only seen in the 2AW4 crystal structure of
vacant E.coli ribosome (2). In other crystal structures the distance between the H89
and GAC RNA varies widely (it even exceeds in some cases 10 Å) (3, 4). The
experimental structures show a wide range of positions sampling a set of more
inward and more outward structures with respect to the A-site of the large subunit
and H89. The range of observed positions agrees with the anisotropic flexibility
direction predicted by MD. We conclude that the X-ray observed flexibility of the
GAC RNA originates from the 3WJ and includes also the H42 stem region below
the 3WJ and above the conserved H42-H97 tertiary interaction. The GAC rRNA
could undergo large-scale rapid thermal fluctuations or structural adaptations to
facilitate gliding of the tRNA to H89, which leads it into its functional destinations
(A/A state) (5), see the Figure. The H42-44 rRNA region is not fully relaxed in
Figure: Left - Large ribosomal subunit (RNA in tan, proteins in cyan, pdb code 2WRO2) from Thermus termophilus including tRNA in the A/T state (in red,
2WRN2). The GAC 3WJ in the X-ray data is in purple, open and closed geometries of the junction occurring in the MD simulations are shown in green and blue,
respectively. Right - Detailed view on coordinated hypothetical movement of the tRNA and GAC 3WJ. The closed MD geometry of the GAC 3WJ forms contact
with H89 (marked by ellipse), which might be important for guiding the tRNA to H89 (in yellow).
1071
the ribosome. It is deformed towards the body of the large subunit by some of the
surrounding elements. The simulations suggest that the L10 protein can regulate
or contribute to this deformation. The intrinsic flexibility of the GAC 3WJ will be
briefly compared with intrinsic flexibility of RNA from other flexible parts of the
large subunit, namely the elbow region at the base of the A-site finger and the bottom part of the L1 stalk.
References
1. I. Besseova, K. Reblova, N. B. Leontis, and J. Sponer. Nucleic Acids Res 38, 6247-6264
(2010).
2. B. S. Schuwirth, M. A. Borovinskaya, C. W. Hau, W. Zhang, A. Villa-Sanjurjo,
J. M. Holton, and J. H. D. Cate. Science 310, 827-834 (2005).
3. T. M. Schmeing, R. M. Voorhees, A. C. Kelley, Y. G. Gao, F. V. Murphy, J. R. Weir, and
V. Ramakrishnan. Science 326, 688-694 (2009).
4. D. J. Klein, P. B. Moore, and T. A. Steitz. J Mol Biol 340, 141-177 (2004).
5. K. Y. Sanbonmatsu, S. Joseph, and C. S. Tung. Proc Natl Acad Sci USA 102, 15854-15859
(2005).
103
Ribosomal Paleontology and Resurrection: Molecular
Fossils from Before Coded Protein
The origins and early development of the translation machinery remain imprinted
in the extant ribosome (1-3), in sequences, folding, and function. To mine the
information contained within the ribosome, we are developing new methods of
molecular paleontology. We are developing experimental and computational tools
Loren Dean Williams*
Chiaolong Hsiao
Jessica C. Bowman
Chad R. Bernier
Jessica Peters
Dana M. Schneider
Eric O’Neill
NASA Astrobiology Institute and the
Center for Ribosomal Origins and
Evolution at the Georgia
Institute of Technology,
School of Chemistry and
Biochemistry,
Georgia Institute of
Technology, Atlanta,
GA, 30332-0400
*[email protected]
The large subunit as an onion.
1072
to understand and recapitulate fundamental steps in the origin and evolution of the
ribosome, and to estimate the relative ages of ribosomal components. We are biochemically resurrecting working models of ancestral ribosomes. We are developing
timelines for critical steps in the evolutionary history of the ribosome. The results
of these studies will help provide keys to understanding the origin of proteins and
RNA, and the origin of life. We use structure- and sequence-based comparisons of
the Large Subunits (LSUs) of Haloarcula marismortui and Thermus thermophilus.
These are the highest resolution ribosome structures available, and represent the
two primary branches of the evolutionary tree. Using an onion analogy, we have
sectioned the superimposed bacterial and archaeal LSUs into concentric shells,
using the sites of peptidyl transfer as the centers (4). This spherical approximation
allows shell-by-shell dissections and comparisons that clearly capture significant
information along the evolutionary timeline. The results support the notion that
ever-older molecular fossils are revealed as one bores toward the center of the LSU
onion. The conformations and interactions of both RNA and proteins change over
time. The frequency with which macromolecules assume regular secondary structural elements, such as A-form helices containing Watson-Crick base pairs (RNA)
and α-helices and β-sheets (protein), increases with time. The conformations of the
oldest ribosomal protein components suggest they are molecular fossils of the noncoded peptide ancestors of ribosomal proteins. We hypothesize that abbreviated
lengths and mixed sequences in the ancestral state proscribed secondary structure,
which is indeed nearly absent near the centers of the LSU onions.
References
1.
2.
3.
4.
104
William A. Cantara*
Yann Bilbille
Jia Kim
Andrzej Malkiewicz
Paul F. Agris
The RNA Institute,
Department of Biological Sciences,
University at Albany, SUNY,
Albany, NY 12222
*[email protected]
C. R. Woese. RNA 7, 1055-1067 (2001).
G. E. Fox. Cold Spring Harb Perspect Biol 2, a003483 (2010).
K. Bokov and S. V. Steinberg. Nature 457, 977-980 (2009).
C. Hsiao, S. Mohan, B. K. Kalahar, and L. D. Williams. Mol Biol Evol 26, 2415-2425 (2009).
Anticodon Loop Modifications Modulate Structural
Flexibility in E. coli tRNAArg1,2 that Lacks a U-turn
Conformation in Solution
Three of the six codons that are decoded by tRNAArg in E. coli are read by the
isoacceptors tRNAArg1,2. The anticodon stem and loop domain (ASLArgICG) of these
isoacceptors differ only in the identity of the residue at position 32 in the as either
2-thiocytidine (s2C32) or cytidine for tRNAArg1 and tRNAArg2, respectively. These
isoacceptors also contain important modifications at positions 34 (inosine, I34) and 37
(2-methyladenosine, m2A37). To investigate the roles of the modifications in proper
folding of the ASLArgICG, six ASLArgICG constructs differing in their array of modifications were analyzed by biophysical spectroscopic methods as well as functional
binding assays. Thermal denaturation and circular dichroism spectroscopy showed
that the modifications contribute competing thermodynamic and base stacking
properties. Spectroscopic methods indicated that ASLArgICG modifications contributed significant differences in structural properties. However, restrained molecular
dynamics calculations of the ASLArgICG structures from NMR spectroscopy clearly
showed that the equilibrium solution conformations of the ASLs are nearly identical,
but do not possess the invariant U-turn structure needed for binding to the ribosomal
A-site. Yet, all of the ASLArg constructs were able to bind to the ribosome in the presence of the cognate CGU codon. The m2A37 modification restricts binding to CGC,
while both s2C32 and m2A37 restrict binding to CGA. Taken together, the results
suggest that chemical modifications modulate the flexibility of the loop, allowing
induced conformations on the ribosome that can restrict binding to specific codons.
This research is supported by NSF grant number 53855.
1073
Structural Characteristics of E. coli YrdC Suggest a
Role in the Enzymatic Biosynthesis of the tRNA
Modification N6-Threonylcarbamoyladenosine
Nucleoside modifications are vital for the proper structure and function of tRNA.
The N6-threonylcarbamoyladenosine modification at position 37 (t6A37), 3’-adjacent
to the anticodon, of many tRNA species ensures the accurate recognition of several
ANN codons by increasing codon affinity and enhancing ribosome binding (1, 2).
Considerable data exists on the biophysical aspects of t6A37, however, the biosynthesis pathway of this hypermodified base is only partially understood (3). This pathway
requires ATP, threonine, a carbon source, and possibly the universal protein family
YrdC/Sua5, which has been shown to be involved in t6A37 biosynthesis (4, 5). To
further investigate this possibility, we examined the interaction of E. coli YrdC with
the heptadecamer anticodon stem loop of tRNA lysine (ASLLys). As determined by
mass spectrometry analysis, NMR, and quenching of intrinsic fluorescence, YrdC
bound unmodified ASLLys with high affinity (Kd = 0.27 ± 0.20 µM), t6A37-modified
ASLLys with significantly lower affinity (Kd = 1.36 ± 0.39 µM), and showed specificity toward threonine and ATP. YrdC also preferentially binds threonine over other
amino acids tested by STD-NMR. Our studies of the YrdC-ASLLys interaction by
NMR, CD, and fluorescence of 2-aminopuine at position 37 of ASLLys, indicated
no structural change in the RNA. Therefore, catalytic function appears to be limited
under these in vitro conditions and YrdC is most likely a subunit of a t6A37 synthetase
complex. Further characterization of the protein-protein and protein-RNA interactions will provide information to determine the role of YrdC in t6A37 biosynthesis.
105
Kimberly A. Harris1,2
Victoria Jones1
Yann Bilbille1
Paul F. Agris2
1Department
of Molecular & Structural
Biochemistry, North Carolina State
University, Raleigh, NC, USA 27695
2The
RNA Institute,
University at Albany -SUNY,
Albany, NY, USA 12222
References
1.
2.
3.
4.
5.
S. Nishimura. Prog Nucleic Acid Res Mol Biol 12, 49-85 (1972).
P. F. Agris. Nucleic Acids Res 32, 223-238 (2004).
J. W. Stuart, et al. Biochemistry 39, 13396-13404 (2000).
A. Körner and D. Söll. FEBS Lett 39, 301-306 (1974).
B. El Yacoubi, et al. Nucleic Acids Res 37, 2894-2909 (2009).
A Structural Method with Single Atom Resolution
for Investigating the Secondary Structure of
RNA: the Deuterium Kinetic Isotope Effect/Hydroxyl
Radical Cleavage Experiment
The hydroxyl radical is widely used as a high-resolution footprinting agent for
DNA (1) and RNA (2). The hydroxyl radical abstracts a hydrogen atom from the
sugar-phosphate backbone, generating a carbon-based radical, which ultimately
leads to a strand break. Substituting deuterium for hydrogen on the ribose results in
a kinetic isotope effect if abstraction of that hydrogen atom by the hydroxyl radical leads to a strand break. The deuterium kinetic isotope effect (KIE) correlates
well with the solvent accessibility of the deoxyribose hydrogen atoms in DNA (3).
RNA, due to its single-stranded nature, adopts a multitude of secondary and tertiary
structures. This results in a non-uniform exposure of its backbone, making it a very
attractive system to study using the deuterium KIE/hydroxyl radical experiment.
We applied this experiment to the sarcin-ricin loop (SRL) RNA molecule, a structurally well-defined component of the large subunit of ribosomal RNA. SRL plays
a critical role in the activation of elongation factor Tu (a GTPase) leading to the
hydrolysis of GTP during translation, which is speculated to be the reason for its
universal conservation (4).
We utilized a one-pot enzymatic protocol to synthesize specifically deuterated ATP
and GTP (5). We then use in vitro transcription to incorporate these deuterated
106
Shakti Ingle*
Robert Azad
Thomas D. Tullius
Boston University,
Boston MA 02215
*[email protected]
1074
nucleotides into the 29-mer SRL RNA molecule. We report the observation of a
substantial deuterium kinetic isotope effect on hydroxyl radical cleavage of SRL
RNA for dideuteration at the C5’ position. This experiment provides direct evidence of abstraction by the hydroxyl radical of hydrogen atoms from the ribose
C5’ position in RNA. We also observe isotope effects for deuteration at the C4’
position, although to a lesser extent. Interestingly, we observed different apparent
KIEs for the same deuterated nucleotide at different positions in the SRL sequence,
suggesting that the extent of reaction with hydroxyl radical depends on the local
structural (and perhaps dynamic) environment. We conclude that the deuterium
KIE/hydroxyl radical experiment provides a way to investigate the solvent exposure of the ribose backbone of RNA at the level of single hydrogen atoms.
This research is supported by NSF Award MCB-0843265.
References
1. T. D. Tullius and B. A. Dombroski. Science 230, 679-681 (1985).
2. J. A. Latham and T. R. Cech. Science 245, 276-282 (1989).
3. B. Balasubramanian, W. K. Pogozelski, and T. D. Tullius. Proc Natl Acad Sci USA 95,
9738-9743 (1998).
4. R. M. Voorhees, T. M. Schmeing, A. C. Kelley, and V. Ramakrishnan. Science 330, 835-838
(2010).
5. T. J. Tolbert and J. R. Williamson. J Am Chem Soc 118, 7929-7940 (1996).
107
Lena Dang1*
Susan Pieniazek1
Anne M. Baranger2
David L. Beveridge1
1Department
of Chemistry, Wesleyan
University, Middletown, Connecticut
2Department
Illinois at Urbana-Champaign, Illinois
*[email protected]
Insight into Transition States in Protein-RNA
Recognition through MD Simulations
Protein-RNA recognition plays an important role in many post transcriptionalgene-expression-regulation, such as RNA modification, transportation, translation
and degradation. U1A, a member of RNA Recognition Motif (RRM) containing
protein family, is a component of an RNA-protein complex called U1A snRNP
involved in pre-mRNA splicing. RRM proteins’ abilities to recognize and bind to
their cognate RNA sequence involves various nonbonding forces, such as electrostatic attraction, hydrogen bonding, aromatic stacking and van de Waals interactions. Results from our Molecular Dynamics (MD) studies on RRM containing
proteins (U1A, SXL) and their cognate RNA sequences indicate that significant
structural adaptations in both proteins and RNA sequences are required for binding. Simulations on mutant proteins demonstrate that electrostatics is the dominating force during the initial steps of the binding event. RMSD and RMSF analyses
of the simulations indicate the free RMM containing proteins are more flexible
1075
than the protein-RNA complex for both U1A and SXL. Regions of the protein
that undergo the greatest flexibility include N- and C-termini, loops and helixes.
Covariance analyses indicate that a strong connectivity in a network of amino acids
separated by long distances can be used to explain the mechanism of conformational change upon protein-RNA binding. PCA analyses and 3D-RMSD spaces are
constructed to further identify the transition states of the binding events.
RNASTEPS, an Online Database of
Sequence-dependent Deformability of RNA
Helical Regions
The RNASTEPS database, located at http://rnasteps.rutgers.edu, is a repository of
the base-pair steps found in the double-helical regions of 656 RNA crystal structures
of 3.5 Å or better resolution. The database includes a variety of structural parameters and molecular images, computed with the 3DNA software package (1, 2) and
known to be useful for characterizing and understanding the sequence-dependent
spatial arrangements of the DNA and RNA base pairs and base-pair steps. The
data can be accessed by searches of Leontis-Westhof base-pair patterns and Protein
Data Bank and Nucleic Acid Database structure identifiers. The site also includes a
repository of the average values and covariance of the rigid-body parameters characterizing well represented base-pair steps, the knowledge-based elastic potentials
derived from these data (3), and other measures of RNA step deformability, such
as the conformational volume, base-pair overlap, and root-mean-square deviation
of atomic coordinates. The collective information provides a useful benchmark for
RNA force-field development. The cumulative data account for the measured persistence length of double-stranded RNA (4) in simulations using the approach of
Czapla et al. (5). An example of the distributions and ‘energies’ extracted from the
roll and twist angles of 1274 GG·CC steps and the corresponding molecular superposition of these steps is shown below:
108
Mauricio Esguerra
Wilma K. Olson
Department of Chemistry &
Chemical Biology, BioMaPS Institute for
Quantitative Biology, Rutgers, the State
University of New Jersey,
Piscataway, NJ 08854, USA
[email protected]
[email protected]
1076
References
1. X-L. Lu and W. K. Olson. Nucleic Acids Research 31, 5108-5121 (2003).
2. X-L. Lu and W. K. Olson. Nature Protocols 3, 1213-1227 (2008).
3. W. K. Olson, A. A. Gorin, X-J. Lu, and L. M. Hock, and V. B. Zhurkin. Proc Natl Acad Sci,
95, 11163-11168 (1998).
4. J. A. Abels, F. Moreno-Herrero, T. van der Heijden, C. Dekker, and N. H. Dekker. Biophysical
Journal 88, 2737-2744 (2005).
5. L. Czapla, D. Swigon, and W. K. Olson. Journal of Chemical Theory and Computation 2,
685-695 (2006)
109
Kamila Reblova*
Judit E. Sponer
Nada Spackova
Ivana Besseova
Jiri Sponer**
Institute of Biophysics, Academy of
Sciences of the Czech Republic,
Kralovopolska 135, CZ-61265,
Brno, Czech Republic
*[email protected]
**[email protected]
A-minor I vs A-minor 0 Tertiary Interactions in
RNA Kink-turns. Molecular Dynamics and
Quantum Chemical Analysis
The second tertiary contact in RNA kink-turns is represented by A-minor interaction between adenine of the second A•G base pair in the NC-stem and the first
canonical base pair of the C-stem. Kvnown kink-turn structures possess either
A-minor I (A-I) or A-minor 0 (A-0) interaction (Figure). Bioinformatics data show
that kink-turns with A-I in the available X-ray structures keep primarily G=C pair
in the first C-stem position during evolution while the inverted base pair (C=G) is
basically not realized. In contrast, kink-turns with A-0 in the observed structures
alternate G=C and C=G base pairs in sequences. Molecular dynamics simulations
of X-ray structures of kink-turns reveal that the A-I interaction with G=C base pair
(A-I/G=C triple) is stable while inversion of the canonical base pair (A-I/C=G)
leads either to kink-turn disruption or rearrangement to A-0/C=G (1). The A-0/
G=C initial configuration tends to transform to the A-I/G=C arrangement. Finally,
simulations testing the A-0/C=G arrangement lead either to disruption of the structure (after unsuccessful transition to the A-I configuration) or are stable. Thus, the
A-I/G=C arrangement appears to be intrinsically preferred by kink-turns while formation of the A-0 interaction is likely related to the context. In addition, as shown
for ribosomal Kink-turn 15, A-0 may be supported by additional interactions that
do not belong to the kink-turn signature interactions. Quantum-chemical calculations explain how a delicate balance of various intermolecular interactions plays
a decisive role in determining the dynamic behavior and stability of the various
A-minor patterns in kink-turns.
Figure: Tertiary interactions (A-0 and A-I) stabilizing kink-turn structures.
Reference
1. K. Reblova, J. E. Sponer, N. Spackova, I. Besseova, and J. Sponer. Submitted.
1077
On the Geometry, Electronic Structure and
Hydrolytic Stability
of the As-DNA Backbone
High level quantum chemical calculations have been applied to investigate the
geometry and electronic properties of the arsenate analogue of the DNA backbone (1). The optimized geometries as well as hyperconjugation effects along the
C3‘-O3‘-X-O5‘-C5‘ linkage (X=P,As) exhibit a remarkable similarity for both
arsenates and phosphates. This suggests that arsenates – if present – might serve as
a potential substitute for phosphates in the DNA-backbone.
On the other hand, our calculations predict that neither steric hindrance nor less polar
solvent medium is able to reduce the otherwise high hydrolysis rate of arsenateesters (2). These results question the stability of As-DNA not only in aqueous but
also in non-aqueous environments.
110
Arnost Mladek1
Jiri Sponer1
Bobby G. Sumpter2
Miguel Fuentes-Cabrera2*
Judit E. Sponer1**
1Institute
of Biophysics, Academy of
References
1. A. Mladek, J. Sponer, B. G. Sumpter, M. Fuentes-Cabrera, and J. E. Sponer. J Phys Chem
Lett, In press. http://pubs.acs.org/doi/full/10.1021/jz200015n
2. A. Mladek, J. Sponer, B. G. Sumpter, M. Fuentes-Cabrera, and J. E. Sponer. Submitted.
Kralovopolska 135,
CZ-61265, Brno, Czech Republic
2Center
for Nanophase Materials
Sciences, and Computer Sciences and
Mathematics Division, Oak Ridge
National Laboratory, Oak Ridge, P. O.
Box 2008,
Oak Ridge, TN 37831-6494, USA
*[email protected]
**[email protected]
1078
111
Vojtěch Mlýnský1
Pavel Banáš1
Nils G. Walter3
Jirˇí Šponer2*
Michal Otyepka1**
1Regional
Centre of Advanced
Technologies and Material,
Department of Physical Chemistry,
Faculty of Science, Palacky University,
Olomouc, Czech Republic
2Institute
Brno, Czech Republic
3Department
of Chemistry, Single
Molecule Analysis Group University of
Michigan Ann Arbor, MI, USA
*[email protected]
**[email protected]
Role of Protonation States of G8 and A38
Nucleobases in Structure Stabilization and Catalysis
of the Hairpin Ribozyme
The hairpin ribozyme is a prominent member of the group of small RNA enzymes
(ribozymes) because it does not require metal ions to achieve catalysis of the reversible, site-specific cleavage of its RNA substrate (1). Guanine 8 (G8) and adenine
38 (A38) were identified by biochemical and structural experiments as the key
participants involved in cleavage. Despite broad efforts, their exact roles in catalysis remains disputed (2). We have carried out classical molecular dynamics (MD)
simulations (3, 4) in explicit solvent on 50-150 ns timescales with various protonation states of G8 and A38. The MD simulations reveal that a canonical G8 agrees
well with the crystal structures while a deprotonated G8 distorts the active site. The
G8 enol tautomer is structurally well tolerated, causing only local rearrangements
in the active site. In most simulations, the canonical A38 departs from the scissile
phosphate and substantially perturbs the active site. The Protonated A38H+ is more
consistent with the crystallography data (5). MD simulations support the idea that
A38H+ is the dominant form in the crystals, grown at pH 6. This finding is also in
agreement with recent NAIM experimental data (6). The MD simulations were also
used to find geometries of potential reactive states bearing G8 and A38 of various
protonation states in the active site. These geometries were further analyzed by
hybrid quantum-mechanical/molecular mechanical (QM/MM) methods to evaluate
the energy along the reaction pathway in order to find the most probable reaction
mechanism. The DFT MPW1K/6-31+G(d,p) method was used for the QM region
and the parm99 force field for MM region, while the communication between these
both layers was realized via electronic embedding (7, 8). The quality of our DFT
method was tested against reference CCSD(T)/CBS calculations. The mean unassigned error of the DFT method was found to be below 1.0 kcal·mol-1. The calculated
activation barriers are in good agreement with experimental data (20-21 kcal·mol-1)
for the systems with canonical G8 and both canonical/protonated forms of A38/
A38H+ (19.6 kcal·mol-1 and 20.1 kcal·mol-1, respectively). The initial nucleophile
attack of the A-1(2’-OH) group on the scissile phosphate is the rate-limiting step
along the reaction path. The G8-enol tautomer increases the overall reaction barrier
by 4.7 kcal·mol-1. On the other hand, our preliminary data indicate that the presence
of an unprotonated G8- (together with A38H+) seems to significantly decrease the
activation barrier. Protonated A38H+ does not significantly affect the overall activation barrier (20.1 kcal·mol-1), but decreases the activation barrier of the exocyclic
cleavage step by 7.7 kcal·mol-1. In the reaction path where a protonated A38H+
acts as the general acid, the product is 9.5 kcal·mol-1 higher in energy compared to
the product in the reaction where a nonbridging oxygen of the scissile phosphate
acts as proton donor and A38H+ is not directly involved in cleavage chemistry.
This work was supported by the Grant Agency of the Academy of Sciences of the
Czech Republic (grant IAA400040802), by the Grant Agency of the Czech Republic (grants 203/09/H046, 203/09/1476, P208/11/1822 and P301/11/P558), by
Ministry of Youth, Sport and Education of Czech Republic (grants LC512 and
MSM6198959216), by Student Project PrF_2010_025 of Palacky University. This
work was supported also by the Operational Program Research and Development
for Innovations - European Social Fund (CZ.1.05/2.1.00/03.0058) and NIH grant
2R01 GM062357 to NGW.
References
1.
2.
3.
4.
M. J. Fedor. Annu Rev Biophys 38, 271-299 (2009).
T. J. Wilson and D. M. Lilley. RNA 17, 213-221 (2011).
P. Sklenovský and M. Otyepka. J Biomol Struct Dyn 27, 521-540 (2010).
M. A. Ditzler, M. Otyepka, J. Šponer, and N. G. Walter. Acc Chem Res 43, 40-47 (2010).
1079
5. V. Mlýnský, P. Banáš, D. Hollas, K. Réblová, N. G. Walter, J. Šponer, and M. Otyepka.
J Phys Chem B 114, 6642-6652 (2010).
6. I. T. Suydam, S. D. Levandoski, and S. A. Strobel. Biochemistry 49, 3723-3732 (2010).
7. P. Banáš, L. Rulíšek, V. Hánošová, D. Svozil, N.G. Walter, J. Šponer, and M. Otyepka.
J Phys Chem B 112, 11177-11187 (2008).
8. P. Banáš, P. Jurečka, N. G. Walter, J. Šponer, and M. Otyepka. Methods 49, 202-216
(2009).
Synthesis of a Disubstituted Purine Analog and its
Binding Interactions with Xanthine Phosphoribosyl
Transferase (xpt) Riboswitch
RNA has the remarkable ability to form 3-dimensional structures that parallel the
structural complexity of proteins. Highly structured RNA molecules present excellent targets for new classes of antibiotics. Riboswitches are folded regions in the
untranslated region of mRNA that control gene expression upon binding to small
ligands (1). Our lab is involved in the synthesis of guanine analogs with modifications at both the C2 and C6 position of the purine ring. These analogs have the
potential of tightly binding to the 5’-untranslated region of xanthine phosphoribosyl transferase (xpt) riboswitch mRNA (2). High-resolution structure of xpt riboswitch shows that there maybe additional binding space (white ovals in the figure
below) where it may be possible to add functional groups without significantly
disrupting ligand binding to the existing pocket (3). Modification at the C2 and C6
position of the purine ring can facilitate additional H-bonds with nucleotides near
the binding pocket of xpt mRNA potentially yielding stronger ligand-mRNA interactions. Current work in our lab is focused on the synthesis and characterization of
2-acetamido-6-hydrazone-purine. Binding affinity of analogs to riboswitch mRNA
is tested using in-line probing with P-32 labeled mRNA and an in vivo GFP-based
reporter system.
References
1. W. C. Winkler and R. R. Breaker. Annu Rev Microbiol 59, 487-517 (2005).
2. J. N. Kim, K. F. Blount, I. Puskarz, J. Lim, K. H. Link, and R. R. Breaker. ACS Chem Biol
4, 915-927 (2009).
3. R. T. Batey, S. D. Gilbert, and R. K. Montange. Nature, 432, 411-415 (2004).
112
Angela Potenza
Rachit Neupane
Emily McLaughlin
Swapan S. Jain*
Bard College,
Annandale on Hudson, NY 12504
*[email protected]
1080
113
Alexander M. Andrianov1*
Ivan V. Anishchenko2
Mikhail A. Kisel1
Vasiliy A. Nikolayevich1
Vladimir F. Eremin3
Alexander V. Tuzikov2
1Institute
of Bioorganic Chemistry,
National Academy of Sciences of Belarus,
Kuprevich Street 5/2,
220141 Minsk, Republic of Belarus
2 United
Institute of Informatics Problems,
Surganov Street 6,
220012 Minsk, Republic of Belarus,
3The
Republican Research and Practical
Center for Epidemiology
and Microbiology,
Filimonova Street 23,
*[email protected]
Computer-Aided Design of Novel HIV-1 Entry
Inhibitors, Their Synthesis and Trials: Glycolipids
Against the Envelope gp120 V3 Loop
The HIV-1 V3 loop plays a central role in the biology of the HIV-1 envelope glycoprotein gp120 as a principal target for neutralizing antibodies, and as a major determinant in the switch from the non-syncytium-inducing to the syncytium-inducing
form of HIV-1 that is associated with accelerated disease progression. HIV-1 cell
entry is mediated by the sequential interactions of gp120 with the receptor CD4 and
a co-receptor, usually CCR5 or CXCR4, depending on the individual virion. The
V3 loop is critically involved in this process. Because of the exceptional role of the
V3 loop in the viral neutralization and cell tropism, one of the actual problems is
that of identifying chemical compounds able to block this functionally crucial site
of gp120. According to empirical observations, glycolipid β-galactosylceramide
(β-GalCer) forming on the surface of some susceptible host cells the primary receptor for HIV-1 alternative to CD4 exhibits a strong attraction to the V3 loop and, for
this reason, may be involved in anti-HIV-1 drug studies. In the light of these observations, the use of bioinformatics tools for imitating the process of making the V3/
glycolipid complexes may provide a structural rationale for the design of efficient
blockers of the functionally important V3 sites.
The objects of this study were to generate the 3D structure model for the complex
of V3 with β-GalCer and, based on the calculation data, to design its water soluble
analogs that could efficiently mask the HIV-1 V3 loop followed by their synthesis and
medical trials. To this effect, the following problems were solved: (i) 3D structures for
the consensus amino acid sequences of the HIV-1 subtypes A and B V3 loops were
computed by homology modeling and simulated annealing; (ii) spatial structures of
β-GalCer, as well as of a series of its modified forms were determined by quantum
chemistry and molecular dynamics simulations; (iii) supramolecular ensembles of
these glycolipids with V3 were built by molecular docking methodology and energy
characteristics describing their stability were estimated by molecular dynamics computations; (iv) synthesis of β-GalCer derivatives that, according to the designed data,
give rise to the stable complexes with V3 was performed, and (v) testing of these compounds for antiviral activity was carried out. From the structural data obtained, the
Phe and Arg/Gln amino acids of the gp120 immunogenic crest were revealed to play
a key role in forming the complexes of glycolipids with V3 by specific interactions
with the galactose residue and sphingosine base respectively. And at the same time,
the sugar hydroxyl groups form the H-bonds with the nearby polar atoms of the V3
backbone. Two water soluble analogs of β-GalCer were also found to display a high
affinity to V3 close to that of the native glycolipid. This inference results from the values of binding free energy evaluated for the calculated structures and coincides with
the experimental data on the complexes of gp120 with β-GalCer. The above theoretical findings are in keeping with those of medical trials of the synthesized molecules,
which testify to their anti-HIV-1 activity against the virus subtypes A and B isolates.
As a matter of record, the molecules constructed here are supposed to present the
promising basic structures for the rational design of novel potent HIV-1 entry
inhibitors that could neutralize the majority of circulating indigenous strains. For
some of the details of the methodlogies of current drug designs employed here
please consult the full length research articles (1-4).
References
1. A. M. Andrianov and I. V. Anishchenko. J Biomol Struct Dyn 27, 179-193 (2009).
2. A. M. Andrianov. J Biomol Struct Dyn 26, 445-454 (2009).
1081
Disclosure of Conserved Structural Motifs
in the HIV-1 Third Variable (V3) Loop by
Comparative Analysis of 3D V3
Structures for Different Virus Subtypes
The V3 loop on gp120 from HIV-1 is a focus of many research groups involved
in anti-AIDS drug development because this region of the protein is the principal target for neutralizing antibodies and determines the preference of the virus
for T-lymphocytes or primary macrophages. Although the V3 loop is a promising
target for anti-HIV-1 drug design, its high sequence variability is a major complicating factor. Nevertheless, the occurrence of highly conserved residues within the
V3 loop allows one to suggest that they may preserve their conformational states
in different HIV-1 strains and, therefore, should be promising targets for designing
new anti-HIV drugs. In this connection, the issue of whether these conserved amino
acids may help to keep the local protein structure and form the structurally rigid
segments of V3 exhibiting the HIV-1 vulnerable spots is very relevant. One of the
plausible ways to answer this question consists of examining the V3 structures for
their consensus sequences corresponding to the HIV-1 group M subtypes responsible for the AIDS pandemic followed by disclosing the patterns in the 3D arrangement of the variable V3 loops. Because of the deficiency of experimental data on
the V3 structures, these studies may be performed by homology modeling using the
high-resolution X-ray and NMR-based V3 models as the templates.
In this work, the 3D structural models for the consensus amino-acid sequences of the V3
loops from the HIV-1 subtypes A, B, C, and D were generated by bioinformatics tools
to reveal common structural motifs in this functionally important portion of the gp120
envelope protein. To this effect, the most preferable 3D structures of V3 were computed
by homology modeling and simulated annealing methods and compared with each
other, as well as with those determined previously by X-ray diffraction and NMR spectroscopy. Besides, the simulated V3 structures were also exposed to molecular dynamics computations, the findings of which were analyzed in conjunction with the data on
the conserved elements of V3 that were obtained by collation of its static models.
As a matter of record, despite the high sequence mutability of the V3 loop, its segments
3-7, 15-20 and 28-32 were shown to form the structurally invariant sites, which include
amino acids critical for cell tropism. Moreover, the biologically meaningful residues
of the identified conserved stretches were also shown to reside in β-turns of the V3
polypeptide chain. In this connection, these structural motifs were suggested to be used
by the virus as docking sites for specific and efficacious interactions with receptors of
macrophages and T-lymphocytes. Therefore, the structurally invariant V3 sites found
here represent potential HIV-1 weak points most suitable for therapeutic intervention.
In the light of the findings obtained, the strategy for anti-HIV-1 drug discovery
aimed at the identification of co-receptor antagonists that are able to efficiently mask
the structural motifs of the V3 loop, which are conserved in different virus subtypes, is highly challenging. To overcome this problem, an integrated computational
approach involving theoretical procedures, such as homology modeling, molecular
docking, molecular dynamics, QSAR modeling and free energy calculations, should
be of great assistance in the design of novel, potent and broad antiviral agents. For
some of the details of the methodlogies of current drug designs employed here please
consult the full length research articles (1-4).
Acknowledgment
This study was supported by grants from the Union State of Russia and Belarus
(scientific program SKIF-GRID; No 4U-S/07-111), as well as from the Belarusian
Foundation for Basic Research (project X10-017).
114
Alexander M. Andrianov1*
Ivan V. Anishchenko2
Alexander V. Tuzikov2
1Institute
of Bioorganic Chemistry,
Kuprevich Street 5/2,
2 United
Institute of Informatics Problems ,
Surganov Street 6,
*[email protected]
1082
References
1. A. M. Andrianov and I. V. Anishchenko. J Biomol Struct Dyn 27, 179-193 (2009).
2. A. M. Andrianov. J Biomol Struct Dyn 26, 445-454 (2009).
3. T. T. Chang, H. J. Huang, K. J. Lee, H. W. Yu, H. Y. Chen, F. J. Tsai, M. F. Sun, and C. Y.
C. Chen. J Biomol Struct Dyn 28, 309-321 (2010).
115
Nikolai B. Ulyanov*
Richard Tjhen
Christophe Guilbert
Thomas L. James
Department of Pharmaceutical Chemistry,
University of California, San Francisco,
CA 94158-2517, USA
*[email protected]
Hypothetical Mechanism of the Kissing to Extended
Dimer Conversion for the HIV-1 Stem-Loop 1 RNA
Two homologous copies of genomic RNA are packaged as a dimer into the HIV-1
virions (reviewed in (1-3)). The initiation of the dimer formation, which is believed
to occur in the cytoplasm of infected cells, has been mapped to a stem-loop SL1
with a palindromic apical loop within the 5’-untranslated region of the HIV-1
RNA. SL1 RNA, its structural features and sequence are strongly conserved among
HIV-1 isolates; deletion of SL1 significantly impairs packaging of RNA into the
virions. RNA extracted from immature HIV-1 virions is thermally unstable as a
dimer; the dimer stability increases with virus maturation, which is a complex process associated with the viral protease activity (4, 5). In vitro, SL1 RNA can form
either a metastable kissing dimer (KD), which is kept together by a 6-bp duplex
formed by the palindromic loops, or a stable extended dimer (ED). KD SL1 RNA
can be converted into ED by the viral nucleocapsid protein NCp7 (6), a proteolytic
fragment of the Gag polyprotein. This conversion can occur without disruption of
the kissing interaction between the palindromic loops (7), which is present in both
KD and ED forms. While it is generally accepted that RNA chaperone properties
of NCp7 are responsible for the KD-to-ED conversion, the detailed mechanism of
the conversion is not known. Indeed, NCp7 destabilizes duplexes only moderately
(8), however, 12 base pairs in each SL1 stem need to be broken and re-arranged
during the conversion; further, only two NCp7 molecules per SL1 dimer are sufficient for the complete conversion (7). Here, we propose a mechanism for the KDto-ED conversion that does not require simultaneous dissociation of all base pairs
in SL1 stems. This hypothetical mechanism involves formation of an RNA analog
of the Holliday junction intermediate between the two stems of the SL1 dimer
1083
and a following branch migration towards the palindromic duplex. According to
this model, the torsional stress accumulated due to the stem rotation caused by the
branch migration is absorbed by the single-stranded purines flanking the palindromic sequence. The NCp7 role is to bring the two stems together by neutralizing the
charges on the phosphate groups and to facilitate formation of the initial cross-over,
possibly at the level of the G-rich internal loop. We will present the models of the
intermediate structures calculated with the miniCarlo program (9).
This research is supported in part by the California HIV/AIDS Research Program
award ID09-SF-030.
References
1. J. C. Paillart, M. Shehu-Xhilaga, R. Marquet, and J. Mak. Nat Rev Microbiol 2, 461-72
(2004).
2. M. D. Moore and W.-S. Hu. AIDS Rev 11, 91-102 (2009).
3. S. F. Johnson and A. Telesnitsky. PLoS Pathog 6, e1001007 (2010).
4. W. Fu, R. J. Gorelick, and A. Rein. J Virol 68, 5013-18 (1994).
5. R. Song, J. Kafaie, L. Yang, and M. Laughrea. J Mol Biol 371, 1084-98 (2007).
6. D. Muriaux, H. De Rocquigny, B.-P. Roques, and J. Paoletti. J Biol Chem 271, 33686-92
(1996).
7. A. Mujeeb, N. B. Ulyanov, S. Georgantis, I. Smirnov, J. Chung, T. G. Parslow, and
T. L. James. Nucl Acids Res 35, 2026-34 (2007).
8. M. A. Urbaneja, M. Wu, J. R. Casas-Finet, and R. L. Karpel. J Mol Biol 318, 749-64
(2002).
9. 9. V. B. Zhurkin, N. B. Ulyanov, A. A. Gorin, and R. L. Jernigan. Proc Natl Acad Sci USA
88, 7046-50 (1991).
Probing the Molecular Determinants of HIV
Alternative Splicing: NMR and Thermodynamic
Studies of UP1/ESS3
Alternative splicing of the human immunodeficiency virus type-1 (HIV-1) genomic
RNA is necessary to produce the complete viral protein complement, and aberrations in the splicing pattern impairs HIV-1 replication (1-2). The cellular protein,
hnRNP A1 (A1), regulates splicing activity at several highly conserved 3’ alternative
splice sites (ssA2, ssA3, and ssA7) by binding 5’-UAG-3’ motifs embedded within
regions containing higher-order RNA structure (3). The biophysical determinants of
A1/splice site recognition remain poorly defined in HIV-1; thus precluding a detailed
understanding of the molecular basis of the splicing pattern. Here, the first 3D structure of an HIV-1 splicing regulatory RNA element, exon splicing silencer 3 (ESS3,
located at ssA7), has been determined by solution 2H-edited NMR spectroscopy and
restrained molecular dynamic simulations (see below). ESS3 adopts a 27-nucleotide
hairpin loop where the first 20 base pairs form an A-helical structure. The helix is
interrupted by a pH sensitive A+C base pair that is conserved across several HIV-1
isolates. The seven nucleotide hairpin contains the high affinity A1 responsive
5’-UAGU-3’ epitope, and a proximal 5’-GAU-3’ motif. The NMR structure shows
that the heptaloop adopts a preformed conformation stabilized by base stacking and
non-canonical interactions. Significantly, the apex of the loop is quasi-symmetric
where UA dinucleotide steps from the 5’-UAGU-3’ and 5’-GAU-3’ motifs stack
on opposite sides of the hairpin - thereby providing a possible A1 binding platform.
To further probe the biophysical determinants of high-affinity A1/ESS3 recognition, the thermodynamic profile (∆G°298K, ∆H°298K, ∆S°298K, and Kd) was measured
via ITC for a C-terminal A1 deletion mutant (UP1). UP1 interacts with ESS3 via
an enthalpically driven process - giving rise to a complex with nanomolar affinity.
The ESS3 binding interface of UP1 was mapped via 15N-1H HSQC titrations. The
results show that the UP1/ESS3 binding interface is broad and involves regions not
116
Blanton S. Tolbert*
Clay Mishler
Jeff Levengood
Prashant Rajan
Department of Chemistry and
Biochemistry, Miami University,
Oxford, OH 45056
*[email protected]
1084
restricted to the beta-pleated sheet. Taken together, we present the first quantitative
study of a host factor/HIV-1 splicing RNA element.
This research has been supported by start up funds from Miami University, College
of Arts and Sciences.
References
1. C. M. Stoltzfus. Advances in Virus Research 7 (1), 1-40 (2009).
2. C. M. Stoltzfus and J. M. Madsen. Current HIV Research 4(1), 43-55 (2006).
3. C. Branlant, et al. Frontiers in Bioscience 14, 2714-2729 (2009).
117
Peter E. Nielsen
Department of Cellular and Molecular
Medicine, Faculty of Health Sciences
& Department of Medicinal Chemistry,
Faculty of Pharmaceutical Sciences,
University of Copenhagen, Denmark
[email protected]
Peptide Nucleic Acid (PNA) – Artificial DNA
with Many Faces
The pseudo-peptidic DNA mimic PNA (peptide nucleic acid) discovered twenty
years ago is still a subject of considerable interest in a range of scientific disciplines
from prebiotic chemistry (origin of life) to drug discovery. With recent examples
form these two very different areas, some of the faces of PNA will be illustrated.
As both as first step towards a minimal chemical mimic of a peptidyl transferase
translation system as well as in a prebiotic context of the evolution of translational
mechanisms we have designed systems that allow PNA directed aminoacyl transfer (translation mimic). The properties and characteristic of these systems will be
presented. Antisense PNA peptide conjugates targeting essential bacterial genes
(such as acpP and ftsZ) have shown promise as potential antibacterials. However,
improved bacterial delivery vehicles (which are compatible with safe and effective
in vivo administration) are still highly warranted. Progress in discovering novel
delivery peptides and in unravelling the mechanism of bacterial uptake of antibacterial PNA peptide conjugates will be presented.
1085
Sequence Specific Targeting Duplex DNA by Artificial
DNA Analogs
Although many natural proteins are capable of targeting duplex DNA (dsDNA)
in a sequence-specific manner, our ability to design de novo proteins with desired
sequence specificity are very limited, at best. That is why the ability of the Peptide Nucleic Acid (PNA) to sequence specifically recognize dsDNA, discovered
20 years ago by Peter Nielsen with colleagues (1), has attracted such considerable
interest. During the past years, the basic understanding of the process of dsDNA
invasion by pyrimidine bis-PNAs and various applications of the phenomenon have
been elucidated. As a result, novel approaches for detecting short (about 20-bplong) signature sites on genomes have been developed in our laboratory. In particular, a PD-loop-based method of pathogen detection has been developed, which
makes it possible to distinguish not only different bacterial species but also to discriminate drug sensitive versus drug resistant strains (2). The next step would be
to extend the approach to human cells, which would open the way for even more
promising applications. Some encouraging data in this direction will be presented.
Other promising applications of bis-PNAs include their use in DNA detection with
solid-state nanopores.
A serious limitation of generic PNAs with respect to targeting duplex DNA consists
in requirement for the homopurine nature of the DNA strand, which is captured by
bis- PNA via the triplex formation. Therefore, various chemical modifications of
the backbone and the bases have been tested in recent years, which could allow to
lift this sequence limitation. Two modifications are of greatest interest: pseudocomplementary PNAs and γ-PNAs. Chiral γ-PNAs with some cytosines replaced
with G-clamp bases, proposed by Danith Ly with colleagues, have proved to be
especially promising. The capability of γ-PNA to invade dsDNA in a sequenceunrestricted manned has been demonstrated. The γ-PNA makes it possible the
exceedingly specific DNA capturing in the duplex form (3). This opens up a very
interesting opportunity of capturing the chromatin in its native form for further
investigation of higher-level chromatin structures.
Over the years, the ability of synthetic DNA analogs to invade dsDNA has been
considered as something purely artificial, without any parallels in vivo. The situation has radically changed recently after discovery of the CRISPR (Clustered
Regulatory Interspaced Short Palindromic Repeat), a bacterial immune system.
It has been shown that in the CRISPR pathway small (about 40-nt-long) singlestranded RNAs recognize the complementary DNA strand within dsDNA via a still
unknown mechanism with the help of special proteins of the CRISPR system (4).
Advancements in the field of dsDNA recognition by artificial DNA analogs not
only provides with new tools for DNA analysis but also can prove to be of great
help in understanding new important biological mechanisms.
References
1.
2.
3.
4.
P. E. Nielsen, M. Egholm, R. H. Berg, and O. Buchardt. Science 254, 1497-500 (1991).
I. Smolina, N. Miller, and M. Frank-Kamenetskii. Artificial DNA 1, 76-82 (2010).
H. Kuhn, B. Sahu, D. H. Ly, and M. D. Frank-Kamenetskii. Artificial DNA 1, 45-53 (2010)
J. E.Garneau et al. Nature 468, 67-71 (2010).
118
Maxim D. Frank-Kamenetskii*
Irina Smolina
Department of Biomedical Engineering,
Boston University.
Boston, MA 02215
*[email protected]
1086
119
Karl Börjesson1
Søren Preus2
Kristine Kilså2
Afaf H. El-Sagheer3,4
Tom Brown3
Bo Albinsson1
L. Marcus Wilhelmsson1*
1Department
of Chemical and Biological
Development and Applications of the Fluorescent
Tricyclic Cytosine Family – The First Nucleic Acid
Base Analogue FRET–pair
The tricyclic cytosine family, tC, tCO, and tCnitro, has been increasingly used in
biophysical and biochemical applications (1). The two fluorescent members of
this family, tC and tCO, both have unique properties among fluorescent base analogues (2, 3). Furthermore, the three tricyclic base analogues all form stable base
pairs with guanine and give minimal perturbations to the native structure of DNA.
Importantly, we have recently utilized tCO as a donor and developed tCnitro as an
acceptor and, thus, established the first nucleic acid base analogue FRET-pair (4).
This pair enables accurate distinction between distance- and orientation-changes in
nucleic acid systems using FRET (see Figure). In combination with the favorable
base pairing properties this will facilitate detailed studies of nucleic acid structures
and structural changes. Moreover, the placement of the FRET-pair inside the base
stack will be a great advantage in studies where other (biomacro)molecules interact
with the nucleic acid.
2Department
Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen, Denmark
3School
Southampton, Highfield, Southampton,
SO17 1BJ, United Kingdom
4Chemistry
Branch, Department of
Science and Mathematics, Faculty of
Petroleum and Mining Engineering,
Suez Canal University, Suez, Egypt
*[email protected]
Figure: Efficiency of energy transfer for the base analogue FRET-pair tCO-tCnitro estimated using
decreases in tCO emission (green circles) and tCO average lifetimes (purple squares) as the two analogues are separated by 2-13 bases in a DNA duplex.
This research is funded by the Swedish Research Council and Olle Engkvist
Byggmästare Foundation.
References
1. L. M. Wilhelmsson. Q Rev Biophys 43(2), 159-183 (2010).
2. P. Sandin, L. M. Wilhelmsson, P. Lincoln, V. E. C. Powers, T. Brown, and B. Albinsson.
Nucleic Acids Res 33, 5019-5025 (2005).
3. P. Sandin, K. Börjesson, H. Li, J. Mårtensson, T. Brown, L. M. Wilhelmsson, and B. Albinsson.
Nucleic Acids Res 36, 157-167 (2008).
4. K. Börjesson, S. Preus, A. H. El-Sagheer, T. Brown, B. Albinsson, L. M. Wilhelmsson. J Am
Chem Soc 131, 4288-4293 (2009).
1087
Two-step Construction of Base-modified Nucleic Acids
for Electrochemical DNA Sensing
Electrochemical DNA sensing strategies, oriented towards development of novel
biosensors and bioassays, use various label-free (based on intrinsic DNA electroactivity) as well as label-based detection principles (1). The latter appear better
suited for sequence-specific DNA analysis when reliable recognition of a specific
nucleotide sequence among others, or when a nucleotide at a specific position (as in
single nucloeotide polymorphism typing), is desirable. Labeled nucleic acids have
usually been prepared via multistep chemical synthesis of oligonucleotides (ON).
We have introduced a novel two-step construction of modified ON, relying in direct
aqueous cross-coupling of halogenated deoxynucleoside triphosphates (dNTP) with
various functional groups and enzymatic incorporation of the nucleotide conjugates
into ON by DNA polymerases (2-6) or by terminal nucleotidyl transferase (TdT)
(7). Compared to the fully chemical ON synthesis, this approach is more versatile,
allowing – once a set of modified dNTPs is available – an easy and instant construction of labeled ON bearing various tags (or their combinations) at specific positions
in the nucloeotide sequence using standard molecular biological tools.
Modified dNTPs, applicable as substrates for DNA polymerases or the TdT enzyme,
were substituted with electrochemically active labels at 7-position of 7-deazapurines
or 5-position of pyrimidines (8). The base-coupled tags include ferrocene (2),
3-nitro- or 3-aminophenyl groups (3) [M(bpy)3]2+ (M = Ru or Os) complexes (4),
tetrathiafulvalene (5) as well as alkylsulfanylphenyl moieties possessing protected
mercaptogroup (6). The correponding nucleotides were successfully incorporated
into ONs, resuling in ON probes with distinct electrochemical properties owing to
redox processes of the labels occuring at diverse potentials. Moreover, in some cases
the electronic communication between the tags and nucleobases made their redox
potentials responding to the conjugate nucleobase and to incorporation into DNA,
thus offering another type of information to gain. We demonstrated applications of
the modified dNTPs and ONs in sensing DNA hybridization, SNP typing and monitoring DNA-protein interactions. Further applications of the proposed strategy in
nanotechnology, ON self-assembly, chemical biology and biosensing is anticipated.
120
Miroslav Fojta1*
Petra Horakova1
Hana Pivonkova1
Ludek Havran1
Peter Sebest1
Jan Spacek1
Petr Orsag1
Milan Vrabel2
Hana Macickova-Cahova2
Lubica Kalachova2
Jan Riedl2
Veronika Raindlova2
Michal Hocek2
1Institute
CZ-61265 Brno, Czech Republic
2Institute
of Organic Chemistry and
Biochemistry, Academy of Sciences of
the Czech Republic, Gilead Sciences &
IOCB Research Center,
This work was supported by the ASCR (Z4 055 0506, Z5 004 0507 and Z5 004
0702) GA ASCR (IAA400040901), GACR (203/09/0317, P301/11/2076) and
MEYS CR (LC06035, LC512).
CZ-16610 Prague 6, Czech Republic
References
1. Labuda, A. M. O. Brett, G. Evtugyn, M. Fojta, M. Mascini, M. Ozsoz, I. Palchetti, E. Palecek,
and J. Wang. Pure Appl Chem 82, 1161-1187 (2010).
2. P. Brazdilova, M. Vrabel, R. Pohl, H. Pivonkova, L. Havran, M. Hocek, and M. Fojta. Chem
Eur J 13, 9527-9533 (2007).
3. H. Cahova, L. Havran, P. Brazdilova, H. Pivonkova, R. Pohl, M. Fojta, and M. Hocek.
Angew Chem Int Ed 47, 2059-2062 (2008).
4. M. Vrabel, P. Horakova, H. Pivonkova, L. Kalachova, H. Cernocka, H. Cahova, R. Pohl,
P. Sebest, L. Havran, M. Hocek, and M. Fojta. Chem Eur J 15, 1144-1154 (2009).
5. J. Riedl, P. Horakova, P. Sebest, R. Pohl, L. Havran, M. Fojta, and M. Hocek. Eur J Org
Chem 3519-3525 (2009).
6. H. Macickova-Cahova, R. Pohl, P. Horakova, L. Havran, J. Spacek, M. Fojta, and M. Hocek.
Chem Eur J in press (2011).
7. P. Horakova, H. Macicková-Cahova, H. Pivonkova, J. Spacek, L. Havran, M. Hocek, and
M. Fojta. Org Biomol Chem 9, 1366-1371 (2011).
8. M. Hocek and M. Fojta. Org Biomol Chem 6, 2233-2241 (2008).
*[email protected]
1088
121
Alexander Rich
Ky Lowenhaupt
Jin-Ah Kwon
Department of Biology,
Massachusetts Institute of Technology,
Cambridge, MA 02139
122
Alpana Ray*
Srijita Dhar
Arvind Shakya
Bimal K. Ray
Department of Veterinary Pathobiology,
University of Missouri,
Columbia, MO 65211
*[email protected]
Roles of Z-DNA Binding Proteins in
Immunity & Infection
Four different Z-DNA binding proteins have been identified and characterized.
Two are normally present in higher eukaryotes. This includes the editing enzyme
double-stranded RNA adenosine deaminase (ADAR1) and the double-stranded
DNA cytoplasmic receptor of the innate immune system which is active in stimulating interferon production. Each of these proteins has Z-DNA binding domains
that are important for certain aspects of their activity.
Two other Z-DNA binding proteins have been identified. One of these is the E3L
protein found in pox viruses. In particular vaccinia E3L has been studied, and it
plays an active role in the pathogenesis of pox virus infections. Another protein
identified in zebra fish and gold fish is a component of the detection system for
alerting the cell that a virus has infected it.
All four of these proteins have a similar protein fold. The possibility of other classes
of Z-DNA binding proteins will be discussed.
Role of Z-DNA Forming Silencer in the Regulation
of Human ADAM-12 Gene Expression
ADAM-12 is a member of novel multifunctional ADAM family of proteins. Expression of ADAM-12 is upregulated in many human cancers, arthritis and cardiac
hypertrophy. The multidomain structure composed of a prodomain, a metalloproteinase, disintegrin-like, epidermal growth factor-like, cysteine-rich and transmembrane domains and a cytoplasmic tail allows ADAM-12 to promote matrix
degradation, cell-cell adhesion and intracellular signaling capacities (1). Basal
expression of ADAM-12 is very low in adult tissues but rises markedly in response
to certain physiological cues, such as during pregnancy in the placenta, during
development in neonatal skeletal muscle and bone and in regenerating muscle
(2, 3). We have identified a highly conserved negative regulatory element (NRE) at
the 5′-UTR of human ADAM-12 gene, which acts as a transcriptional repressor (4).
The NRE contains a stretch of dinucleotide-repeat sequence that is able to adopt
a Z-DNA conformation both in vitro and in vivo. Substitution of the dinucleotiderepeat-element with a non-Z-DNA-forming sequence inhibits NRE function. We
have detected Z-DNA-binding protein activity in several tissues where ADAM-12
expression is low while no such activity was seen in the placenta where ADAM-12
expression is high. These observations suggest that interaction of Z-DNA-binding
proteins with ADAM-12 NRE is critical for transcriptional repression of ADAM-12.
We further show that the Z-DNA forming transcriptional repressor element, by
interacting with Z-DNA-binding proteins, is involved in the maintenance of constitutive low-level expression of human ADAM-12. Together these results provide a
new insight on the regulatory role of Z-DNA-binding proteins which could be used
as therapeutic targets for down-regulation of ADAM-12.
This research has been supported by grants from U.S. Army Medical research and
Material Command and University of Missouri.
References
1. J. M. White. Current Opinion in Cell Biology 15, 598-606 (2003).
2. B. J. Gilpin, F. Loechel, M. G. Mattei, E. Engvall, R. Albrechtsen, and U. M. Wewer.
Journal of Biological Chemistry 273, 157-166 (1998).
3. N. Kawaguchi et al. Journal of Cell Science 116, 3893-3904 (2003).
4. B. K. Ray, S. Dhar, A. Shakya, and A. Ray. Proceedings of the National Academy of
Sciences of the United States of America 108, 103-108 (2011).
1089
Efficient B-Z DNA Conformational Transition
Induced by Ru(II) Complexes
The left-handed Z-DNA plays important role in biological processes, it can regulate
gene expression and involve in DNA processing event such as genetic instability
(1). This may provides target for the prevention and treatment of some human diseases (2). However, Z-DNA is a higher energy conformation than B-former, it is a
transient structure and intrinsically instable (3). Z-DNA is rarely formed without
the help of high salt concentrations or negative supercoiling. It was found in d(CG)n
polymer at high salt concentrations (2.5 M NaCl) (4). Recently, the designs of
molecules that can induce transition from B to Z conformations and stabilize the
Z-DNA have received considerable attention (5).
In the present work, two new
ruthenium
complexes
[Ru(tpy)
(pnt)]2+ (1) and Ru(dmtpy)(pnt)]2+
(2) have been synthesized (tpy =
2,2’:6’,2”-terpyridine, dmtpy =
5,5''-dimethyl-2,2':6',2"-terpyridine,
pnt = 2-(1,10-phenanthrolin-2-yl)naphtho[1,2-e][1,2,4] triazine). Two
ruthenium complexes interact with
DNA by an intercalation binding
mode (6-8). More interesting, they
can efficiently convert the B-form of
DNA into the Z conformation under
physiological salt condition in micromole concentration. This induced
left-handed helix is extraordinary
stable against high temperature. The
mechanism is proposed that complexes insert to DNA base pairs and
distort DNA helix structure. Meanwhile, the Ru(II) center reduces electrostatic repulsion of phosphate backbone in the zigzag path of Z-DNA and locks
the left-handed conformer irreversible to right-handed helix.
Acknowledgements
We gratefully acknowledge the supports of the 973 Program, the National Natural Science Foundation of China, the Program for New Century Excellent Talents
in University, the Guangdong Provincial Natural Science Foundation and Sun
Yat-Sen University.
References
1.
2.
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5.
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A. Rich and S. Zhang. Nat Rev Genet 4, 566-572 (2003).
J. Geng, C. Zhao, J. Ren, and X. Qu. Chem Commun 46, 7187-7189 (2010).
F. M. Pohl and T. M. Jovin. J Mol Biol 67, 375-396 (1972).
I. Doi, G. Tsuji, K. Kawakami, O. Nakagawa, Y. Taniguchi, and S. Sasaki. Chem Eur J 16,
11993-11999 (2010).
6. Y. Dalyan, I. Vardanyan, A. Chavushyan, and G. Balayan. J Biomol Struct Dyn 28, 123-131
(2010).
7. B. Jin, H. M. Lee, and S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010).
8. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim. J Biomol Struct Dyn 28, 421-430 (2010).
Lv-Ying Li
Hui Chao
Li-Ping Weng
Liang-Nian Ji*
123
MOE Key Laboratory of
Bioinorganic and Synthetic
Chemistry, State Key
Laboratory of Optoelectronic Materials
and Technologies, MOE Key Laboratory
of Gene Engineering, School of
Chemistry and Chemical Engineering,
Sun Yat-Sen University, Guangzhou
510275, P. R. China
*[email protected]
1090
124
Katherine Castor
Roxanne Kieltyka
Pablo Englebienne
Nathanael Weill
Johans Fakhoury
Johanna Mancini
Nicole Avakyan
Anthony Mittermaier
Chantal Autexier
Nicolas Moitessier
Hanadi F. Sleiman*
McGill University, Montreal,
Quebec, Canada H3A2K6
*[email protected]
Platinum(II) Phenanthroimidazoles for G-quadruplex
Targeting: The Effect of Structure on Binding
Affinity, Selectivity and Telomerase Inhibition
G-quadruplexes have gained recognition as viable targets for chemotherapeutic
drug design, based on their ability to interfere with cancer cell proliferation (1, 2).
These higher order DNA structures, held together by Hoogsteen hydrogen bonds,
result from the folding of a guanine (G) rich DNA sequence in the presence of
potassium or sodium cations (3). G-quadruplex forming sequences have been identified throughout the human genome in telomeres, promoter regions of oncogenes,
nuclease hypersensitivity regions and untranslated regions of RNA (4, 5). From
this list, one highly investigated G-quadruplex target for drug design is the telomere. Small molecules that promote the folding of the human telomeric sequence,
(TTAGGG)n, into a G-quadruplex structure can result in biologically relevant phenomena, such as the loss of telomere integrity through disruption of the shelterin
complex of proteins (6, 7) or the prevention of telomere elongation by the reverse
transcriptase enzyme telomerase (8). Both of these events have profound effects on
cancer cell proliferation thereby accomplishing one of the main goals of chemotherapy, to halt tumor growth.
We have previously shown that platinum phenanthroimidazole-based binders are
good stabilizers of the intermolecular T4G4T4 G-quadruplex motif (9). These complexes possess optimal geometries for targeting this structure and are substitutionally
inert. Their synthesis is facile and highly modular, lending itself to the ready generation of compound libraries to maximize affinity and selectivity. However, upon initial studies involving the binding of the phenyl [1] and naphthyl [2] derivatives to the
intramolecular motif based on the human telomere, we discovered that a significant
twist within the ligand itself may prevent favorable π-stacking interactions of the
naphthyl [2] ligand with the G-quadruplex. We hypothesized that incorporating an
internal hydrogen bond within our phenanthroimidazole ligands may reduce the twist,
resulting in a planar ligand surface for optimal overlap with the G-quadruplex.
Through the use of molecular modeling, circular dichroism, and fluorescence displacement assays, we have shown that phenanthroimidazole platinum(II) complexes
template and stabilize G-quadruplex forming sequences based on the human telomeric repeat, (TTAGGG)n with the greatest stabilization from the indoyl [3] derivative (G4DC50 = 0.53 µM). However, while the incorporation of the internal hydrogen
bond does increase binding strength to the quadruplex motif, it also tends to reduce
selectivity between quadruplex and duplex DNA. We found that the introduction
of a chloride modification to the phenanthroimidazole core, as well as a sidearm
with protonable sites restores selectivity while also increasing binding strength to
the quadruplex motif (G4DC50 = 0.28 µM for 6). We also show complexes 1-4 are
able to inhibit telomerase through the TRAP-LIG assay, thus verifying that phenanthroimidazole-based platinum(II) complexes can elicit antiproliferative effects and
act as telomere disruption agents.
1091
References
1. A. De Cian, L. Lacroix, C. Douarre, N. Temime-Smaali, C. Trentesaux, J. F. Riou, and
J. L. Mergny. Biochimie 90, 131-155 (2008).
2. Y. Qin and L. H. Hurley. Biochimie 90, 1149-1171 (2008).
3. A. N. Lane, J. B. Chaires, R. D. Gray, and J. O. Trent. Nucleic acids research 36, 5482-5515
(2008).
4. S. Balasubramanian and S. Neidle. Current Opinion in Chemical Biology 13, 345-353
(2009).
5. J. L. Huppert. Biochimie 90, 1140-1148 (2008).
6. H. Tahara, K. Shin-ya, H. Seimiya, H. Yamada, T. Tsuruo, and T. Ide. Oncogene 25,
1955-1966 (2006).
7. R. Rodriguez, S. Müller, J. A. Yeoman, C. Trentesaux, J. F. Riou, and S. Balasubramanian.
Journal of the American Chemical Society 130, 15758-15759 (2008).
8. T. Tauchi, K. Shin-Ya, G. Sashida, M. Sumi, S. Okabe, J. H. Ohyashiki, and K. Ohyashiki.
Oncogene 25, 5719-5725 (2006).
9. R. Kieltyka, J. Fakhoury, N. Moitessier, and H. F. Sleiman. Chemistry-A European Journal
14, 1145-1154 (2008).
G-Quadruplex, Telomere and Telomerase
Telomeres serve as protective caps at the ends of linear eukaryotic chromosomes,
playing a crucial role in cell survival and proliferation. Tandem repeats of the
sequence TTAGGG constitute the human telomeres, with pendent G-rich single
strands of 100–200 nt at the 3’ ends. The propensity of these G-rich overhangs to
form G-quadruplexes, and the inhibitory effects of such structures on the catalytic
activity of the enzyme telomerase, have led to a growing interest in the study of
telomeric G-quadruplexes and the development of specific telomeric quadruplexstabilizing ligands as anticancer drugs. Previously, it has been reported that human
telomeric DNA sequences could adopt in different experimental conditions four
different intramolecular G-quadruplexes each involving three G-tetrad layers,
namely, Na+ solution antiparallel-stranded basket form (1), K+ crystal parallelstranded propeller form (2), K+ solution (3 + 1) Form 1 (3-5), and K+ solution
(3 + 1) Form 2 (6). Here we report novel intramolecular G-quadruplex structures
adopted by canonical (TTAGGG) (7) and variant (CTAGGG, TAGGG) (8, 9) fourrepeat human telomeric sequences in K+ solution, which surprisingly utilize only
two of the three contiguous guanines from successive G-tracts for G-tetrad core
formation. Structural elucidation of these oligonucleotides revealed extensive base
Lim Kah Wai
Phan Anh Tuân
125
School of Physical and Mathematical
Sciences and School of Biological
Sciences, Nanyang Technological
University, Singapore
[email protected]
[email protected]
1092
pairing and stacking interactions in the loops, indicating that the overall G-quadruplex topology of a G-rich sequence is defined not only by maximizing the number
of G-tetrads but also by maximizing all possible interactions in the loops. On the
other hand, promoter G-quadruplex formation represents an alternative approach of
selective gene regulation at the transcriptional level. The promoter for the catalytic
subunit of human telomerase, hTERT, contains many guanine-rich stretches on the
same DNA strand suitable for targeting (10-12). We show here that one particular
G-rich sequence in this region coexists in two G-quadruplex conformations (11),
each of which comprises several robust structural motifs. Recurrence of structural
elements in the structures presented suggests a “cut-and-paste” principle for the
design and prediction of G-quadruplex topologies, for which different elements
could be extracted from one G-quadruplex and inserted into another.
This research was supported by grants from Singapore Biomedical Research Council,
Singapore Ministry of Education, and Nanyang Technological University.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
126
Herry Martadinata
Anh Tuan Phan
School of Physical and Mathematical
Sciences, School of Biological Sciences,
Nanyang Technological University,
Singapore 637371
[email protected]
[email protected]
Y. Wang and D. J. Patel. Structure 1, 263-282 (1993).
G. N. Parkinson, M. P. Lee, and S. Neidle. Nature 417, 876-880 (2002).
Y. Xu, Y. Noguchi, and H. Sugiyama. Bioorg Med Chem 14, 5584-5591 (2006).
A. Ambrus, D. Chen, J. Dai, T. Bialis, R. A. Jones, and D. Yang. Nucleic Acids Res 34,
2723-2735 (2006).
K. N. Luu, A. T. Phan, V. Kuryavyi, L. Lacroix, and D. J. Patel. J Am Chem Soc 128, 9963-9970 (2006).
A. T. Phan, K. N. Luu, and D. J. Patel. Nucleic Acids Res 34, 5715-5719 (2006).
K. W. Lim, S. Amrane, S. Bouaziz, W. Xu, Y. Mu, D. J. Patel, K. N. Luu, and A. T. Phan.
J Am Chem Soc 131, 4301-4309 (2009).
K. W. Lim, P. Alberti, A. Guedin, L. Lacroix, J. F. Riou, N. J. Royle, J. L. Mergny, and
A. T. Phan. Nucleic Acids Res 37, 6239-6248 (2009).
L. Hu, K. W. Lim, S. Bouaziz, and A. T. Phan. J Am Chem Soc 131, 16824-16831 (2009).
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(2009).
K. W. Lim, L. Lacroix, D. J. Yue, J. K. Lim, J. M. Lim, and A. T. Phan. J Am Chem Soc
132, 12331-12342 (2010).
E. Micheli, M. Martufi, S. Cacchione, P. De Santis, and M. Savino. Biophys Chem 153,
43-53 (2010).
Structure of Human Telomeric RNA G-quadruplexes
Telomeres, which are located at the chromosomal ends, act as protective caps that
prevent chromosome loss and degradation. Telomeres had always been thought to
be transcriptionally silent until the recent finding that they could be transcribed into
RNA molecules with lengths ranging from 100 to 9000 nt (1, 2). It has further been
shown that telomeric-repeat-containing RNA (TERRA) perform various cellular
regulatory functions, such as regulation of telomere length, inhibition of telomerase,
telomeric heterochromatin formation, and telomere protection (3-7).
Our structural studies showed that human TERRA sequences formed propeller-type
parallel-stranded RNA G-quadruplexes. We have determined the NMR-based solution structure of a dimeric propeller-type RNA G-quadruplex formed by the 12-nt
human TERRA sequence r(UAGGGUUAGGGU). We also observed the stacking of two such propeller-type G-quadruplex blocks for the 10-nt human TERRA
sequence r(GGGUUAGGGU) and a higher-order G-quadruplex structure for the
9-nt human TERRA sequence r(GGGUUAGGG).
Ribonuclease protection assay was used to investigate the structures formed by long
human TERRA (9,10). We found that G-quadruplexes comprising four and eight
UUAGGG repeats were most resistant to RNase T1 digestion, presumably with
the former adopting an all-parallel-stranded conformation and the latter forming a
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structure with two tandemly stacked G-quadruplex subunits each containing three
G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA
sequences consisting of different stacking interfaces between the two G-quadruplex
subunits, i.e. 5’-5’, 3’-3’, 3’-5’, and 5’-3’, demonstrated stacking feasibility for
all but the 5’-3’ arrangement. A continuous stacking of the loop bases from one
G-quadruplex subunit to the next was observed for the 5’-5’ stacking conformation. Based on the results, we propose a “beads-on-a-string”-like arrangement along
human TERRA (11), whereby each bead is made up of either four or eight UUAGGG
repeats in a one- or two-block G-quadruplex arrangement, respectively (10).
References
1. C. M. Azzalin, P. Reichenbach, L. Khoriauli, E. Giulotto, and J. Lingner. Science 318,
798-801 (2007).
2. S. Schoeftner and M. A. Blasco. Nat Cell Biol 10, 228-236 (2008).
3. B. Horard and E. Gilson. Nat Cell Biol 10, 113-115 (2008).
4. B. Luke and J. Lingner. EMBO J 28, 2503-2510 (2009).
5. S. Feuerhan, N. Iglesias, A. Panza, A. Porro, and J. Linger. FEBS Letter 584, 3812-3818
(2010).
6. Z. Deng, A. E. Campbell, and P. M. Lieberman. Cell Cycle 9, 69-74 (2010).
7. I. L. de Silanes, M. S. d’Alcontres, and M. A. Blasco. Nat Comm 1, 1-9 (2010).
8. H. Martadinata and A.T. Phan. J Am Chem Soc 131, 2570-2578 (2009).
9. A. Randall, J. D. Griffith. J Biol Chem 284, 13980-13986 (2009).
10. H. Martadinata, B. Heddi, K. W. Lim, and A. T. Phan (2011) submitted.
11. H. Yu, D. Miyoshi, and N. Sugimoto. J Am Chem Soc 128, 15461-15468 (2006).
Structural Transition in the Human Telomeric
DNA Sequence d[(TTAGGG)4] upon interaction
With Putative Anticancer Agents, Sanguinarine
And Ellipticine
Even though there have been several studies of interaction between DNA and antcancer agents (1-4), not very much is known about their interactions with the telomeric regions of chromosomes. Guanine rich sequences fold into a non-canonical
structure known as G-quartet in the presence of appropriate salt concentration.
G-quartets stack on one another to form G-quadruplex. G-rich sequences are found
in the telomeric regions of chromosomes and play an important role in chromosome duplication. They are potential targets for anticancer drugs (5). Here we have
reported the structural transition of G-quadruplex induced by two putative anticancer agents, Sanguinarine (SGR) and Ellipticine (ELP), from plant sources.
SGR binds with the human telomeric DNA sequence d[(TTAGGG)4] (H24) in
the presence of K+ with a 2:1 binding stoichiometry possibly via the end stacking
mechanism. Studies based on CD spectroscopy have indicated that at higher concentration (above 20 µM) SGR induces a structural alteration in H24. The structure
of H24 changes from the mixed Type-I conformation to the Na+ -conformation
(6). ELP also binds with H24. At lower concentrations (100 nM) it binds with 3:2
127
Saptaparni Ghosh1*
Suman Kalyan Pradhan1,2
Gautam Basu3
Dipak Dasgupta1*
1Biophysics
Division, Saha Institute
of Nuclear Physics, Block-AF, Sector-I,
Bidhannagar, Kolkata 700064, India
2Section
of Molecular Biology,
UC San Diego, 9500 Gilman,
Dr. La Jolla, CA 92093 (present address)
3Department
of Biophysics, Bose
Institute, P-1/12 CIT Scheme VIIM,
Kolkata 700054, India
* [email protected]
* [email protected]
1094
stoichiometry (ELP : H24) as suggested from Jobs’ Plot based on fluorescence
measurements. But at higher ellipticine concentration (8 µM), it binds with a stoichiometry of 2:1 (ELP : H24) and a relatively lower affinity. CD spectroscopic
studies suggest that at higher concentrations of drug, H24 undergoes a structural
change from the mixed Type-I conformation to the Na+ -conformation. Similar
studies from other group (7) along with our results suggest that G-quadruplex interacting drugs probably induce a structural change at higher drug concentration. It
could be a plausible molecular mechanism of the action of G-quadruplex binding
drugs that interferes with normal biological activities.
References
1. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky, V. F. Pismensky,
V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26, 701-718 (2009).
2. G. Singhal and M. R. Rajeswari. J Biomol Struct Dyn 26, 625-636 (2009).
4. H. M. Lee, B. Jin, S. W. Han, and S. K. Kim, J Biomol Struct Dyn 28, 421-430 (2010).
5. S. Ghosh, P. Majumder, S. K. Pradhan, and D. Dasgupta. Biochim Biophys Acta 1799,
795-809 (2010).
6. S. K. Pradhan, D. Dasgupta, and G. Basu. Biochem Biophys Res Comun (in press).
7. R. D. Gray, J. Li, and J. B. Chaires. J Phys Chem B 113, 2676-2683 (2009).
128
John A. Tainer1,2
1Lawrence
Berkeley National
Laboratory, Berkeley, CA, USA
2Department
of Molecular Biology,
The Skaggs Institute for Chemical
Biology, The Scripps Research Institute,
La Jolla, CA 92037, USA
[email protected]
Dynamic XPD & Mre11-Rad50-Nbs1 DNA
Repair Complexes: Disease-causing Mutations and
Biological Outcomes
DNA ends at breaks, replication forks and telomeres are paradoxically often critically
controlled for repair and integrity by a single trimeric complex of Mre11-Rad50Nbs1 (MRN) dimers. MRN heterohexamer acts in key sensing, signaling, regulation, and effector responses to DNA double-strand breaks including ATM activation,
homologous recombinational repair, microhomology-mediated end joining and, in
some organisms, non-homologous end joining. Our results suggest that this is possible because each MRN subunit can exist in three or more distinct states; thus, the
trimer of MRN dimers can exist in a stunningly large number of states. MRN can
therefore act as a molecular machine that effectively assesses optimal responses and
signals pathway choice based upon its states as set by cell status and the nature of the
DNA damage. Diverse bulky lesions that distort double helical DNA are repaired by
nucleotide excision repair (NER) orchestrated by the TFIIH complex enzymes: the
XPB and XPD helicases and CAK kinase. Combined with mapping of XP patient
mutations, detailed structural analyses provide a framework for integrating and unifying the rich biochemical and cellular information that has accumulated on NER
over nearly forty years of study. This integration resolves puzzles regarding XP helicase functions and indicates that XP helicase positions and activities within TFIIH
detect and verify damage, select damaged strand for incision, and coordinate repair
with transcription and cell cycle through CAK signaling. Overall this concept that
allosteric changes coordinate repair with replication, transcription, and cell cycle by
coupling conformations to kinase activities provides opportunities to develop ligand
master keys to cell biology and improved therapeutic interventions (1-4).
References
1. R. S. Williams, G. Moncalian, J. S. Williams, Y. Yamada, O. Limbo, D. S. Shin,
L. M. Groocock, D. Cahill, C. Hitomi, G. Guenther, D. Moiani, J. P. Carney, P. Russell, and
J. A. Tainer. Cell 135, 97-109 (2008).
2. L. Fan, J. O. Fuss, Q. J. Cheng, A. S. Arvai, M. Hammel, V. A. Roberts, P. K. Cooper, and
J. A. Tainer. Cell 133, 789-800 (2008).
3. R. S. Williams, G. E. Dodson, O. Limbo, Y. Yamada, J. S. Williams, G. Guenther, S. Classen,
J. N. M. Glover, H. Iwasaki, P. Russell, and J. A. Tainer. Cell 139, 87-99 (2009).
4. E. A. Rahal, L. A. Henricksen, Y. Li, R. S. Williams, J. A. Tainer, and K. Dixon. Cell Cycle
9, 2866-2877 (2010).
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Search of Damaged Bases by DNA Repair Enzymes:
Random Walks in One and Three Dimensions
Many DNA-dependent proteins, such as restriction endonucleases, transcription factors, or DNA repair enzymes face the challenge of finding rare specific sequences or
structural elements of DNA in a vast excess of competing non-specific DNA (1, 2).
Proteins may locate targets in DNA using either diffusion in three dimensions of
movement along the DNA contour. The proteins that move along DNA use two
fundamentally different movement mechanisms: directed movement coupled with
ATP hydrolysis and random one-dimensional diffusion driven by Brownian fluctuations. We have developed a new approach to quantitatively analyze the latter
mechanism (3) and used it to study the process of lesion search by several DNA
repair enzymes: Escherichia coli and human uracil DNA glycosylases, 8-oxoguanine-DNA glycosylases, and AP endonucleases. All these enzymes were able to
move along DNA by one-dimensional diffusion over distances up to 80 base pairs,
with the probability of passage decreasing with the increasing travel distance. The
average travel distance was significantly influenced by ionic strength, Mg2+ ions,
and competing non-specific DNA-binding molecules but was barely affected by
crowding agents. Nicks and short gaps in DNA, as well as specifically bound small
ligands, were efficiently overpassed, and DNA strands could be switched during the
search, indicating that the enzymes are able to use hopping, a mode of movement
involving dissociation of the protein–DNA complex and immediate reassociation
of the protein with DNA in the close vicinity of its previous position. Differences
in the behavior of uracil-DNA glycosylase on blunt and hairpin DNA ends was
observed, suggesting that the ends serve neither as points of irreversible loss nor
total reflection of the moving protein. An analytical model has been developed that
describes the one-dimensional random walk of proteins along DNA in terms of
probabilities of the enzyme to move or dissociate at each step.
129
Dmitry O. Zharkov1*
Grigory V. Mechetin1
Boris A. Veytsman2
1SB
RAS Institute of Chemical Biology
and Fundamental Medicine,
Novosibirsk, Russia 630090
2Computational
Materials Science Center,
George Mason University,
Fairfax, VA 22030, USA
*[email protected]
This research has been supported by RFBR (11-04-00807-a) and Presidium of RAS
“Molecular and Cell Biology” program (6.14).
References
1. O. G. Berg, R. B. Winter, and P. H. von Hippel. Biochemistry 27, 6929-6948 (1981).
2. D. O. Zharkov, and A. P. Grollman. Mutat Res 577, 24-54 (2005).
3. V. S. Sidorenko, G. V. Mechetin, G. A. Nevinsky, and D. O. Zharkov. FEBS Lett 582,
410-414 (2008).
130
Kinetic Characterization of the Repair Enzyme OGG1
acting on Triplet Repeat DNA
Triplet repeat sequences, such as CAG/CTG, expand in the human genome to
cause several neurological disorders. Work from other laboratories has implicated
the DNA repair enzyme 8-oxo-7,8-dihydroguanine glycosylase (OGG1) in triplet
repeat expansion (1, 2). OGG1 initiates the base excision repair (BER) process in
mammalian cells by excising the oxidatively damaged nucleobase 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Motivated by the demonstrated involvement of
OGG1 in triplet repeat expansion we initiated a comprehensive kinetic analysis of
the activity of OGG1 on triplet repeat oligonucleotide substrates. These substrates
include canonical CAG/CTG duplexes and also the non-canonical stem-loop hairpins that are proposed to contribute to repeat expansion. We determined the binding affinity, the rate of cleavage of the N-glycosidic bond of 8-oxoG, and the rate
of product release for human OGG1 acting on TNR sequences. Comparison of the
results obtained for the triplet repeats sequences to those obtained for a mixedsequence duplex allowed us to define the contribution of sequence context of the
damage to enzyme activity. We demonstrate that the structure of the DNA substrate
Daniel A. Jarem
Kelly M. Schermerhorn
Nicole R. Wilson
Sarah Delaney*
Bown University,
324 Brook St, Box H,
Providen RI 02912,
*[email protected]
1096
modulates OGG1 activity whereas changes to the sequence composition are well
tolerated. Taken together, these results contribute to our knowledge of the sequence
and structural specificity of OGG1 and, furthermore, define the kinetic parameters
of the event that initiates triplet repeat expansion via BER.
References
1. I. V. Kovtun, Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray. Nature
447, 447-452 (2007).
2. Y. Liu, R. Prasad, W. A. Beard, E. W. Hou, J. K. Horton, C. T. McMurray, and S. H. Wilson.
J Biol Chem 284, 28352-28366 (2009).
131
Reuben S. Harris
Molecular Biology & Biophysics,
University of Minnesota – Twin Cities,
Minneapolis & Saint Paul, MN 55455
[email protected]
Enzyme-catalyzed DNA Cytidine Deamination in
Human Biology
Over the past decade, we have learned that humans have ten DNA cytidine deaminases that catalyze a variety of physiological functions from triggering antibody
gene diversification to providing innate immunity to a broad number of foreign
DNA elements such as viruses and transposons. I will review the biology of these
enzymes and discuss recent biochemical and structural advances that help us to
better appreciate their integral cellular functions (1-4).
This research has been supported by NIH grants R01 GM080437, R01 AI064046,
R03 MH089432 and R21/R33 AI073167.
References
1. S. M. D. Shandilya, M. N. L. Nalam, E. A. Nalivaika, P. J. Gross, J. C. Valesano, K. Shindo,
M. Li, M. Munson, E. Harjes, T. Kouno, H. Matsuo, R. S. Harris, M. Somasundaran, and
C. A. Schiffer. Structure 18, 28-38 (2010).
2. M. D. Stenglein, M. B. Burns, M. Li, J. Lengye, and R. S. Harris. Nature Structural and
Molecular Biology 17, 222-229 (2010).
3. E. W. Refsland, M. D. Stenglein, K. Shindo, J. S. Albin, W. L. Brown, and R. S. Harris.
Nucleic Acids Research 38, 4274-84 (2010).
4. L. S. Shlyakhtenko, A. Y. Lushnikov, M. Li, L. Lackey, R. S. Harris, and Y. L. Lyubchenko.
Journal of Biological Chemistry 286, 3387-95 (2011).
132
Enrico V. Avvedimento
Dept Biologia e Patologia Molecolare
e Cellulare, Facolta’ di Medicina,
Universita’ “Federico II”,
Naples, Italy
The Good and the Bad of DNA Damage DNA
Damage and Epigenetic Marks Drive
Transcription and Repair
Methylation is an epigenetic modification of the DNA, which can silence genes. By
using a sophisticated genetic system, we have induced a break in the double helix of
DNA at a single site in mouse or human cells. This important lesion is repaired by
a precise mechanism: the damaged chromosome pairs and retrieves genetic information from the homologous chromosome partner. The repair of the double strand
break leaves a series of methyl groups on the DNA base C (methylation) in a fraction of the repaired molecules flanking the break and, as consequence, the underlying genetic information may be packed and silenced. DNA methylation represents
a scar marking damaged and repaired DNA. This processi s a strong evolutionary
adaptive response used by the cell to protect the genome. Packed chromatin DNA
is more resistant to damage, and silencing protects the cell from expressing a damaged gene, since the repair may introduce some changes in the code.
During these experiments we found that selective and local DNA damage is an essential step governing transcription of genes. Estrogens are hormones that associated to
1097
the specific receptor penetrate into chromatin-DNA and bind a specific DNA sequence
present in several places in the chromosomes. We have found that the receptor bound
to the hormone induces oxidation and single strand breaks in the DNA, where the
receptor binds. The relaxed DNA chromatin bends and loops out bringing in close
proximity non-contiguous regions of chromosomes. The loops of DNA promote the
association of the receptor-hormone and RNA polymerase II. This enzyme normally
is poised for activation of transcription at 5’ end of genes across the genome. As soon
as the RNA polymerase touches the receptor, the enzyme receives directions and
starts transcription.
We found that DNA untwisting, which renders the strands flexible, is caused by
nicks in the DNA induced by the receptor. These “holes” open an entry site for
RNA polymerase, which now can find the bound receptor and eventually are sealed
rapidly by repair enzymes. At the end of transcription, the DNA is packed back into
condensed chromatin. This mechanism was unexpected, since “nicking” of DNA
is rather a dangerous event, which shall be quickly repaired. However, the price
to pay for estrogen, sex differentiation, and maintenance of the gnetic information
may be high, because repeated nicks and sealing events may result in imperfect
repair and change of the genetic code. This mechanism may explain the occurrence of breast cancer, which is considered strictly dependent on estrogens. Also,
it suggests that transcription is a costly process that deteriorate the whole genomic
machine in the long run.
133
Substrate Interactions of a Human DNA
Alkyltransferase
Human cells contain DNA alkyltransferases that protect genomic integrity under
normal conditions but also defend tumor cells against chemotherapeutic alkylating agents. Here we explore how structural features of the DNA substrate affect
the binding and repair activities of the human O6-alkylguanine-DNA alkyltransferase (AGT).
Manana Melikishvili
Lance Hellman
Michael G. Fried*
To perform its repair functions, AGT partitions between adduct-containing sites
and the large excess of adduct-free genomic DNA. Cooperative binding results in
an all-or-nothing association pattern on short templates. The apparent binding site
size Sapp (mean = 4.39 ± 0.02 bp) oscillates with template length. Oscillations in
cooperativity factor ω have the same frequency but are of opposite phase to Sapp so
the most stable complexes occur at the highest packing densities. At high binding
densities the site size (~4 bp/protein) is much smaller than the contour length (~8 bp)
occupied in crystalline 1:1 complexes. A model in which protein molecules overlap
along the DNA contour has been proposed; this model predicts that optimal proteinprotein contacts will occur when the DNA is torsionally relaxed. Supporting this
prediction, competition assays show that AGT binds relaxed DNAs in preference to
negatively-supercoiled forms and topoisomerase assays show that AGT binding is
accompanied by a small-but-measurable net unwinding (7.14 ± 0.33 deg/protein).
These results predict that AGT will partition in favor of torsionally-relaxed, relatively protein-free DNA structures like those near replication forks.
Department of Molecular and Cellular
AGT must also function at telomeres, where G-rich sequences have the potential to
form quadruplex structures and where methylation at the O6 position of guanines
interferes with quadruplex formation. AGT interactions with small unimolecular
quadruplexes are characterized by reduced binding stoichiometries, affinities and
O6-methyl G repair activities when compared to those with linear DNAs. Thus,
AGT may function best at telomeres when quadruplex formation is inhibited by
helicases or other telomere-binding proteins.
Biochemistry, University of Kentucky,
741 S. Limestone St., Lexington,
KY 40536
*[email protected]
1098
This work was supported by NIH Grant GM070662 to MGF.
Model of AGT-DNA complex with double-stranded DNA. The repeating unit of
this model is one molecule of AGT (colors) plus 4 base-pairs of DNA (black); the
coordinates were derived from PDB file 1T38. Repeating units were juxtaposed
with preservation of B-DNA helical parameters (separation = 3.4 Å, twist = 34.6
degrees) between base-pairs of adjacent units. The result is a 3-start helical array
(left) with important contacts between proteins n and n + 3 (right).
134
Indrani Kar
Rajagopal Chattopadhyaya*
Bose Institute,
P-1/12, C.I.T. Scheme VIIM,
Calcutta 700054, India
*[email protected]
Terminalia chebula Extract Enhances Fenton-reaction
Mediated Nucleoside Damage
Antioxidant and reactive oxygen species scavenging properties of 70% methanolin-water extracts of fruits of the plants Terminalia chebula, Terminalia bellirica and
Emblica officinalis (respectively Harra, Bahera and Amala in Hindi) were reported
by another group in our institute (1). The efficacy of these and other plant extracts
in prolonging lives of mice suffering from laboratory-induced cancer (ascitis)
were tested by the same group, though hitherto unpublished (2). These plants are
already documented in India for their medicinal use for several other diseases (3),
and popular as an ayurvedic tonic called ‘trifala’. We have investigated the effect
of including these extracts in concentrations 30 µg/ml, 60 µg/ml and 90 µg/ml
along with 1 mM deoxynucleoside concentrations taken as standard Fenton-reaction
mediated in vitro damage assays (4-6). For all four deoxynucleosides, it is found
that the fruit extract enhances the amount of Fenton-reaction mediated damage,
amount of enhancement increasing with amount of plant extract added. It is likely
that these plants prove beneficial in cancer due to this enhancement of nucleoside
damage rather than its reduction (as scavengers).
References
1. B. Hazra, R. Sarkar, S. Biswas, and N. Mandal. B M C Complementary and Alternative
Medicine 10, 20 (2010).
2. S. Biswas and N. Mandal. private communication to R.C. (2008).
3. P. Kaushik and A. K. Dhiman. Medicinal Plants and Raw Drugs of India, 255-257, 254-255
and 484-486, Dehradun, Bishen Singh and Mahendra Pal Singh (1999).
4. Y. Luo, E. S. Henle, and S. Linn. J Biol Chem 271, 21167-21176 (1996).
5. E. S. Henle, Y. Luo, W. Gassmann, and S. Linn. J Biol Chem 271, 21177-21186 (1996).
6. Y. Luo, E. Henle, R. Chattopadhyaya, R. Jin, and S. Linn. Methods in Enzymology 234,
51-59 (1994).
1099
Characterization of erp Operator DNA Binding by
Borrelia burgdorferi Protein BpaB
BpaB is an erp Operator DNA binding protein expressed by Borrelia burgdorferi,
the causative agent of Lyme disease. BpaB and 2 other transcription regulatory proteins, EbfC and BpuR proteins control erp transcription levels. Erp proteins bind
host factor H, a regulator of complement activation. By doing so they help protect
the bacterium from the alternative pathway that involves complement-mediated
killing. Transcription and expression of Erp proteins occurs only upon association
with mammalian host blood or tissue, when multiple environmental signals, including temperature induce expression.
BpaB has recently been identified as an erp transcription repressor and competes
for binding to erp Operator DNA with a second B. burgdorferi DNA-binding protein, EbfC (1). Mutagenesis of erp Operator DNA and EMSA binding specificity measurements show that BpaB binds within a 15 bp region just 5’ of the -35
sequence on erp operator 2 DNA. DNA Footprinting experiments also indicate that
BpaB binds initially within the 15 bp erp DNA region after which it then initiates
nonspecific binding to flanking regions. BpaB binds initially at a stoichiometry of
1, to a region of erp Operator DNA that contains the 15 bp binding region. Further
complex formations observed on EMSA gels indicated that 1 protein was being
added in a step wise fashion. Ultracentrifugation equilibrium analysis indicated
that up to 13 proteins can attach to the same 23 bp erp DNA oligomer thereby supporting footprinting results. Similar analysis also showed that 24 proteins are capable of binding to a 50 bp erp dsDNA oligomer. EMSA binding assays indicate a
Kd/dissociation constant of ~0.25 µM for binding to 50 bp erp dsDNA. Experiments
are under way to determine if BpaB binding to erp operator DNA is cooperative.
135
Claire Adams*
Manana Melikishvili
Mike Fried
Brian Stevenson
Department of Microbiology,
Immunology and Molecular
Genetics, University of Kentucky,
College of Medicine, Lexington,
Kentucky 40536-0298
*[email protected]
Reference
1. L. H. Burns, C. A. Adams, S. P. Riley, B. L. Jutras, A. Bowman, A. M. Chenail,
A. E. Cooley, L. A. Haselhorst, A. M. Moore, K. Babb, M. G. Fried, and B. Stevenson.
Nucleic Acids Research, 38, 1-13 (2010).
136
Characterization of Fpg Protein Binding to DNA
Lesions using Pyrrolocytosine Fluorescence
Reactive oxygen species damage DNA to produce a variety of genotoxic lesions. In
particular, 7,8-dihydro-8-oxoguanine (oxoG) is one of the most common pre-mutagenic products of base oxidation in DNA. OxoG is repaired (1) through excision
by formamidopyrimidine-DNA glycosylase (Fpg) in bacteria or 8-oxoguanineDNA glycosylase (OGG1) in eukaryotes. In addition to its glycosylase activity,
Fpg possesses an AP-lyase activity, which catalyzes sequential elimination of the
3’-phosphate (β-elimination) and the 5’-phosphate (δ-elimination) at the nascent
or pre-formed abasic (AP) site, producing a one-nucleotide gap flanked by two
phosphates (2). The glycosidic bond breakage is initiated by a nucleophilic attack
at C1’ by the Pro-1 residue, resulting in a covalent enzyme–DNA Schiff base intermediate, which then rearranges and undergoes elimination. The three-dimensional
structure of E. coli Fpg shows that DNA binding is accompanied with drastic conformational changes, including DNA bending, eversion of oxoG from DNA, and
insertion of Met-73, Arg-108 and Phe-110 residues into DNA (3, 4).
In our previous studies we have used quench-flow technique to show that the
kinetics of processing of oxoG and AP site lesions by Fpg from E. coli involves a
burst and a stationary phases. The data from stopped-flow kinetics with tryptophan
and 2-aminopurine fluorescence detection revealed that both the protein and the
Nikita A. Kuznetsov
Olga S. Fedorova*
Institute of Chemical Biology and
Fundamental Medicine, Novosibirsk
630090, Russia
*[email protected]
C H3
(A)
N
H
(B)
oxoG/pyrC
N
N
O
O
O
O
Fluorescence pyrC
1100
0.1
F/pyrC
AP/pyrC
1
10
100
Time, s
Figure 1: Structure of pyrC (A) and its fluorescence traces in the Fpg catalytic cycles with oxoG-,
AP- and tetrahydrofurane (F) containing DNA-substrates. Stopped-flow fluorescence kinetics demonstrated the multi-step character of lesion recognition (Figure 1B). Thermodynamic parameters of each
recognition step were found by analysis of fluorescence traces at different temperatures.
damaged DNA undergo extensive conformational changes in the course of DNA
substrate binding and cleavage (5, 6). It was concluded that the cleaved product
formation is initially reversible. We have also applied mass spectrometry with electrospray ionization to follow appearance and disappearance of transient covalent
intermediates between Fpg and the substrate DNA (6, 7). The overall rate-limiting
step of the enzymatic reaction seemed to be the release of Fpg from its adduct with
the 4-oxo-2-pentenal remnant of the deoxyribose moiety formed as a result of DNA
strand cleavage by β,δ-elmination.
To gain a deeper insight into mechanism by which Fpg protein recognizes DNA
lesions we have studied the changes both in fluorescence of pyrrolocytosine (pyrC)
(Fig. 1A) and in FRET of Trp/pyrC donor/acceptor pair. PyrC was placed opposite
damaged nucleotides in DNA strand.
Acknowledgements
This work was supported by Grants from the RFBR (10-04-00070), Siberian Division of the Russian Academy of Sciences (28, 48), Russian Ministry of Education
and Sciences (02.740.11.0079, NS-3185.2010.04, MK-1304.2010.04).
References
1. N. A. Timofeyeva, V. V. Koval, D. G. Knorre, D. O. Zharkov, M. K. Saparbaev,
A. A. Ishchenko, and O. S. Fedorova. J Biomol Struct Dyn 26, 637-652 (2009).
2. D. O. Zharkov, G. Shoham, and A. P. Grollman. DNA Repair 2, 839-862 (2003).
3. R. Gilboa, D. O. Zharkov, G. Golan, A. S. Fernandes, S. E. Gerchman, E. Matz,
J. H. Kycia, A. P. Grollman, and G. Shoham. J Biol Chem 277, 19811-19816 (2002).
4. V. V. Koval, N. A. Kuznetsov, D. O. Zharkov, A. A. Ishchenko, K. T. Douglas,
G. A. Nevinsky, and O. S. Fedorova. Nucleic Acids Res 32, 926-935 (2004).
5. N. A. Kuznetsov, V. V. Koval, D. O. Zharkov, Y. N. Vorobjev, G. A. Nevinsky,
K. T. Douglas, and O. S. Fedorova. Biochemistry 46, 424-435 (2007).
6. N. A. Kuznetsov, D. O. Zharkov, V. V. Koval, M. Buckle, and O. S. Fedorova. Biochemistry
48, 11335-11343 (2009).
7. V. V. Koval, N. A. Kuznetsov, A. A. Ishchenko, M. K. Saparbaev, O. S. Fedorova. Mutation
Res 685, 3-10 (2010).
1101
Dynamical Allosterism in the Mechanism of Action
of DNA Mismatch Repair Protein MutS
The multidomain protein T. aquaticus MutS and its prokaryotic and eukaryotic
homologs recognize DNA replication errors and initiate mismatch repair (MMR).
MutS actions are fueled by ATP binding and hydrolysis, which modulate its interactions with DNA and other proteins in the MMR pathway. The DNA binding
and ATPase activities are allosterically coupled over a distance of ~70 Å, and the
molecular mechanism of coupling has not been clarified. To address this problem, all-atom molecular dynamics (MD) simulations of ~150 ns including explicit
solvent were performed on two key complexes—ATP-bound and ATP-free
MutS•DNA(+T bulge). We used principal component analysis (PCA) in fluctuation space to assess ATP ligand-induced changes in MutS structure and dynamics.
The MD calculated ensembles of thermally accessible structures showed markedly
small differences between the two complexes. However, analysis of the covariance
of dynamical fluctuations revealed a number of potentially significant inter-residue
and inter-domain couplings. Moreover, PCA analysis revealed clusters of correlated atomic fluctuations linking the DNA and nucleotide binding sites, especially
in the ATP-bound MutS•DNA(+T) complex. These results support the idea that
allosterism between the nucleotide and DNA binding sites in MutS can occur via
ligand-induced changes in motion, i.e., dynamical allosterism.
137
Susan N. Pieniazek*
Manju M. Hingorani
D. L. Beveridge
*[email protected]
This work was supported by the NSF (MCB-1022203 to M.M.H) and the NIH
(GM-076490 to D.L.B). S.N.P. was supported by a NIH NRSA Postdoctoral
Fellowship (F32-GM-87101).
138
Kinetics of the Initial Step of Base Excision
Repair in Trinucleotide Repeats
Dynamic mutations arising from trinucleotide repeat expansion cause a number of
grave hereditary neurodegenerative diseases and possibly contribute to neurodegeneration during normal aging. In the case of terminally differentiated neurons
such expansion can be caused by DNA base excision repair (BER). This hypothesis
was confirmed by experiments in which expansion of CAG triplets characteristic
of Huntington disease was initiated in the course of normal repair of the damaged
base 8-oxoguanine (8-oxoG) (1). Yet it is unclear how such a DNA base lesion and
its repair might cause the expansion. Besides, there is extremely little information
is available on efficiency of BER enzyme and their substrate specificity when DNA
substrate contains trinucleotide repeats. Thus it would be very useful to gain kinetic
parameters of reaction involving DNA glycosylase, the main participants of BER,
and such DNA substrates. First of all, we were interested in how the position of
8-oxoG in a substrate containing a run of CAG repeats can influence on its excision rate by human 8-oxoguanine glycosylase (OGG1). For this purpose we have
determined rate constants of 8-oxoG excision (k2) by OGG1 enzyme from CAGsubstrate depending on the position of the damaged triplet. Also for these substrates
we have determined the rate constants of DNA product release from a complex with
OGG1 (k3). We have then compared the rates of 8-oxoG between purified enzyme
and nuclear extract of the human hepatoma cell line (LICH). We have observed
that k2 of purified OGG1 and the rate of excision in the extracts exhibited a similar
dependence on the position of the lesion in the substrate. Therefore we suggest that
the reaction rate in the cell extract is limited not by the product release stage, as in
the case of pure protein, but by the catalytic step of reaction, as shown previously
for the activity of OGG1 in the presence of AP-endonuclease APEX1 (2).
A. V. Endutkin*
A. G. Derevyanko
D. O. Zharkov
SB RAS Institute of Chemical Biology
and Fundamental Medicine,
Novosibirsk, Russia
*[email protected]
1102
Figure 1: Dependence of the excision rate constant (k2) of oxoG from CAG substrates by purified
OGG1 (black bars) and the excision rate (v) of oxoG in LICH extracts (gray bars) on the position of
oxoG in the substrate containing seven CAG trinucleotides. The number in the name of the substrate
corresponds to the damaged trinucleotide.
In the case of substrates containing 8-oxoG in CGG repeats, typical for fragile
X syndrome, the kinetic parameters were comparable with those for the CAGsubstrates. Given that potentially not only repair of 8-oxoG can lead to expansion
of triplets, we have obtained the values of Michaelis constant and catalytic constants for the reaction of uracil excision by human uracil-DNA glycosylase (UNG)
from CAG-run substrates containing a single uracil, the product of cytosine deamination. Remarkably, despite the strict dependence of both constants on the position
of the damaged triplet in the substrate, the specificity constant (kcat/KM) did not
significantly change.
This work was supported in part by Russian Foundation for Basic Research (10-0491058-PICS_a).
References
447, 447-452 (2007).
2. V. S. Sidorenko, G. A. Nevinsky, and D. O. Zharkov. DNA Repair 6(3), 317-328 (2007).
1103
Post-synthetic Generation of Fapy-dG Lesion within
Oligodeoxynucleotides: Differential Anomeric Impacts
on DNA Duplex Properties
Cellular DNA is a target for oxidative stress arising from exposure to environmental
and endogenous sources that account for thousands of oxidatively damaged bases
per day. The prominent oxidative lesion, imidazole ring opened N6-(2-Deoxya,b-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine
(Fapy-dG) is one of the most common lesions of this type (1). Its formation has
been reported under various experimental conditions, and its accumulation is associated with progression of many age-related diseases and cancer (2, 3). Structural
and thermodynamic studies of this lesion require development of universal and
reliable synthetic strategy to incorporate cognate Fapy-dG site-specifically within
any oligodeoxynucleotide sequence. We elaborated the scheme that consists of a
two-step post-synthetic treatment of the modified nitropyrimidine oligonucleotide
and does not require purification of the intermediate product (4). This approach
has been successfully applied to the preparation of isotopically labeled Fapy-dG in
DNA for subsequent solution state NMR studies. We demonstrated that the lesion
exists in DNA in a several slowly interconverting anomeric and rotameric forms
(4). The anomeric forms of the Fapy-dG containing synthetic oligonucleotides have
been successfully separated using ion-exchange HPLC. The resultant anomeric
duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and β-anomers.
References
1. M. Dizdaroglu, G. Kirkali, and P. Jaruga. Free Radical Biol Med 45, 1610-1621 (2008).
2. J. Q. Wang, W. R. Markesbery, and M. A. Lovell. J Neurochem 96, 825-832 (2006).
3. D. C. Malins, N. L. Polissar, and S. J. Gunselman. Proc Natl Acad Sci USA 93, 2557-2563
(1996).
4. M. Lukin, C. A. S. A. Minetti, D. P. Remeta, S. Attaluri, F. Johnson, K. J. Breslauer, and
C. de los Santos. Nucl Acids Res 2011 (in press).
139
Mark Lukin1,*
Conceição A. S. A. Minetti2
David P. Remeta2
Sivaprasad Attaluri1
Francis Johnson1
Kenneth J. Breslauer2
Carlos de los Santos1
1Department
of Pharmacological
Sciences, School of Medicine, Stony
Brook University, Stony Brook,
New York 11794-8651
2Department
of Chemistry and Chemical
Biology, Rutgers – The State University
of New Jersey, Piscataway, New Jersey
08854, USA
*[email protected]
140
Base Excision Repair of Trinucleotide Repeat DNA
The expansion of trinucleotide repeat DNA leads to a multitude of neurodegenerative
disorders, where CAG/CTG expansion leads to the onset of Huntington’s Disease
(1). Interestingly, in mouse model studies, 8-oxo-7,8-dihydroguanine glycosylase
(OGG1) has been implicated in the expansion mechanism of CAG/CTG repeats in
the huntingtin gene (2). OGG1, a glycosylase enzyme, is responsible for initiating
the base excision repair (BER) pathway by removal of an 8-oxo-7,8-dihydroguanine (8-oxoG) lesion. AP endonuclease (APE1) processes the abasic site created
by OGG1 to cleave the DNA backbone and further the BER pathway; polymerase
β and DNA ligase then complete the repair event. In vitro, it has been shown that
DNA containing CAG or CTG trinucleotide repeats has the ability to form non-B
type conformations; more specifically they have been shown to adopt hairpin conformations (3). Recent work done in our laboratory has shown that the guanine located
in the loop region of these DNA hairpins is highly susceptible to oxidation (4). Given
these observations, the aim of this work is to characterize the BER pathway on trinucleotide repeat hairpin and duplex DNA constructs. Here we have examined the activity of
OGG1 and APE1 on trinucleotide repeat substrates containing an 8-oxoG lesion.
References
1. Huntington Disease Collaborative Research Group, Cell 72, 971-983, (1993).
447, 447-452, (2007).
3. A. M. Gacy, G. Goellner, N. Juranic, S. Macura, C. T. McMurray. Cell 81, 533-540 (1995).
4. D. Jarem, N. Wilson, and S. Delaney. Biochemistry 48, 6655-6663, (2009).
Kelly Schermerhorn*
Sarah Delaney
Brown University,
Providence, RI 02912
*[email protected]
1104
141
Khiem V. Nguyen
Cynthia J. Burrows*
University of Utah, Salt Lake City,
UT 84112
*[email protected]
A Prebiotic Role for Oxidized Purine
Nucleotides as Mimics of Flavins in
Cyclobutane Pyrimidine Dimer Repair
The “RNA World” hypothesis suggests that ancient life evolved from the catalytic
chemistry of RNA oligomers. Though RNA has been found to catalyze a wide
range of chemical reactions including ligation, hydrolysis, and replication (1), our
understanding of RNA catalysis of electron transfer processes is still limited. Today,
protein enzymes require functional molecules such as flavin, nicotinamide or pterins to promote redox reactions. How can RNA catalyze redox reactions while these
complex molecules were not likely present in the primitive world?
We hypothesize that oxidized purine nucleotides may predate flavin as redox
cofactors that assisted RNA in redox reactions. Using a model of electron transfer
through double-stranded oligonucleotides, we investigated redox-active purines
as mimics of flavin in repairing the photochemical lesion cyclobutane pyrimidine
dimer (CPD). The repair efficiency was studied in duplexes with different positions
of oxidized purines relative to a thymine dimer. We found that photorepair of the
CPD was dependent upon the 5’ vs. 3’ orientation as well as the strand location
and base pair context. Compared with previous studies (2-4), this data is consistent
with a reductive repair mechanism in which thymine dimer repair is triggered by
accepting one electron from the photoexcited state of the purine. Similarly, oxidized purines were able to photorepair the uracil CPD in both a DNA/RNA hybrid
duplex and a RNA/RNA duplex, though the efficiency was less than the repair of
thymine dimer in the DNA/DNA duplex.
The similarity of purine and flavin chemistries suggests that nature may have
adopted these simple nucleotide derivatives as redox cofactors prior to the evolution of modern enzyme cofactors.
References
1.
2.
3.
4.
142
Craig J. Yennie*
Sarah Delaney
Brown University,
*[email protected]
X. Chen, N. Li, and A.D. Ellington. Chem Biodiversity 4, 633-655 (2007).
D. J-F. Chinnapen and D. Sen. Proc Natl Acad Sci USA 101, 65-69 (2004).
M. R. Holman, T. Ito, and S. E. Rokita. J Am Chem Soc 129, 6-7 (2006).
M. A. O’Neill and J. K. Barton. Proc Natl Acad Sci USA 99, 16543-16550 (2002).
Incorporation of Oxidized Guanine
Nucleotides into DNA
An oxidized product of guanine that is detected following exposure of DNA to one
of several oxidizing agents is 8-oxo-7,8-dihydro-guanine (8-oxoGua). 8-oxoGua is
present in genomic DNA at steady-state levels of ~1-10 per 107 bases and is a biomarker for several diseases (1). In order to prevent the genetic effects caused by the
presence of 8-oxoGua, cells are equipped with several repair enzymes. Mammalian
cells have a glycosylase/AP lyase, OGG1 (MutM in E. coli), that excise 8-oxoGua
from duplex DNA when it is paired with cytosine. A second glycosylase, MYH
(MutY in E. coli), removes adenine from an OG:A mispair. Cells are also equipped
with a phosphatase, MTH1 (MutT in E. coli), that can convert 8-oxodGuoTP to
8-oxodGuoMP; this action removes 8-oxodGuoTP from the nucleotide pool in
order to prevent incorporation during DNA replication. The importance of removing 8-oxodGuoTP from the nucleotide pool is underscored by the fact that E. coli
lacking MutT, have a 100-10,000-fold higher mutation rate compared to wild type
E. coli (2, 3). This dramatic increase in mutation rate in the absence of MutT indicates
that the nucleotide pool represents a biologically significant source of 8-oxoGua.
Indeed, the incorporation of 8-oxodGuoTP by several bacterial and mammalian
1105
polymerases has been examined (4, 5). Not only has 8-oxoGua been shown to be
mutagenic when replicated in DNA, it has also been shown to be chemically labile
towards further oxidation (6, 7). Several oxidized 8-oxoGua lesions have been
identified such as spiroiminodihydantoin and guanidinohydantoin. Many of these
oxidized lesions have been shown to be potently toxic and mutagenic when replicated in vitro and in vivo. The aim of this investigation is to determine the extent
to which the nucleotide pool serves as a source of these oxidized lesions and their
ability to be substrates for DNA polymerases.
References
1. H. J. Helbock, K.B. Beckman, M. K. Shigenaga, P. Walter, A. A. Woodall, H. C. Yeo, and
B. N. Ames. Proc Natl Acad Sci USA, 95, 288-293 (1998).
2. H. Maki and M. Sekiguchi. Nature, 355, 273-275 (1992).
3. M. L. Michaels, C. Cruz, A. P. Grollmam, and J. H. Miller. Proc Natl Acad Sci USA 89,
7022-7025 (1992).
4. H. J. Einolf, N. Schnetz-Boutaud, and P. F. Geungerich. Biochemistry 37, 13300-13312 (1998).
5. J. W. Hanes, D. M. Thal, and K. A. Johnson. J Biol Chem 281, 36241-36248 (2006).
6. W. L. Neeley and J. M. Essigmann. Chem Res Toxicol 19, 491-505 (2006).
7. J. C. Niles, J. S. Wishnok, and S. R. Tannenbaum. Nitric Oxide 14, 109-121(2006).
143
Investigation of Trinucleotide Repeats in Nucleosome
Core Particles
Both prokaryotic and eukaryotic genomes have incredible potential to grow, shrink,
and change. One such mechanism of change is through the expansion of trinucleotide
repeats (TNRs). TNRs occur throughout the genome and their expansion can affect
varied cellular processes such as gene expression, mRNA processing, and protein
folding. In particular, TNR expansion has been linked to several neurodegenerative diseases, such as Huntington’s Disease, Myotonic Dystrophy (both caused by
expansion of CAG•CTG repeats), and Fragile X Mental Retardation (caused by
expansion of CGG•CCG repeats) (1). These expansions are ascribed to formation
of non-canonical structures in the repeat region, leading to DNA polymerase slippage during replication. DNA repair also appears to play a role in expansion (2).
TNR structure and expansion has been studied both in vivo and in vitro. Work
with both oligonucleotides and TNRs incorporated into plasmids indicates the
potential to form several stable non-canonical secondary structures (e.g. hairpins)
(1). However, genomic DNA is packaged into chromatin. Previous studies have
shown that disease-length, expanded CAG•CTG repeats readily incorporate into
the basic unit of chromatin packing, the nucleosome core particle (NCP), composed of 146 base pairs of DNA wrapped around a core of eight histone proteins
(3). However, long CGG•CCG repeats exclude NCP formation. Here, we seek
to investigate the properties associated with shorter repeats (those that have not
expanded) incorporated into NCPs. To assess the global interactions involved
in interaction between the TNRs and the histone core, we performed competitive nucleosome exchanges to determine the efficiency with which various TNR
sequences incorporate into NCPs. It is not yet known what structure TNRs will
adopt in this context but understanding that structure is important to understanding how TNRs expand in the genome.
This research has been supported by National Institute of Environmental Health
Sciences (NIEHS) award ES019296. C.B.V. is supported by a NDSEG fellowship.
References
1. R. R. Sinden. Am J Hum Genet 64, 346-353 (1999).
2. C. T. McMurray. Nat Rev Genet 11, 786-799 (2010).
3. Y.-H. Wang. Cancer Lett 232, 70-78 (2006).
Catherine Burke Volle
Sarah Delaney*
Biology and Biochemistry and
Brown University,
*[email protected]
1106
144
Amalia Ávila-Figueroa
Douglas Cattie
Sarah Delaney*
Brown University,
Providence, RI 02906, USA
*[email protected]
Structure and Reactivity of Triplet Repeat Sequences
Associated with Neurodegenerative Disorders
Expansion of triplet repeat DNA is implicated in several neurodegenerative disorders,
including Huntington’s Disease (HD). Expansion of the triplet repeat polymorphism is
strongly dependent on repeat length, with longer repeats being more likely to expand
across generations. While the mechanism of expansion for HD remains unknown,
formation of non-B DNA structures by a repetitive (CAG)/(CTG) motif has been proposed to facilitate expansion. Persistence of the aforementioned non-B DNA structures
during events such as DNA replication and/or repair could influence the likelihood of
expansion to occur (1-5).
We studied the structural properties as well as the reactivity of a (CAG)10 non-B
hairpin construct and a series of complementary (CTG)n strands of variable length
and sequence composition. A molecular beacon methodology was employed to
monitor the behavior of the (CAG)10 hairpin by labeling with a 5’-fluorophore and
a 3’-quencher functionalities (6). Modifications in the structure and base composition for the series of complementary hairpins have a profound effect on the stability
of the (CAG)10 hairpin as seen by fluorescence and UV-Vis optical melting assays.
Time-resolved electrophoretic assays also revealed that structural differences can
alter the kinetics of hairpin-duplex conversion. These studies show that structure
and base composition at distinct sites within these stem-loop DNA conformations
influences molecular recognition between hairpins and modulates conversion to
duplex.
This work was supported by National Institute of Environmental Health Sciences
(R01ES019296). A.A.F. was supported by a National Science Foundation Graduate Research Fellowship.
References
1.
2.
3.
4.
5.
6.
145
Daniel A. Jarem*
Nicole R. Wilson
Kelly M. Schermerhorn
Sarah Delaney
324 Brook Street, Box H,
Brown University, Providence,
RI 02912
*[email protected]
S. M. Mirkin. Nature 447, 932-940 (2007).
I. V. Kovtun and C. T. McMurray. DNA Repair 6, 517-529 (2007).
C. T. McMurray. Nat Rev Genet 11, 786-799 (2010).
A. L. Castel, J. D. Cleary, and C. E. Pearson. Nat Rev Mol Cell Bio 11, 165-170 (2010).
J. Zhao, A. Bacolla, G. Wang, and K. M. Vasquez. Cell Mol Life Sci 67, 43-62 (2010).
A. A. Figueroa and S. Delaney. J Biol Chem 285, 14648-14657 (2010).
Substrate Binding, Catalytic Activity and Product
Release by Human 8-Oxo-7,8-dihydroguanine
Glycosylase (hOGG1) are Modulated by the
Structural Context of 8-Oxo-7,8-dihydroguanine in a
CAG Trinucleotide Repeat Sequence
The DNA repair protein human 8-oxo-7,8-dihydroguanine glycosylase (hOGG1)
initiates base excision repair (BER) in mammalian cells by removing the oxidized guanine base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA (1). Interestingly, OGG1 has been implicated in expansion of the trinucleotide repeat (TNR)
sequence CAG/CTG and this expansion represents the molecular basis of several
neurodegenerative disorders (2). Furthermore, in addition to the duplex conformation, CAG/CTG sequences have been shown to adopt non-B conformations such
as stem-loop hairpins (3). Via a long-patch BER (LP-BER) pathway it has been
shown in vitro that the ability of these repeat regions to form hairpins during BER
can result in a flap that is refractory to flap endonuclease 1 (FEN1). Expansion of
the TNR DNA is a consequence of the persistence of a trapped hairpin that can be
ligated into the duplex (4, 5).
We reported previously that hairpins that may form during this BER expansion
mechanism contain hot spots for oxidative damage when treated with peroxynitrite
(ONOO−) (6). Therefore, TNR substrates containing site-specifically incorporated
8-oxoG were then synthesized to define the kinetic parameters of hOGG1 activity on duplex and hairpin structures. In this work we first used an electrophoretic
mobility shift assay to determine the KD for hOGG1 binding to hairpin and duplex
substrates in which the position of the 8-oxoG was varied. Second, the rate at which
hOGG1 catalyzes excision of 8-oxoG was quantified by performing single-turnover
experiments. Third, multiple-turnover experiments were used to define the rate of
product release for hOGG1 acting on the hairpin and duplex substrates. As a benchmark for hOGG1 activity, the data obtained for the TNR substrates were compared
to those obtained for a mixed-sequence duplex.
We find that hOGG1 binding, activity and product release for TNR duplexes is
indistinguishable from the mixed-sequence control, indicating the BER can be initiated by hOGG1 just as efficiently in TNR regions of DNA as elsewhere in the
genome. Interestingly, the activity of hOGG1 is modulated by the structure of the
DNA substrate. For the hairpin substrates, hOGG1 has a reduced affinity, excises
8-oxoG at a slower rate, and releases the DNA product faster as compared to the
corresponding TNR duplex substrates.
References
1. S. David, V. O’Shea, and S. Kundu. Nature 447, 941-950 (2007).
2. A. López Castel, J. D. Cleary, and C. E. Pearson. Nat Rev Mol Cell Biol 11, 165-170
(2010).
3. A. Gacy, G. Goellner, N. Juranić, S. Macura, and C. McMurray. Cell 81, 533-540 (1995).
447, 447-452 (2007).
5. Y. Liu, R. Prasad, W. Beard, E. Hou, J. Horton, C. McMurray, and S. Wilson. J Biol Chem
284, 28352-28366 (2009).
6. D. Jarem, N. Wilson, and S. Delaney. Biochemistry 48, 6655-6663 (2009).
1107
1108
Functional Nuclear Architecture Studied by
High-resolution Microscopy
146
Thomas Cremer1*
Marion Cremer1
Yolanda Markaki1
Barbara Hübner1
Katharina Austen1
Daniel Smets1
Jacques Rouquette1
Manuel Gunkel2
Sven Beichmanis2
Rainer Kaufmann2
Heinrich Leonhardt1
Lothar Schermelleh1
Stanislav Fakan1
Christoph Cremer2**
1LMU
Biocenter, Department of
Biology II, Ludwig Maximilians
University (LMU),
82152 Martinsried, Germany
2Kirchhoff-Institute
for Physics and
BioQuant Center, University of
Heidelberg, 69120 Heidelberg, Germany
*[email protected]
**[email protected]
Recent developments of 4D (space-time) live cell microscopy (1), 3D light optical nanoscopy with resolution beyond the classical Abbe limit (2-6) and advanced
electron microscopic approaches (7, 8) complement each other in studies of the
functional nuclear architecture (for review see 9). The results of these studies
support the chromosome territory-interchromatin compartment model of nuclear
architecture (10-13).
Chromosome territories (CTs) occupy distinct regions of the nuclear space. They
are built up from a network of interconnected chromatin domains with a DNAcontent in the order of 1 Mb, termed ~1Mb CDs. The structure of ~1Mb CDs has
not yet been resolved, but we argue that each ~1Mb CD is built up from a series of
more or less compacted chromatin loop domains with a DNA content in the order
of 100 kb, termed ~100 kb CDs. Moreover, several ~1Mb CDs can cluster together
and form larger CDs. During S-phase replicating ~1Mb CDs act as replication
foci. Direct contacts between neighboring ~1Mb CDs provide ample opportunities
for interactions in cis (within a given CT) or trans (between neighboring CTs),
including the formation of intra- and interchromosomal rearrangements. Beyond
the nucleosome and the 10 nm thick, ‘beads on a string’ chromatin fiber, we lack
indisputable, quantitative evidence for possible hierarchies of higher order chromatin fibers and loops. In particular, we do not know the extent of quantitative
variation possible with regard to the size distributions and extent of intermingling
of fibers and loops at different hierarchical levels in different cell types of different species exposed to different internal or external stimuli. To which extent
interactions between giant chromatin loops expanding from widely separated CTs
are involved in the relocalization of genes to specialized subnuclear compartements (“gene kissing”), is still controversially discussed. Based on our current
knowledge, we expect that such events are mostly driven by locally constrained
Brownian movements.
The perichromatin region (PR) represents a 100-200 nm thick layer of decondensed chromatin, located at the periphery of CDs. It constitutes the nuclear
compartment for transcription, splicing, DNA-replication and possibly also
DNA-repair (6 - 8, 9). The PR is in direct contact with the interchromatincompartment (IC), which forms an interconnected 3D system of IC-channels
(width <400 nm) and IC-lacunas (width >400 nm). It starts/ends with small
channels at the nuclear pores and expands both between CTs and throughout the
interior of CTs. Accordingly the 3D organization of a CT can be compared with
a sponge built up from a 3D chromatin network permeated by the IC (Figure 1)
(6, 13). It should be noted that constrained Brownian movements of CDs in
the nucleus of living cells (1) result in continuous changes of the width of IC
channels providing dynamic opportunities for normal or pathological interactions. The interior of IC lacunas is free of chromatin and harbors nuclear bodies
and splicing speckles (6, 7). This structural organization allows direct functional
interactions between the IC and the PR, such as the delivery of splicing components from splicing speckles to sites of co-transcriptional splicing (Figure 1).
Although individual proteins may be able to diffuse through the whole nuclear
space, including heterochromatic domains, it still seems to be a valid possibility
that the IC and the PR represent specific nuclear compartments for the intranuclear traffic of protein complexes involved in nuclear functions and the export
ribonucleoprotein complexes.
1109
Figure 1: This 2D cartoon shows a simplified scheme of the more complex 3D nuclear architecture. CDs are not drawn to scale (adapted from Figure 4 in
T. Cremer and M. Cremer, 2010).
References
1. H. Strickfaden. Nucleus 1, 284-297 (2010).
2. L. Schermelleh, et al. Science 320, 1332-1336 (2008).
3. M. Gunkel, et al. Biotechnol J 4, 927-938 (2009).
4. D. Baddeley, et al. Nucleic Acids Res 38, e8 1-11 (2010).
5. C. Cremer, et al. In: Nanoscopy and Multidimensional Optical Fluorescence Microscopy
(A. Diaspro, Edit.) Taylor & Francis, pp. 3/1 - 3/35 (2010).
6. Y. Markaki, et al. Cold Spring Harb Sym Quant Biol 75, in press (2011).
7. J. Rouquette, et al. Chromosome Res 17, 801-810 (2009).
8. J. Niedojadlo, et al. Exp Cell Res 317 433-444 (2011).
9. J. Rouquette, et al. Int Rev Cell Mol Biol 282, 1-90 (2010).
10. T. Cremer, et al. Crit Rev Eukar Gene 10, 179-212 (2000).
11. T. Cremer, and C. Cremer. Nat Rev Genet 2, 292-301 (2001).
12. C. Lanctôt, et al. Nat Rev Genet 8, 104-115 (2007).
13. T. Cremer and M. Cremer Cold Spring Harb Perspect Biol 2 a003889 (2010).
147
How is a Long Strand of DNA Compacted
into a Chromosome?
Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division (1). A long strand of
DNA is wrapped around the core histone and forms a nucleosome. The nucleosome
has long been assumed to be folded into 30-nm chromatin fibers (Figure A) (1).
However, it remains unclear how the nucleosome or 30-nm chromatin fiber is organized into mitotic chromosomes, although it is well known that condensins and
topoisomerase IIα are implicated in this process (2-4). When we observed frozen
hydrated (vitrified) human mitotic cells using cryo-electron microscopy, which
enables direct high-resolution imaging of the cellular structures in a close-to-native state, we did not find any higher order structures, or even 30-nm chromatin
Kazuhiro Maeshima
Biological Macromolecules Laboratory
Structural Biology Center,
National Institute of Genetics,
Mishima, Japan
[email protected]
1110
fibers, but just a uniform disordered texture of the chromosome (Figure B) (5, 6).
To further investigate the structure of mitotic chromosome, we performed small
angle x-ray scattering or SAXS, which can detect regular internal structures in noncrystalline materials in solution. Mitotic chromosomes purified from HeLa cells were
exposed to the synchrotron radiation beam at SPring-8 in Japan. Again, the results
were striking: no structural peaks larger than 11-nm were detected. Therefore, we
propose that the nucleosome fibers exist in a highly disordered, interdigitated state
like a “polymer melt” that undergoes local dynamic movement (Figure B) (5, 6).
We also postulate that a similar state exists in active interphase nuclei, resulting in
several advantages in the transcription and DNA replication processes (6, 7). The
possible genomic organization in the mitotic chromosomes and nuclei is discussed.
References
1. B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. Molecular Biology of
the Cell, Garland, NY. (2007).
2. J. R. Swedlow and T. Hirano. Mol Cell 11, 557-569 (2003).
3. S. Ohta, L. Wood, J. C. Bukowski-Wills, J. Rappsilber, and W. C. Earnshaw. Curr Opin Cell
Biol 23, 114-121 (2010).
4. K. Maeshima, S. Hihara, and H. Takata. Cold Spring Harb Symp Quant Biol 75 (2011),
in press.
5. M. Eltsov, K. M. Maclellan, K. Maeshima, A. S. Frangakis, and J. Dubochet. Proc Natl Acad
Sci USA 105, 19732-19737 (2008).
6. K. Maeshima, S. Hihara, and M. Eltsov. Curr Opin Cell Biol 22, 291-297 (2010).
7. E. Fussner, R. W. Ching, and D. P. Bazett-Jones. Trends Biochem Sci 36, 1-6 (2011).
1111
A Genomic Code for Nucleosome Positioning from
Archaebacteria to Man
Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA
from interacting with most DNA binding proteins. We have discovered that genomes
care where their nucleosomes are located on average, and that genomes manifest
this care by encoding an additional layer of genetic information, superimposed on
top of other kinds of regulatory and coding information that were previously recognized. The physical basis of the nucleosome DNA sequences preferences lies in
the sequence-dependent mechanics of DNA itself. We have an increasingly good
ability to read this nucleosome positioning information and predict the in vivo locations of nucleosomes. Our results suggest that genomes utilize this nucleosome
positioning code to facilitate specific chromosome functions, including to define
the next higher level of chromosome structure itself. Comparisons across diverse
organisms suggests that basic aspects of this nucleosome positioning code are conserved from archaebacteria to man. While we now have a good theoretical and
experimental understanding of the approximate locations of nucleosomes in vivo,
many aspects of chromosome structure and function hinge on knowing nucleosome
locations to basepair resolution; and current experimental mapping methods do not
come close to achieving such accuracy. I will discuss a new experimental approach
to obtaining nucleosome maps with true basepair resolution, and novel discoveries
resulting from the use of this methodology.
148
Jonathan Widom
Department of Molecular Biosciences
and Department of Chemistry,
Northwestern University,
Evanston, IL 60208-3500
[email protected]
149
Computational Approach to Unravel Molecular
Events During Vitiligo
Vitiligo is an acquired depigmenting autoimmune disorder characterized by the
loss of functional melanocytes from the epidermis. The pathomechanism of the
disease is unpredictable because of multifactorial and overlapping mechanisms.
Vitiligo involves complex interaction of environmental and genetic factors that
ultimately contribute to melanocyte destruction, resulting in the characteristic
depigmented lesions. There is no agreement about the pathomechanisms involved
in the disappearance of melanocytes to form the characteristic achromic lesions.
Several hypothesis have been developed for pathogenesis, but none of the hypothesis enlighten the path followed during the disorder. Therefore to construct a map of
molecular interactions of the vitiligo disease is a valuable resource for research in
this area. In this paper, we present a comprehensive pathway map of vitiligo, based
on the protein-protein interaction network using String 8.0. The map reveals that
the overall architecture of the pathway is a bow-tie structure with several feedback
loops. The map is created using CellDesigner software that enables us to graphically represent interactions using a well defined and consistent graphical notation,
and to store it in Systems Biology Markup Language (SBML).
Key words: Vitiligo; CellDesigner; String; Protein-Protein Interaction Network.
Khushhali Menaria
Department of Bioinformatics, Maulana
Azad National Institute of Technology,
Bhopal (M.P.)
[email protected]
[email protected]
1112
150
Ekaterina E. Khrameeva*
Andrey A. Mironov
Mikhail S. Gelfand
Institute for Information Transmission
Problems, Russian Academy of Sciences,
Bolshoy Karetny per. 19, Moscow,
127994, Russia
*[email protected]
The Impact of Interchromosomal Associations on the
Functional State of the Human Genome
The chromatin state is one of the main determinants of transcription rate in eukaryotes. The chromatin is described as a fiber made up of an array of nucleosomes
which consist of core histone proteins wrapped around by DNA double helix
(1). Dynamic chromatin movements and interactions play a crucial role in gene
regulation. Adjacent genes can be packed together in distinct chromatin domains
that ensure coordinated gene expression. Evidence is emerging that distant genes
located on different chromosomes also can interact, and their proximity might be
essential for coordinated regulation. However, it has been unclear whether these
interchromosomal associations are exceptional, or occur frequently.
We systematically analyzed genome-wide interchromosomal interactions in the
nuclei of human cells. 3D data from (2) were associated with the results of several
high-throughput studies of the chromatin functional state (3). All pairs of regions
from different chromosomes were divided into groups according to their proximity,
and the distribution of various chromatin marks was calculated within these groups
and then compared between the groups.
The results show that, indeed, gene regions that are spatially close tend to have
similar patterns of histone modifications, methylation state, open or closed chromatin state, and expression level. Spatially close genome domains tend to have similar
chromatin state and to be coregulated and coexpressed.
Moreover, we found that interacting domains may produce transcripts composed
of sequence segments coming from two different chromosomes. We analyzed chimeric transcripts as determined by genome mapping of paired-read RNA-Seq data
(4, 5) and observed that the frequency of pairs mapping to two different genome
loci is higher among spatially proximal regions. We suggest that these transcripts
might be formed by trans-splicing.
In summary, interchromosomal associations seem to be much more common than
previously believed, and they likely play important roles in the regulation of gene
expression.
This research has been supported by State contracts, grants of the Russian Foundation
of Basic Research, programs “Molecular and Cellular Biology” and “Basic Science
for Medicine” of the Russian Academy of Sciences.
References
1. A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. 3. J Biomol Struct Dyn
27, 713-724 (2010).
2. Lieberman-Aiden, N. L. van Berkum, L. Williams, M. Imakaev, T. Ragoczy, et al. Science
326, 289-293 (2009).
3. ENCODE Project Consortium Science 306, 636-640 (2004).
4. A. G. Xu, L. He, Z. Li, Y. Xu, M. Li, et al. PLoS Comp Biol 6(7), e1000843 (2010).
5. M. F. Berger, J. Z. Levin, K. Vijayendran, A. Sivachenko, X. Adiconis, et al. Genome Res.
20, 413-427 (2010).
1113
Mapping Oxidative Damage at High Resolution
Throughout an Entire Genome
Oxidative damage to DNA has been proposed to be a major contributing factor to
the process of ageing. Accumulation of oxidative damage has been associated with
many degenerative diseases. Determining the locations of vulnerability to oxidative damage on a genome-wide scale will be pivotal to understanding the ageing
process. One type of oxidative damage is produced by Reactive Oxygen species
(ROS), primarily in the form of hydroxyl radicals, which abstract deoxyribose
hydrogens along the DNA backbone, resulting in strand breaks. Regions of chromatin that encode essential genes that are highly expressed often are found in areas
of relatively low chromatin condensation, and so may be more susceptible to ROS
attack. These genomic regions are bound by numerous proteins, including transcription factors, and their accessibility is governed by nucleosome positioning.
We are developing a new method to biochemically process oxidatively damaged
genomic DNA to make it suitable for high-throughput sequencing, in order to map
oxidative damage throughout a genome at single-nucleotide resolution. Our initial
experiments on a well-studied model system (1) demonstrate that this new method
successfully identifies sites of oxidative damage. We will use these quantitative
maps to determine which regions of a genome are particularly susceptible to oxidative damage, and which are resistant, and relate these damage maps to the underlying genes and functional regions of the genome. These studies will provide a view
at unprecedented resolution of the spectrum of oxidative damage in the genome, to
facilitate a deeper understanding of the relationship of DNA damage to ageing.
Reference
1. L. M. Ottinger and T. D. Tullius. J Am Chem Soc 122, 5901-5902 (2000).
151
Sarah Bernard1
Cheryl Chiang1
Stephen C. J. Parker2
Elliott H. Margulies2
Thomas D. Tullius1,3*
1Department
of Chemistry,
Boston University,
Boston, MA 02215
2Genome
Informatics Section,
Genome Technology Branch,
National Human Genome Research Institute, National Institutes of Health,
Bethesda, MD 20892
3Program
in Bioinformatics,
Boston University,
Boston, MA 02215
*[email protected]
152
Three-Dimensional Genome Architecture Predicts the
Distribution of Chromosomal Alterations in
Human Cancers
Over the last decade, novel technologies have exposed how cancer genomes are
riddled with mutations, including everything from single nucleotide substitutions
through large-scale copy-number alterations. Progress towards deciphering highly
contorted cancer genomes lies in a better understanding of the forces and mutational mechanisms behind patterns of genomic alterations in cancer.
Unequivocally establishing a connection between genomic alterations and threedimensional genome structure in cancer has up to this point been limited by our ability to measure three-dimensional structure of DNA, and the resolution with which
we are able to observe genomic alterations in cancer. Fortunately, both of these
have become available in the last two years. Array-based technology now allows
determination of somatic copy number alterations (SCNAs) in cancer at much
higher resolutions and throughput than microscopy-based methods (1). Similarly
doing away with microscopy, the 3C technique and its descendants (2), use a biochemical approach for high resolution determination of three-dimensional genome
Geoffrey Fudenberg1*
Leonid Mirny1,2,3
1Harvard
University Graduate
Biophysics Program,
Cambridge, MA 02138
2Departmetn
of Physics, Massachusetts
Institute of Technology
3Harvard-MIT
Division of Health
Sciences and Technology
*[email protected]
1114
architecture across a population of cells. In 3C, DNA is crosslinked, digested, and
ligated; ligation products are then read using sequencing to determine pairs of DNA
fragments which are close in space. Pairing SCNA data with HiC data allows us to
examine the role of three-dimensional chromosomal architecture in the formation
of somatic structural alterations.
By constructing a heatmap (or matrix) of Somatic Copy-Number Alterations
(SCNAs) from (1), we demonstrate the influence of three-dimensional genome
structure as determined by Hi-C (2) on the distribution of SCNAs. Towards this
end, we developed a Maximum Likelihood framework and permutation procedures for statistically justified comparisons of heatmaps. These statistical techniques establish a connection between intra-chromosomal genomic alterations
in cancer and the three-dimensional genomic architecture. Furthermore, the
strength of this link is bolstered when we account for purifying selection. Our
result provides evidence for a mutational mechanism of chromosomal alterations,
and emphasizes the importance of spatial chromatin organization in addition to
forces of natural selection for genome function. Finally, our work displays the
potential of novel genomic technologies by connecting chromosomal alterations
in cancer with three-dimensional genomic architecture at previously impossible
megabase resolution.
References
1. R. Beroukhim et al. The landscape of somatic copy-number alteration across human cancers,
Nature 463, 899-905 (2010).
2. E. Lieberman-Aiden, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome, Science 326, 289-293 (2009).
153
Nicholas B. Hammond1
Clare C. Rittschof2
Thomas D. Tullius3
Boston University,
Boston, MA 02215
[email protected]
[email protected]
[email protected]
High-Throughput DNA Structural Analysis by
Hydroxyl Radical Cleavage and Capillary
Electrophoresis
DNA structure affects protein binding and rates of transcription, and directly informs
cellular processes (e.g., 1). Since DNA structure depends on local base composition
(2), our long-term goal, in concert with the ENCODE Project, is to identify regions
within the human genome with evolutionarily conserved structure. We report here
on our efforts to add to the OH Radical Cleavage Intensity Database (ORChID) (3),
to improve our algorithm for predicting DNA structure, by adding cleavage data on
relatively long (300-400 base pair) DNA sequences. This necessitates development
of an accurate, high-throughput method for DNA shape determination.
Our lab has previously developed a method to map DNA structure using hydroxyl
radical cleavage patterns (4). Hydroxyl radicals create single-stranded breaks in
DNA. The frequency of damage at a particular site depends on the local structure, because of sequence-dependent variation in backbone solvent accessibility.
We use this technique, coupled with the high-throughput analytical capabilities
of capillary electrophoresis, to quickly evaluate the structural variation of longer
DNA sequences than was previously possible using standard gel electrophoresis.
Cleavage patterns at single nucleotide resolution are quantified from the fluorescence trace of the capillary electrophoresis instrument using the open-source program CAFA (capillary automated footprinting analysis) (5). This high-throughput
method enables fast and accurate acquisition of DNA structural information for
improvement of the ORChID algorithm.
1115
References
1. S. C. J. Parker, L. Hansen, H. O. Abaan, T. D. Tullius, and E. H. Margulies. Science, 324,
389-392 (2009).
2. R. E. Dickerson and H. R. Drew. J Mol Biol 78, 2179-2183 (1981).
3. J. A. Greenbaum, B. Pang, and T. D. Tullius. Genome Res 17, 947-953 (2007).
4. W. J. Dixon, J. J. Hayes, J. R. Levin, M. F. Weidner, B. A. Dombroski, and T. D. Tullius.
Methods Enzymo 208, 380-413 (1991).
5. S. Mitra, I. V. Shcherbakova, R. B. Altman, M. Brenowitz, and A. Laederach. Nucleic Acids
Res 36, e63 (2008).
154
When Replication Meets Transcription
The replication fork and RNA polymerase share the same DNA template making
occasional collisions between the two machineries inevitable. In the case of head-on
collisions, the front edge of RNA polymerase collides with the lagging strand synthesis components of the replication fork. In the co-directional case, in contrast,
the rear edge of RNA polymerase meets with the leading strand synthesis components of the replication fork. The first data suggesting that head-on collisions can
occur both in vitro and in vivo resulting in replication fork stalling were obtained
more than two decades ago. The mechanisms responsible for these events remained
obscure. My lab got involved in these studies as a result of two purely serendipitous
developments. First, while attempting to prove that H-DNA formed by d(G)n•d(C)n
runs can stall DNA replication in vivo, we found that it is, in fact, transcription in
a specific direction through those runs that causes fork stalling (1). Recently, we
showed that unusually stable rG/dC hybrids created during transcription of those
runs trigger the formation of R-loops, which, in turn, stall replication forks moving
co-directionally (2). Second, while attempting to understand the nature of some
obscure replication stall sites in a bacterial plasmid, we found that direct physical
collisions between transcription and replication apparata led to profound fork stalling (3). Furthermore, even when RNA polymerase has not cleared the promoter,
its front edge represented a formidable obstacle for the replication forks moving
toward it (4). Finally, we found that RNA polymerase trapped or backtracked at the
terminator sequences stalls co-directional replication forks (4). It would be fair to
say that these data revitalized the field resulting in many spectacular structural and
functional discoveries to be discussed at this session.
References
1. M. M. Krasilnikova, G. M. Samadashwily, A. S. Krasilnikov, and S. M. Mirkin Transcription through a simple DNA repeat blocks replication elongation. EMBO J 17, 5095-5102
(1998).
2. B. P. Belotserkovskii, et al. Mechanisms and implications of transcription blockage by
guanine-rich DNA sequences. Proc Natl Acad Sci USA 107, 12816-12821 (2010).
3. E. V. Mirkin and S. M. Mirkin. Mechanisms of transcription-replication collisions in bacteria. Mol Cell Biol 25, 888-895 (2005).
4. E. V. Mirkin, D. Castro Roa, E. Nudler, and S. M. Mirkin. Transcription regulatory
elements are punctuation marks for DNA replication. Proc Natl Acad Sci USA 103:
7276-7781 (2006).
Sergei M. Mirkin
Department of Biology,
Tufts University,
Meford, MA 02143
[email protected]
1116
155
Anne Langlois de Septenville
Stéphane Duigou
Hasna Boubakri
Enrique Viguera
Bénédicte Michel*
CNRS, Centre de Génétique
Moléculaire, UPR3404, Gif-sur-Yvette,
F-91198, France
*[email protected]
Essential Role of the Rep, UvrD and DinG
Helicases and of the RecBC Recombination
Complex upon Replication – Transcription
Collisions in the E. coli Chromosome
We are interested in the consequences of replication fork arrest in bacteria. We
characterized several different reactions occurring at blocked forks prior to replication restart, which depend on the cause of arrest (reviewed in 1). Recently, we used
Escherichia coli strains carrying inverted ribosomal operons (rrn) in order to analyze replication arrest caused by replication-transcription collisions. We identified
three helicases required for replication upon head-on collision with transcription
complexes: the Rep, UvrD and DinG helicases (2). Replication arrest sites could
be directly visualized in ribosomal operons facing replication (provided that Rep
or DinG was inactivated), and the inactivation of any combination of two of these
three accessory replicative helicases was highly detrimental for viability. All three
helicases are involved in RNA Pol removal, and DinG has an additional function
of R-loop removal.
In E. coli, rep uvrD and rep uvrD dinG recF mutants grow poorly particularly on
rich medium (multiple replication forks conditions). We isolated mutations that
suppress these growth defects; all map in RNA polymerase genes and presumably
facilitate RNA Pol dislodging from DNA, which supports the idea that the Rep,
UvrD and DinG helicases also facilitate replication across transcribed regions in
wild-type E. coli cells (3).
In addition, increasing replication-transcription collisions by inverting an rrn
operon creates a requirement for the recombination complex RecBC, specific for
the degradation and the recombinational repair of DNA double-strand ends. However, the key homologous recombination protein RecA is not required, suggesting
a specific role for RecBC. The role of recombination proteins upon replicationtranscription collisions will be discussed.
References
1. B. Michel, H. Boubakri, M. Le Masson, Z. Baharoglu, and R. Lestini. DNA Repair 6,
967-980 (2007).
2. H. Boubakri, A. Langlois de Septenville, E. Viguera, and B. Michel. EMBO J 29, 145-157
(2010).
3. Z. Baharoglu, R. Lestini, S. Duigou, and B. Michel. Mol Microbiol 77, 324-336 (2010).
1117
A Topological View of Transcription-Replication
Collisions
Positive supercoiling builds-up ahead of progressing forks and DNA topoisomerases
counteract this effect allowing a smooth advance (1). But the processivity of helicases outcomes topoisomerases and as completion of replication approaches the
DNA ends up with an excess of positive supercoiling. This supercoiling in the
unreplicated portion can migrate to the replicated portion by swivelling the forks.
Here it gives rise to right-handed catenanes once replication is over (2). In bacteria,
topoisomerase IV (Topo IV) is responsible for the decatenation of sister duplexes
(3), but Topo IV is more efficient in the elimination of left-handed crosses (4). This
apparent contradiction is known as the Topo IV paradox (5) and recent data suggests that supercoiling of the newly made sister duplexes plays an essential role to
solve it (6, 7). The scenario changes significantly if transcription and replication
forks progress in opposite directions. The accumulation of positive supercoiling in
between would slow down the advancing forks and this might allow topoisomerases
to cope helicases in order to maintain the replicon negatively supercoiled up to the
end. If negative supercoiling ahead of the forks migrates to the replicated portion,
it would give rise to left-handed catenanes once replication is over. Among other
consequences, it is known this enhances the formation of intermolecular knots during replication (8). Here we combined classical genetics with high-resolution twodimensional agarose gel electrophoresis and atomic force microscopy to investigate
this problem in bacterial plasmids and yeast minichromosomes as prokaryotic and
eukaryotic model systems, respectively.
This research was supported by grants BFU2008-00408/BMC to JBS and BFU200762670 to PH from the Spanish Ministerio de Ciencia e Innovación.
References
1. J. B. Schvartzman and A. Stasiak. EMBO Rep 5, 256-261, (2004).
2. J. J. Champoux. Annu Rev Biochem 70, 369-413, (2001).
156
Virginia López
Estefanía Monturus de
Carandini
María-Luisa Martínez-Robles
Pablo Hernández
Dora B. Krimer
Jorge B. Schvartzman*
Department of Cell Proliferation &
Development, Centro de Investigaciones
Biológicas (CSIC), Ramiro de Maeztu 9,
Madrid 28040, Spain
*[email protected]
1118
3. E. L. Zechiedrich and N. R. Cozzarelli. Gene Dev 9, 2859-2869, (1995).
4. N. J. Crisona, T. R. Strick, D. Bensimon, V. Croquette, and N. R. Cozzarelli. Genes Dev 14,
2881-2892, (2000).
5. K. C. Neuman, G. Charvin, D. Bensimon, and V. Croquette. Proc Natl Acad Sci USA 106,
6986-6991, (2009).
6. J. Baxter, N. Sen, V. Lopez-Martinez, M. E. Monturus de Carandini, J. B. Schvartzman,
J. F. Diffley, and L. Aragon. Science in press, (2011).
7. M. L. Martinez-Robles, G. Witz, P. Hernandez, J. B. Schvartzman, A. Stasiak, and
D. B. Krimer. Nucleic Acids Res 37, 5126-5137, (2009).
8. J. M. Sogo, A. Stasiak, M. L. Martinez-Robles, D. B. Krimer, P. Hernandez, and
J. B. Schvartzman. J Mol Biol 286, 637-643, (1999).
157
Boris P. Belotserkovskii1*
Richard Liu1
Shayon Saleh1
Silvia Tornaletti2
Maria M. Krasilnikova3
Sergei M. Mirkin4
Philip C. Hanawalt1
1Department
of Biology, Stanford
University, Stanford, CA 94305
2Department
of Anatomy and Cell
Biology, University of Florida, FL 32610
3Department
of Biochemistry and
Molecular Biology, Penn State University,
University Park, PA 16802
4Department
of Biology, Tufts
University, Medford, MA 02155
*[email protected]
Transcription Blockage by Guanine-Rich DNA
Sequences and Possible Effects on Transcription
of Nascent RNA Binding to DNA
Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and
with genomic instability. An important example is sequences containing G-rich
homopurine-homopyrimidine stretches, for which unusual transcriptional behavior
is implicated in regulation of immunogenesis and in other processes, such as genomic
translocations and telomere function. To elucidate the mechanism of the effect of
these sequences on transcription we have studied T7 RNA polymerase transcription
of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, length- and supercoiling-dependent
manner. Based upon the effects of various sequence modifications, solution conditions and substitution of inosine or deazaguanosine for guanosine, we conclude that
transcription blockage is due to formation of unusually stable RNA/DNA hybrids,
which could be further exacerbated by triplex formation (1). These structures
are likely responsible for transcription-dependent replication blockage by G-rich
sequences in vivo (2). We have also analyzed the general effect of stable nascent
RNA binding to DNA theoretically, and we conclude that such anchoring could
interfere with transcription downstream from the anchoring point (3).
Supported by a grant CA77712 from the National Cancer Institute, NIH
References
1. A. Gabrielian. J Biomol Struct Dyn 26, 837-838 (2009).
2. B. P. Belotserkovskii, R. Liu, S. Tornaletti, M. M. Krasilnikova, S. M. Mirkin, and
P. C. Hanawalt. Proc Natl Acad Sci 107(29), 12816-12821 (2010).
3. B. P. Belotserkovskii and P. C. Hanawalt. Biophysical Journal (in press).
1119
Transcription-associated Mutagenesis and Top1
Activity in Budding Yeast
High levels of transcription stimulate both homologous recombination and mutagenesis in yeast, and do so by multiple mechanisms (1). Using the CAN1 gene fused
to the GAL1 promoter, a novel, 2-5 bp deletion signature associated with high levels
of transcription has been identified. These deletions accumulate at discrete hotspots
that coincide with short tandem repeats of the same size, and are completely dependent on the activity of Top1 (2, 3). Top1 is a type 1B topoisomerase that nicks
DNA, forming a reversible intermediate with the enzyme covalently attached at the
nick via a 3’ phosphotyrosyl linkage (4). We propose that the deletions reflect the
processing of a Top1 cleavage product generated during the removal of transcription-associated supercoils. To study the genetic control of deletion formation, individual 2-bp deletions hotspots have been transplanted into a frameshift reversion
assay where they can be studied in isolation. In this much more sensitive system,
Top1-dependent deletions are observed even under low-transcription conditions.
Because of the similarity of the Top1-dependent deletion signature to that recently
associated with a failure to remove ribonucleotides (rNMPs) from genomic DNA
(an RNase H2-deficient background; ref. 5), we have explored whether there is any
relationship between the two. We find that rNMP-initiated deletions that accumulate in the absence of RNase H2 require the activity of Top1. This suggests a model
in which presence of an rNMP at the scissile phosphate leads to an irreversible
Top1 cleavage product (6) that is subsequently processed into a deletion intermediate. While all rNMP-initiated deletions appear to require Top1, there are subclasses
of Top1-dependent deletions that are not elevated in the absence of RNase H2. We
propose that rNMP-independent deletions reflect processing of a reversible Top1
cleavage intermediate that becomes trapped on DNA. Finally, we demonstrate that
there is a synergistic relationship between rNMP-associated deletions and high
levels of transcription, suggesting that rNMP levels within genomic DNA can be
influenced by transcriptional status.
This work has been supported by NIH grants GM38464 and GM93197 awarded
to SJR.
References
1. A. Aguilera and B. Gomez-Gonzalez. Nat Rev Genet 9, 204-217 (2008).
2. M. J. Lippert, et al. Proc Natl Acad Sci USA 108, 698-703 (2011).
3. T. Takahashi, G. Burguiere-Slezak, P. A. Van der Kemp, and S. Boiteux. Proc Natl Acad
Sci USA 108, 692-697 (2011).
4. J. C. Wang. Nat Rev Mol Cell Biol 3, 430-440 (2002).
5. S. A. McElhinny, et al. Nat Chem Biol 6, 774-781 (2010).
6. J. Sekiguchi and S. Shuman. Mol Cell 1, 89-97 (1997).
158
Nayun Kim
Jang-Eun Cho
Sue Jinks-Robertson*
Department of Molecular Genetics
and Microbiology,
Duke University Medical Center,
Durham, NC 27710
*[email protected]
1120
159
Roxana E. Georgescu
Nina Y. Yao
Mike O’Donnell
Rockefeller University and Howard
Hughes Medical Institute, 1230 York
Avenue, New York, NY 10021, USA
160
Yayan Zhou
Manju M. Hingorani
Molecular Biology and
Biochemistry Department,
[email protected]
[email protected]
The E. coli Replisome and Use of Clamps to Bypass
Replication Barriers
Chromosomal replicases utilize circular sliding clamps for high-processivity during
replication. Sliding clamps not only bind the chromosomal replicase, but they also
function with all 5 DNA polymerases in E. coli as well as other proteins for repair
and lesion bypass. This presentation focuses on the use of the sliding clamp by the
replicase in crossing barriers that are encountered during replication. Studies have
shown that the replicase, Pol III, can rapidly hop from one sliding clamp to another
clamp in order to transfer to the multiple RNA primers synthesized during lagging
strand replication. However, recent studies indicate that polymerase hopping among
sliding clamps is of more general use, and can allow the polymerase to bypass
lesions and even to circumvent a tightly bound RNA polymerase that it encounters
during replication. We will present studies that demonstrate the how the replisome
deals with collisions with RNA polymerase transcribing either the leading or lagging
strand template. We will also present our findings on how different polymerases
control the use of the clamp to form alternative replisomes for DNA damage avoidance and, when needed, to deal with lesions that are encountered directly. Finally,
we present single-molecule studies that examine replisome action during lagging
strand synthesis and the consequences for the leading strand polymerase.
An Active Clamping Role for PCNA During
Assembly and Function on DNA
Circular clamp proteins enable processive DNA replication by tethering polymerases to the primer-template during DNA replication. Clamps also bind to and
coordinate the functions of several other proteins on DNA, and are therefore essential for many DNA metabolic reactions (1). Clamps are loaded onto DNA in an
ATP-fueled reaction by multi-subunit AAA+ protein complexes known as Clamp
Loaders. The mechanism of action of these proteins is under active investigation,
given their important role in genomic DNA replication, repair and recombination (2). According to our current kinetic model of the S. cerevisiae RFC clamp
loader, ATP binding activates RFC, allowing it to bind and open the PCNA clamp
for entry of primer-template DNA; DNA binding to RFC triggers ATP hydrolysis,
PCNA closure around DNA and release of the PCNA•DNA complex from RFC
(3). The question we are addressing is whether PCNA plays an active role in clamp
assembly. The specific hypothesis is that interactions between positively charged
residues on the inside of the clamp and DNA help trigger clamp closure around
DNA and catalytic turnover of RFC. In order to test this hypothesis, we have generated several PCNA mutants in which individual conserved amino acids have been
substituted with Alanine. Data from transient kinetic analysis of key events during
clamp assembly, including ATP-bound RFC• PCNA•DNA complex association and
dissociation, PCNA opening and closing, as well as phosphate release, indicates
that alteration of a single PCNA-DNA contact can alter rate-limiting steps in the
reaction. Thus, the kinetic data support the hypothesis that PCNA is more than just a
passive ring around DNA—it is an active contributor to the clamp assembly reaction
mechanism and, by extension, possibly other DNA metabolic reactions as well.
This research is supported by funding from NSF and NIH.
References
1. G. L. Moldovan, B. Pfander, and F. Jentsch. Cell 129, 665-679 (2007).
2. M. O’Donnell and J. Kuriyan. Current Opinion in Structural Biology 16, 35-41 (2006).
3. S. Chen, M. K. Levin, M. Sakato, Y. Zhou, and M. M. Hingorani. Journal of Molecular
Biology 388, 431-442 (2009).
1121
Electrostatic Properties of Bacteriophage T7 Early
Promoters Recognized by E. coli RNA Polymerase
During T7 bacteriophage infection, its transcription is closely regulated by two different RNA polymerases. RNA polymerase of E. coli transcribes the early (class
I) genes of T7, and newly made T7 RNA polymerase transcribes the late (class II
and class III) genes. The both enzymes are very specific for their own promoters.
RNA polymerase of E. coli recognized three strong (A1, A2, and A3) and five week
(B, C, D, E, F) promoters in T7 DNA. All these promoters have been earlier characterized by their biochemical properties. Their functional characteristics were shown
to differ from “consensus sequence rule” behavior thus stimulating a search of new
promoter determinants.
Here electrostatic properties of the promoters were studied, and this has been
recently reported to play an important role in protein-nucleic acid recognition (1, 2).
Electrostatic potential distribution around 300 bp- fragments containing the individual promoters was calculated by Coulomb method (3) using the computer program of Sorokin A. A. Electrostatic profiles of three strong and one weak promoters
are shown in Figure 1(A-D). Electrostatic properties in the far upstream region
corresponding to –75 - –100 bp position are of most interest for our task since this
region is known to be involved in electrostatic interaction with E. coli RNA polymerase α-subunit (4).
161
S. G. Kamzolova1
P. M. Beskaravainy1,2,*
A. A. Sorokin1,**
1 Institute
of Biophysics, RAS Pushchino
Moscow Region 142290, Russia
2 Institute
of Theoretical and
Experimental Biophysics, RAS
Pushchino, Moscow Region 142290,
Russia
*[email protected]
**[email protected]
Electrostatic profile of PA1 (Figure 1a) can be characterized by the presence of the
most negatively charged element at –70 - –100 bp and more positive flanking sites.
Figure 1: Distribution of electrostatic potential around PA1(A), PA2(B), PA3(C) and PB(D).
1122
The promoter shares these specific electrostatic features with two E. coli ribosomal
promoters (5). Although electrostatic profiles of PA2 (Figure 1b) and PA3 (Figure 1c)
are not identical, they reveal some common features in the far upstream region and
they share these features with T4 early promoters P114.6 and P73.0 (6). The important feature of their patterns is a continuous rise of electrostatic potential at –80 to
–100 bp with an extended positive peak at –90 bp Thus, PA2 and PA3 are characterized by the presence of the positively charged element in the functionally important
region of promoter DNA. At the same time, this region for PA1 is mostly negative
having no pronounced positively charged area. The difference in electrostatic properties of the tandem T7 promoters can result in a difference in their functional behavior
allowing a high level of gene 1 transcription to be maintained in different conditions
during the phage infection.
Electrostatic properties of all weak T7 promoters (a representative example in
Figure 1D) differ from that of the strong PA1, PA2 and PA3. Their electrostatic upelements are less structurally expressed, some of them being barely distinguishable
from ordinary coding regions.
Thus, there is a good correlation between electrostatic properties of early T7 promoters and their strength. All the results support that the far upstream region of T7
early promoters can be involved in modulation their activities by acting through
electrostatic interaction with E. coli RNA polymerase alpha-subunit.
References
1. S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010).
3. R. V. Polozov, T. R. Dzhelyadin, A. A. Sorokin, N. N. Ivanova, V. S. Sivozhelezov, and
S. G. Kamzolova. J Biomol Struct Dyn 16, 1135-43 (1999).
4. S. G. Kamzolova, V. S. Sivozhelezov, A. A. Sorokin, T. R. Dzhelyadin, N. N. Ivanova, and
R. V. Polozov. J Biomol Struct Dyn 18, 325-334 (2000).
5. S. G. Kamzolova, A. A. Sorokin, P. M. Beskaravainy, and A. A. Osypov. Bioinformatics of genome regulation and structure II. Eds. N. Kolchanov, R. Hofestaedt, Z. Milanesi,
Springer Science Business Media Inc. p. 67-74 (2006).
6. A. A. Sorokin, A. A. Osypov, T. R. Dzhelyadin, P. M. Beskaravainy, and S. G. Kamzolova.
J Bioinf Comp Biol 4, 455-467 (2006).
162
P. M. Beskaravainy1,2,*
A. A. Osypov1
G. G. Krutinin1
E. A. Krutinina1
S. G. Kamzolova1
1Institute
of Biophysics, RAS Pushchino,
2Institute
of Theoretical and
Experimental Biophysics, RAS Pushchino,
*[email protected]
Electrostatic Properties of T7 RNA Polymerase
Specific Promoters
Analysis of electrostatic properties of promoter DNA is a promising source for
yielding information about promoter recognizable elements and their functioning.
Here, electrostatic properties of 17 T7 RNA-polymerase specific promoters where
studied, and this has been recently reported to play an important role in proteinnucleic acid recognition (1, 2). These promoters may be classified into three groups
according to their expression time and functional characteristics of the corresponding genes: class “early” (subgroup of class II), class II and class III (1). All the
promoters have been erlier characterized in detail by their interaction with RNApolymerase and transcription initiation (3, 4). Their sequences are characterized by
comparatively extended consensus sequence (23 bp) with a high level of homology
for all promoters but, despite their considerable sequence similarity, the different
promoter classes differ by their strengths and by many other biochemical properties (3, 4). Thus, the choice of these promoters for our study was motivated by
their unusual functional characteristics differing from “consensus sequence rule”
behavior.
1123
Figure 1: The averaged profiles of electrostatic potential distribution around T7 RNA-polymerase
specific promoters belonging to different functional group.
Electrostatic potential distribution around duble-helical DNA of the promoters was
calculated by the Coulombic method (5) using a new computer program (6).
Electrostatic profiles of the individual promoters were combined into three corresponding groups and the averaged electrostatic profiles of these separate groups
were calculated as indicated in (6). The data obtained are shown in figure 1. The
profiles of the promoters can be characterized by the presence of particular electrostatic elements in the region from –25 bp to +20 bp wich is known to be involved in
contacts with T7 RNA-polymerase. These electrostatic elements are specific for the
different promoter groups. The promoters belonging to class II are characterized
by two valleys at ~ –18 bp to ~ –5 bp and a more positive flanking site at +5 bp. In
contrast, the promoters belonging to class III has the only most expressed negative
fall at ~ –18 bp and two peaks downstream at ~ +5 bp and ~ +20 bp. The promoters
belonging to the “early” group are characterized a more smoothed profile. They are
located within coding region transcribed by the host E. coli RNA-polymerase. This
can account for a more smoothed profile of this group since the variation of average
electrostatic potential is more homogeneous in nonpromoter DNA.
Thus, electrostatic patterns of T7 DNA promoters are distinguished owing to specific motifs wich may be involved as signal elements in differential recognition of
T7 RNA-polymerase specific promoters by the enzyme.
References
1.
2.
3.
4.
S. M. West, R. Rohs, R. S. Mann, and B. Honig. J Biomol Struct Dyn 27, 861-866 (2010).
D. Wang, N. B. Ulyanov, and V. B. Zhurkin. J Biomol Struct Dyn 27, 843-859 (2010).
J. J. Dunn and F. W. Studier. Proc Natl Acad Sci USA 70, 1559-63 (1973).
W. T. McAllister, C. Morris, A. H. Rosenberg, and F. W. Studier. J Mo Biol 153, 527-44
(1981).
S. G. Kamzolova. J Biomol Struct Dyn 16, 1135-43 (1999).
6. A. A. Osypov, G. G. Krutinin, and S. G. Kamzolova. J Bioinf Comp Biol 8, 413-25 (2010).
1124
Strand Exchange Reaction Between Short
Oligonucleotides Promoted by a Derivative
of 1,3 – diazaadamantane
163
Anna Gabrielian1,*
Tatiana N. Bocharova2
Elena A. Smirnova2
Alexander A. Volodin2
Gayane Harutjunyan1
1Research-technological
Center of
Organic & Pharmacological Chemistry
(Armenian National Academy of
Sciences), 26 Azatutian Avenue,
0014 Armenia
2Institute
of Molecular Genetics
(Russian Academy of Sciences), 2,
Kurchatov Square 123182, Moscow,
Russia
*[email protected]
The DNA strand exchange reaction (SER) underlies main pathways of homologous
recombination (HR) and DNA repair in different organisms. These systems of DNA
metabolism closely relate to carcinogenesis and better understanding of interaction of antitumor drugs with their components is indispensable for development of
new approaches to antitumor therapy. Recently this Journal has reported antitumor
intercalators and groove binders interacting with DNA double helix (1-4).
Derivatives of 1,3-diazaadamantane are promising compounds that possess essential antitumor activity on different types of experimental tumors (5). In the present
study the interaction of the 1,3-diazaadamantane derivative HG122 [1′-benzil5,7-dipropil-6-oxyspiro(1,3-diazaadamantane-2,4′-piperidine)] with DNA and its
behavior in the system that simulate SER has been characterized.
Earlier we described an experimental system for the study of SER between short
oligonucleotides promoted by RecA protein from ε.coli and RecA-like human
recombinases (6). RecA protein is a paradigm of the wide class of proteins of HR
systems from different organisms and promotes the central stage of HR – DNA
strand exchange. In our system fluorescent dye-tagged 21-mer oligonucleotide and
unlabelled double stranded 21 bp oligonucleotides were used as single- and double
stranded substrates of SER respectively.
We did not detect any influence of HG122 on the RecA protein promoted DNA
strand exchange. At the same time, it was revealed that this compound by itself
exhibited the capability to facilitate SER in this system. Kinetic curves of the SER
in solution with different concentrations of Hg122 were obtained for various temperatures. The final yield of SER didn’t depend on temperature in the range from
28 to 50°C that argues in favor of thermodynamics equilibrium reached by this
reaction. In separate experiments we found no effect of HG122 on the melting
temperature of double-stranded DNA; therefore, the observed acceleration of SER
in the presence of HG122 can not be explained by trivial effect of destabilization
of the DNA double helix.
Earlier, the DNA strand exchange activity was documented for Cationic Combtype Copolymers (7) and positively charged liposome surfaces (8). We found that
other polycations such as linker histones and cobalt hexamine also exhibit the capability to promote SER (manuscript in preparation). The present results provide us
with the first example of the quite different class of SER facilitating compounds
that contain large lipophilic core.
Figure 1: Structure of HG-122.
1125
Common feature of the strand exchange reactions promoted by polycationic agents
is their low tolerance to heterology between the SER substrates. In contrast to the
reactions promoted by RecA-like proteins that exhibit increased tolerance to heterology between the substrates (6) the tolerance of polycations promoted strand
exchange is comparable to that of the spontaneous SER that proceeds at elevated
temperatures. Hg122 promoted reaction exhibits the tolerance to heterology intermediate between the cases of the SER promoted by polycations and RecA-like
proteins.
Therefore, the present results demonstrate that Hg122 exhibits interesting behavior
and can serve as a model object for further studies of different aspects of DNA
strand exchange reaction. Biomedical implications of this and other similar compounds activities remain to be estimated.
References
1. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky,
V. F. Pismensky, V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26,
701-718 (2009).
5. G. L. Harutjunyan, A. A. Chachoyan, C. E. Ahajanyan, and B. T. Haribjanyan. Chem pharm
J (Russia), 12, 20-24 (1996).
6. A. A. Volodin, T. N. Bocharova, E. A. Smirnova, and R. D. Camerini-Otero. J Biol Chem
284, 1495-1504 (2009).
7. W. J. Kim, Y. Sato, T. Akaike, and A. Maruyama. Nat Mater 2, 815-820 (2003).
8. K. Frykholm, B. Nordén, and F. Westerlund. Langmuir 25,1606-1611 (2009).
164
Transcription Factor - DNA interactions: cis
Regulatory Codes in the Genome
The interactions between sequence-specific transcription factors (TFs) and their
DNA binding sites are an integral part of the gene regulatory networks within cells.
My group developed highly parallel in vitro microarray technology, termed protein
binding microarrays (PBMs), for the characterization of the sequence specificities
of DNA-protein interactions at high resolution. Using universal PBMs, we have
determined the DNA binding specificities of >500 TFs from a wide range of species. These data have permitted us to identify novel TFs and their DNA binding
site motifs, predict the target genes and condition-specific regulatory roles of TFs,
predict tissue-specific transcriptional enhancers, investigate functional divergence
of paralogous TFs within a TF family, investigate the molecular determinants of
TF-DNA ‘recognition’ specificity, and distinguish direct versus indirect TF-DNA
interactions in vivo. Further analyses of TFs and cis regulatory elements are likely
to reveal features of cis regulatory codes important for driving appropriate gene
expression patterns.
This research has been supported by Grants from NIH / NHGRI to M.L.B.
Martha L. Bulyk
1Division
of Genetics,
Department of Medicine
2Dept.
Pathology, Brigham and Women’s
Hospital and Harvard Medical School,
Boston, MA 02115, USA
3Harvard-MIT
Division of Health
Sciences and Technology (HST),
Harvard Medical School, Boston,
MA 02115, USA
[email protected]
1126
165
Ivan V. Kulakovskiy1,3
Yulia A. Medvedeva2,3
Vsevolod J. Makeev2,3*
1Engelhardt
Institute of Molecular
Biology, Russian Academy of Sciences,
Moscow, Russia
2Vavilov
Institute of General Genetics,
Russian Academy of Sciences, Moscow,
Russia
3FSUE
Research Institute of Genetics and
Selection of Industrial Microorganims,
Moscow, Russia
*[email protected]
Cis-Regulatory Modules: Identification in silico and
Understanding of Gene Regulatory Networks
Cis-regulatory modules (CRM) are segments of DNA responsible for tissue- and
time- specific regulation of gene expression (1). The length of CRMs is difficult to
estimate directly but it is believed to vary from several hundreds to several thousands of base pairs. In multicellular eukaryotes CRMs may be located not only in
the upstream vicinity of transcription start sites of the dependent genes but also
at tens of thousands nucleotides upstream or downstream from the transcription
start sites. CRMs contain multiple binding sites for protein factors regulating the
transcription. Identification of CRMs in silico and prediction of their regulatory
function allows one to suggest new regulatory inputs controlling expression of particular genes, which makes a useful introductory step before modeling of cell signaling processes. CRMs make an important component of meaningful non-coding
DNA. Genetic variations overlapping with CRMs contribute to functional disorders associated with non-coding DNA.
Detail investigation of CRM sequences exhibit that transcription factor binding
sites (TFBS) form complex arrangements, probably corresponding to yet unknown
regulatory code of gene expression. The simplest feature found in CRMs is clusters
of binding sites. Eukaryotic gene expression usually is controlled by many inputs
from different regulatory circuits, so a typical CRM contains many binding sites for
different transcription factors (TF), both activators and repressors, thus integrating
different regulatory contributions. Thus, a typical CRM contains many sites for different TFs which are rather densely packed and form a so-called heterotypic cluster
(2). Sites for different TFs in such clusters are often found at specific distances from
each other (3), probably facilitating correct positioning of proteins at DNA necessary for protein-protein interaction, either direct or via adapter proteins (4).
In addition, many CRMs contain many occurrences of the same binding sites,
forming the so-called homotypic clusters of TFs (5, 6). The function of homotypic
clustering is yet unclear; probably it is served for providing for a specific TF-concentration dependence for TF binding (7). The alternative explanation is that this
type of arrangement is needed to facilitate lateral diffusion of binding factor along
DNA to its functional binding position (8). The exceptional form of a homotypic
cluster is a tandem repeat made from sequences, specifically bound by TFs. This
type of arrangement is characterstic for some CRMs in Drosophila (9), and might
serve as an origin of some CRMs in evolution.
Studying DNA sequences of CRMs often help to decipher yet unknown regulatory circuits. Two examples are touched upon: Drosophila development and human
hypoxia response cascade. I’m also going to discuss how CRMs are predicted
in silico, and consequences of site turnover for prediction of CRM and TFBS.
This research has been supported by Presidium of Russian Academy of Sciences
Program in Molecular and Cell Biology.
References
1. E. H. Davidson. Genomic Regulatory Systems. Academic Press, San Diego, USA (2001).
2. B. P. Berman, Y. Nibu, B. D. Pfeiffer, P. Tomancak, S. E. Celniker, M. Levine, G. M. Rubin,
and M. B. Eisen. Proc Natl Acad Sci 99: 757-762, (2002).
3. V. J. Makeev, A. P. Lifanov, A. G. Nazina, and D. A. Papatsenko. 31(20): p. 6016-26
(2003).
4. I. V. Kulakovskiy, A. A. Belostotskiy, and V. J. Makeev. 17th Albany conversation, Albany
NY, USA (2011).
5. A. P. Lifanov, V. J. Makeev, A. G. Nazina, D. A. Papatsenko. Genome Res 13: 579-588,
(2003).
1127
6. V. Gotea, A. Visel, J. M. Westlund, M. A. Nobrega, L. A. Pennacchio, and I. Ovcharenko.
Genome Res 20, 565-77, (2010).
7. D. E. Clyde, M. S. Corado, X. Wu, A. Pare, D. Papatsenko, and S. Small. Nature 426, 849-853,
(2003).
8. A. Tafvizi, F. Huang, A. R. Fersht, L. A. Mirny, and A. M. van Oijen. Proc Natl Acad Sci
USA 108, 563-568, (2011).
9. V. A. Boeva, M. Regnier, D. Papatsenko, and V. Makeev. Bioinformatics 22, 676-684, (2006).
Preferred Pair Distance Templates for Identification
of Functional Binding Sites for Interacting
Transcription Factors
We study a set of transcription factors (TFs) including the hypoxia-inducible factor
1 (HIF-1) involved in regulation of hypoxia response in human cells. We demonstrate that binding sites for a pair of interacting TFs can be found at distances
that are dramatically nonrandom and depend both on selected TFs and a relative
orientation of their binding sites. The set of characteristic intersite distances forms
a preferred pair distance template (PPDT).
We started identification of high-quality binding motifs with the help of our original
computational tool, ChIPMunk. ChiPMunk performs motif discovery integrating
166
Ivan V. Kulakovskiy1,2*
Alexander A. Belostotsky3
Vsevolod J. Makeev2,3
1Laboratory
of Bioinformatics and
System Biology, Engelhardt Institute of
Molecular Biology, Russian Academy
of Sciences, Vavilov str. 32, Moscow
119991, Russia
2Laboratory
of Bioinformatics,
Vavilov Institute of General
Genetics, Russian Academy of
Sciences, Gubkina str. 3, Moscow
119991, Russia
3Laboratory
of Bioinformatics,
Research Institute for Genetics and
Selection of Industrial
Microorganisms, 1st Dorozhny
proezd 1, Moscow 117545, Russia
*[email protected]
Figure 1: Distance distribution between HRE elements (corresponding to the HIF‑1α:ARNT dimer binding) and
Sp1 binding sites. Y axis shows the number of genes for which at least one pair of binding sites is found at the
selected distance (shown at X-axis) within the 10kb upstream promoter region. Panels: orientation of HRE in the
reference to Sp1; (top) direct; (bottom) reverse.
1128
data from different experimental sources (1) including ChIP-chip and ChIP-Seq
experiments (2). Both our own (3) and independent (4) benchmarks have shown
that ChIPMunk can produce high-quality motifs in a reasonable time.
The motifs discovered were used to detect transcription factor binding sites in the
5’ regulatory regions of all human genes (according to the UCSC hg18 assembly).
For TFs involved in the known protein-protein interaction with the HIF1α we have
observed a surprisingly large fraction of binding sites found at specific distances
from HIF1α binding sites (see Figure 1). For some TF pairs the corresponding
PPDTs contained quite large distances, sometimes longer than 200 bp.
Thus, with a help of PPDTs we can correctly identify composite elements (5) in
regulatory segments. Moreover, PPDT can be used as a filter to remove false positive binding sites in human cis-regulatory modules. PPDT also can be used to
assess functional interaction between a pair of DNA binding proteins solely by
means of sequence analysis. Currently we explore how PPDT information can be
used together with gene expression data to identify correct target responding to
hypoxia conditions.
This research is supported by Russian Fund of Basic Research grant 10-0492663-Ind.
References
1. I. V. Kulakovskiy, V. J. Makeev. Biophysics 54, 667-674 (2009).
2. I. V. Kulakovskiy, V. J. Makeev, V. A. Boeva, V. J. Makeev. Bioinformatics 26, 2622-2623
(2010).
3. http://line.imb.ac.ru/ChIPMunk/Supplementary_text.pdf
4. http://cmotifs.tchlab.org/help.html
5. V. Matys, O. V. Kel-Margoulis, E. Fricke, et al., Nucleic Acids Res 34, D108-10 (2006).
167
Konstantin S. Shavkunov
Maria N. Tutukina
Irina S. Masulis
Olga N. Ozoline*
Institute of Cell Biophysics, RAS,
Pushchino Moscow Region, 142290,
Russian Federation
*[email protected]
Promoter Islands: The Novel Elements
In Bacterial Genomes
Seventy-eight “promoter islands” (PIs) were delineated in E. coli genome based
on high density of potential transcription start points, high ability of RNA polymerase (RNAP) binding and paradoxically low transcription efficiency (1). Among
them there are 23 PIs lying inside coding sequences (exemplified in Figure 1A) or
between convergent genes, where promoter activity is not expected. The specific
location and high length of genomic regions (300 bp), containing overlapping
transcription signals, distinguish them from clustered promoter-like sites previously found nearby highly expressed genes. In this study we compared chromatin
structure for PIs and 181 “single” promoters using published ChIP-on-chip data
(2-4) and validated the ability of 16 intragenic PIs + 3 PIs located between convergent genes to form “open” complexes with RNAP in vitro and in vivo.
It turned out that PIs more frequently than “single” promoters associate with nucleoide proteins H-NS and Fis. Interaction with RNAP was registered for all PIs (100%)
if antibodies against σ subunit were used to collect complexes (Figure 1B gray bars)
and only for 43% of “islands” if precipitation was carried out by β-specific reaction
(Figure 1B black symbols), while complexes with “single” promoters were detected
with approximately equal efficiency by these two approaches (~87% and 77%,
respectively). These differences assume certain singularity in the chromatin structure within PIs. Functional analysis testified ability of all analyzed PIs to undergo
local DNA melting upon interaction with RNAP in vitro and 12 “islands” form
transcription bubbles in vivo, thus assuming their ability to start RNA synthesis.
1129
Figure1: (A) PI associated with genes yigF and yigG. Bars represent transcription start points predicted by PlatProm. Triangles and asterisks indicate
positions of transcription bubbles registered in vitro and in vivo, respectively. (B): Sites of interaction with RNAP as registered by ChIP-on-chip technique
(4) with σ- or β-specific antibodies.
However in most cases (75% for analyzed PIs and 75.6% for the total set of 78 PIs)
expected products were not found among the sequenced cDNAs (5). For “single”
promoters this failure was 2-fold lower (35%). Since other bacterial genomes show
approximately the same distribution of PIs, we suppose that they perform evolutionary conserved biological role, not necessarily associated with RNA synthesis.
The research is supported by Grants from Russian Foundation for Basic Research
(10-04-01218).
References
1. K. S. Shavkunov, I. S. Masulis, M. N. Tutukina, A. A. Deev, and O. N. Ozoline. Nucleic
Acids Res 37, 4919-4931 (2009).
2. D. C. Grainger, D. Hurd, M. D. Goldberg, and S. J. W. Busby. Nucleic Acids Res 34:
4642-4652 (2006).
3. D. C. Grainger, H. Aiba, D. Hurd, D. F. Browning, and S. J. W. Busby. Nucleic Acids Res.,
35: 269-278 (2007).
4. N. B. Reppas, J. T. Wade, G. M. Church, and K. Struhl. Mol Cell, 24, 747-757 (2006).
5. J. E. Dornenburg, A. M. DeVita, M. J. Palumbo, and J. T. Wade. mBio 1: e00024-10
(2010).
1130
168
Alexander A. Lomzov1,2,*
Irina V. Khalo1,2
Dmitrii V. Pyshnyi1
1Institute
of Chemical Biology and
Fundamental Medicine SB RAS,
Lavrent’v ave., 8, 630090,
Novosibirsk, Russia
2Novosibirsk
State University, Pirogova
st., 2, 630090, Novosibirsk, Russia
*[email protected]
The Influence of Aliphatic Alcohols on
Oligonucleotide Hybridization
The biological activity of nucleic acids is correlates strongly with their physicochemical properties, in particular their thermodynamic properties. The thermal
stability of nucleic acids depends strongly on the properties of intracellular environment such as ionic strength, polarity of the medium and presence of various
osmolites, compartmentalization and etc (1-3). The influence of monovalent (Na+
and K+) and divalent (Mg2+) cations has been widely studied (4, 5). By contrast the
thermodynamics properties of nucleic acids in the presence of various co-solvents
have not been studied in details previously, excepting the molecular crowding
effect (6). Here we begin the systematic study of the thermodynamics of DNA
duplex formation in the presence of a large variety of co-solvents. In this work
the efficiency of oligodeoxyribolnucleotides hybridization and DNA duplex secondary structure in the presence of various aliphatic alcohols (ethanol, ethylene
glycol, diethylene glycol, 2,2,2-trifluoroethanol, 2-cyanoethanol) in water solution
(10 mM sodium cacodylate, pH 7.2) has been characterized. It has been shown that
the increase of concentration of aliphatic alcohol in solution from 0 to 50% (v/v)
does not change the conformation of DNA helix. We have demonstrated that for
most alcohols (ethanol, cyanoethanol, ethylene glycol and diethylene glycol) rising
of their concentration up to 50% in a aqueous solution lead to monotonous decrease
in melting temperature for DNA duplexes of various length (12, 15 or 20 b.p.)
and GC-content (27–60%). In the case of trifluoroethanol the stability of double
stranded DNA decreases with the increase of the alcohol concentration up to 20 %.
The higher concentrations of trifluoroethanol (up to 75%) resulted in slight stabilization of DNA duplexes. The analysis of the data obtained has shown the possibility of using a simple model for the description of thermal stability of duplexes
in mixed water/alcohol solutions except 2,2,2-trifluoroethanol. The model assume
that the number of solvents molecules (water and co-solvent) which are bound
to DNA changes at helix-to-coil transition. The proposed model describes the
dependence of melting temperatures of oligonucleotide complexes on concentration of ethanol, cyanoethanol, ethylene glycol and diethylene glycol with accuracy
1.1, 1.7, 1.0, 2.2°C, respectively.
The results obtained can be applied in hybridization analysis in various buffer conditions, for example, with the use of exciplexes (7); in studying nucleic acid –
protein interactions and in the development of molecular-imprinted polymers (8).
This research has been supported by Russian Government (P1073), SB RAS, RFBR
and by MCB and FBNN programs of RAS.
References
(2010).
2. I. A. Il’icheva, P. K. Vlasov, N. G. Esipova, and V. G. Tumanyan. J Biomol Struct Dyn
27,677-693 (2010).
3. T. C. Mou, M. C. Shen, T. C. Terwilliger, and D. M. Gray. Biopolymers 70, 637-648
(2003).
4. E. N. Galyuk, R. M. Wartell, Y. M. Dosin, and D. Y. Lando. J Biomol Struct Dyn 26,
517-523 (2009).
5. R. Owczarzy, B. G. Moreira, Y. You, M. A. Behlke, and J. A. Walder. Biochemistry 47,
5336-5353 (2008).
6. D. Miyoshi and N. Sugimoto. Biochimie 90, 1040-1051 (2008).
7. E. V. Bichenkova, A. Gbaj, L. Walsh, H. E. Savage, C. Rogert, A. R. Sardarian, L. L. Etchells, and K. T. Douglas. Org Biomol Chem 5, 1039-1051 (2007).
8. E. V. Dmitrienko, I. A. Pyshnaya, A. V. Rogoza, and D. V. Pyshnyi. Patent of Russian
Federation # 2385889, (2010).
1131
Minor Groove Ligands Based on Dimeric
Bisbenzimidazoles as Inhibitors of
DNA-dependent Enzymes
Recently in this Journal there have been reports of both intercalators and groove
binders interacting with DNA double helix (1-4). We have reported elsewhere that
dimeric bisbenzimidazoles (DB(n)) differing in the length of methylene linkers can
interact with dsDNA in the minor groove (5).
Compounds DB(n) were studied as inhibitors of two DNA-dependent enzymes,
namely, calf thymus DNA topoisomerase I (topo-I) (6) and the catalytic domain
Dnmt3a of murine DNA methyltransferase (MTase) (7). Ligands DB[3, 4, 5, 7, 11]
inhibited activities of topo-I in vitro at 0.5-2.5 μM concentrations. Inhibitory activity
of DB[7] was 50-fold higher than that of camptothecin, a known topo-I inhibitor.
DB[1, 2, 3, 11] reduced in vitro the MTase Dnmt3a activity by 50% at 5-12 μM
concentrations. Bisbenzimidazoles DB[4, 5, 7] were shown to be less effective: 50%
inhibition of MTase activity was observed only at a concentration of 20 μM and
higher. Increased time of incubation of the tested DB[n] with DNA allowed for a
substantial growth of inhibitory activity of the ligands toward both topo-I and MTase
(6, 7). It should be noted that monomeric MB, a shortened DB[n] analogue, only negligibly inhibited both enzymes at 200 μM concentration. We assume that the found
inhibitory activity of DB[n] is explained by their competition with DNA-dependent
enzymes for binding sites on DNA. It was previously shown that the Hoechst 33258
dye containing the same bisbenzimidazole motive as DB[n] bound specifically to two
consecutive AT-pairs [8, 9]. Therefore, we suppose that compounds DB[n] would
bind with the highest specificity to the -(A/T)n-(N)m-(A/T)n- DNA site, where N, a
base pair, n = 2, m = 1-4, which correlates with computer modeling data. Indeed, both
duplex I (a highly effective topo-I cleavage site) and duplex II sensitive to MTase
Dnmt3a contain sequences optimal for DB[n] binding (Figure 2).
169
Alexander A. Ivanov1*
Sergey A. Streltsov2
Victor I. Salyanov2
Olga Yu. Susova1
Elizaveta S. Gromova3
Alexei L. Zhuze2
1Institute
of Carcinogenesis Blokhin
Cancer Research Center Russian Academy
of Medical Sciences, Kashirskoye Shosse
24, Moscow 115478, Russia
2Engelhardt
Institute of Molecular
Biology Russian Academy of
Sciences, Vavilova st. 32, Moscow
119991, Russia
3Chemistry
Department, Moscow State
University, Moscow 119991, Russia
*[email protected]
Figure 1: Structure of dimeric bisbenzimidazoles DB[n], where n = 1, 2, 3, 4, 5, 7, 11.
Figure 2: Oligonucleotide fragments used for studies of DB[n] inhibitory activities. Consecutive
AT-pairs are in bold; potential binding sites of the corresponding ligands are underlined. For duplex I ,
the arrow shows the site cleaved by topo-I and for duplex II, cytosine residues to be methylated.
To summarize, DB[n] were proved to be inhibitors of at least two different DNAdependent enzymes at low micromolar concentrations. Higher biological activity
of DB[n] if compared with monomeric MB is due to the formation of dimeric molecules capable of binding to dsDNA in the form of bidentant ligands.
1132
The work was supported by RFBR (Grants 09-04-01126, 10-04-00809 and 11-0400589) and by the Program of Presidium of RAS on Molecular and Cell Biology.
References
1. N. P. Bazhulina, A. M. Nikitin, S. A. Rodin, A. N. Surovaya, Yu. V. Kravatsky,
V. F. Pismensky, V. S. Archipova, R. Martin, and G. V. Gursky. J Biomol Struct Dyn 26,
701-718 (2009).
5. A. A. Ivanov, V. I. Salyanov, S. A. Streltsov, N. A. Cherepanova, E. S. Gromova, and
A. L. Zhuze. Russ J. Bioorgan Chemistry 37 (2011) (in press).
6. O. Y. Susova, A. A. Ivanov, S. S. Morales Ruiz, E. A. Lesovaya, A. V. Gromyko,
S. A. Streltsov, and A. L. Zhuze. Biochemistry (Moscow) 75, 695-701 (2010).
7. N. A. Cherepanova, A. A. Ivanov, D. V. Maltseva, A. S. Minero, A. V.Gromyko,
S. A. Streltsov, A. L. Zhuze, and E. S. Gromova. J Enzyme Inhib Med Chem (2011) [Epub
ahead of print].
8. P. E. Pjura, K. Grzeskowiak, and R. E. Dickerson. J Mol Biol 197, 257-271 (1987).
9. M. K. Teng, N. Usman, C. A. Frederick, and A. H. Wang. Nucleic Acids Res. 16, 2671-2690
(1988).
170
Poghos O. Vardevanyan1
Ara P. Antonyan1
Mariam A. Shahinyan1
Margarita A. Torosyan2
Armen T. Karapetian2
1Department
of Biophysics of Yerevan
State University, A.Manoogian str. 1,
Yerevan 0025, Armenia
2Physics
Department of Yerevan State
University of Architecture and
Construction, Teryan str. 105, bldg.2,
[email protected]
Effect of Millimeter Electromagnetic Waves
on the Water-Saline Solutions of DNA and
DNA-Ligand Complexes
There have been recently studies of various factors that affect the stability of DNA,
DNA-ligand and RNA-ligand complexes (1-6). In this paper we examine the effect
of radiation and ionic strength on the thermostability of DNA and DNA-ligand
complexes. The effect of non-thermal (∼50 µW/cm2) (50,3 and 64,5 GHz are resonant of water hexagonal structure frequencies) millimeter electromagnetic waves
(MM EMW) on water-saline solutions of DNA and DNA-ligand complexes with
EtBr and Hoechst 33258 is studied. Experimental data showed that radiation
increased the thermostability of the investigated samples. The differences between
the melting temperatures of radiated (Tmr) and non-radiated (Tmnr) DNA and its
complexes with ligands (δTm = Tmr-Tmnr) depended on the ionic strength; the greater
the ionic strength, the smaller is the δTm.
It is known, that the hydration of DNA decreases with increase of the Na+ concentration. Therefore, decrease of the value of δTm at high concentration of Na+ points
to the non significant effect of the radiation of the structure of hydration shell of
DNA and its complexes with the ligands, at these conditions. This conclusion is
supported by our experimental results according to which the radiation of DNA and
DNA-ligand complexes by non-resonant of water hexagonal structure frequency
MMEMW (48.3 GHz) caused no changes in the thermostability of the samples at
all investigated concentrations of Na+.
Experimental results obtained made it possible to suggest that radiation, like ionic
strength of solutions, increases the thermostability of DNA and DNA-ligand complexes.
1133
References
1. A. Borkar, I. Ghosh, and D. Bhattacharyya. J Biomol Struct Dyn 27, 695-712 (2010).
3. G. Singhal and M. R. Rajeswari. J Biomol Struct Dyn 26, 625-636 (2009)
4. B. Jin, H. M. Lee, S. K. Kim. J Biomol Struct Dyn 27, 457-464 (2010).
(2010).
Dynamics of Two Combined Reversible Binding
Ligands to DNA
Kinetic investigations on ligand binding to DNA describe the binding process as a
function of time and allow getting information on rates of formation and dissociation of DNA-ligand complexes. Usually one works on binding kinetics of a single
ligand binding on DNA. For examples of current study of ligand binding to nucleic
acids, see references 1-5.
In this paper binding of two different ligands with DNA is considered under
condtions in which both ligands occupy the same adsorption centers with different
binding constants, and each ligand occupies different number of absoption centers.
The ligand binding and dissociation of the complex can be represented as a discrete
Markov process, and the derived differential equations describe the kinetics of combined binding of various ligands to DNA. Analysis of the data show that depending on
adsorption parameters various modes of ligands binding to DNA may be realized.
Comparison of experimental curves of ligands interacting with DNA as a function
of time with theoretical curves allows to determine the rate constants of formation
and dissociation of DNA-ligand complex It is shown that the kinetics of DNA
occupancy with a ligand of given type depends on the ratios between binding
constant rates and concentration of ligands as well. Depending on their ratios, it
may be possible that nonmonotonic change of number of ligands adsorbed on DNA
over time may lead to inversion of ligand occupancy.
References
5. Y. Dalyan, I. Vardanyan, A. Chavushyan, and G. Balayan. J Biomol Struct Dyn 28,
123-131 (2010).
171
Valery B. Arakelyan1
Gohar G. Hovhannisyan2
Poghos O. Vardevanyan2
1Department
of Molecular Physics
of Yerevan State University,
A.Manooqian str. 1, Yerevan 0025,
Armenia
2Department
of Biophysics of Yerevan
State University, A.Manooqian str. 1,
[email protected]
1134
Investigation of Interaction of Podophyllotoxins
with DNA
172
Ara P. Antonyan1
Anna S. Ryazanova2
Hrachik R. Vardapetyan2*
1Department
of Biophysics, Yerevan
State University, Yerevan, 0025, Armenia
2Biomedical
faculty, Russian-Armenian
(Slavonic) University, Yerevan, 0051,
Armenia
[email protected]
*[email protected]
173
Igor G. Morgunov
Svetlana V. Kamzolova
G. K. Skryabin Institute of B
iochemistry
and Physiology of Microorganisms,
Russian Academy of Sciences, pr-t
Nauki 5, Pushchino, Moscow Region
142290, Russia
[email protected]
The complex formation of podophyllotoxin and its semi synthetic derivative –
etoposide with DNA has been investigated by absorption and thermal denaturation
methods. The data show that these ligands have a destabilizing effect on DNA. The
values of binding constant (Kb) obtained and the number of binding sites (n) of
these ligands with DNA indicate that both ligands bind with DNA by two ways –
specifically with limited sites of binding corresponding to intercalative (or semiintercalative) mechanism and non specifically (external with one chains of DNA)
with unlimited binding sites. For the various ways of binding to nucleic acids see
references 1-5. It has been shown that the value of Kb of etoposide to DNA is higher
than that of podophyllotoxin. The data obtained by these methods reveal that both
ligands interact with DNA and possess a higher affinity with AT sequences.
The data obtained also show that these ligands bind with DNA immediately and the
citotoxicity may be conditioned by inhibition of topoisomerses I and II of mammalian DNA, as well as a destabilization and damage to DNA itself.
References
(2010).
Yarrowia Lipolytica Yeast Possesses an Atypical
Catabolite Repression
Catabolite repression was thoroughly studied in the bacterium Escherihia coli
and, though not so well, in the yeast Saccharomyces cerevisiae (1, 2). Glucose and
related sugars repress the transcription of genes encoding enzymes required for
the utilization of alternative carbon sources. The different sugars produce signals
which modify the conformation of certain proteins that, in turn, directly or through
a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins (3, 4),
but there are different circuits of repression for different groups of genes (2). Catabolite repression allows the respective microorganisms effectively use carbohydrate
substrates, which first assimilate one of the two available substrates (commonly, a
carbohydrate), whereas the assimilation of the other substrate starts only after the
first substrate is fully consumed from the medium. The degree of catabolite repression varies very significantly in microorganisms. For example, glucose suppresses
the expression of invertase in Saccharomyces cerevisiae by 800 times, whereas the
expression of aconitate hydratase, cytochrome oxidase, and isocitrate dehydrogenase, are suppressed not more than by 10 times (1).
glycerol + oleate
GK
0,2
12
8
PC
IL
1135
MS
0,1
4
0
0
6
12
18
24
30
0
6
12
Time (h)
18
24
30
Time (h)
PDH
glucose + oleate
PC
IL
MS
0,2
12
8
0,1
4
0
0
6
12
18
24
0
6
12
Time (h)
18
24
27
Time (h)
glucose + hexadecane
PDH
12
PC
IL
MS
0,2
8
0,1
4
0
0
6
12
18
24
Time (h)
30
36
0
6
12
18
24
Time (h)
30
36
Figure: The growth of Yarrowia lipolytica in the two-substrate medium (left) and key enzymes (write)
in these conditions. Variation of • biomass,  glycerol,  oleic acid,  glucose and  hexadecane
(expressed in g/l); enzyme activity (expressed in U/mg of protein) of GK (glycerolkinase), PC (pyruvate
carboxylase), PDH (pyruvate dehydrogenase), IL (isocitrate lyase) and MS (malate synthase).
The non-conventional yeast Yarrowia lipolytica is an organism of great biotechnological interest due to its ability to excrete organic acids and proteins to the
medium. The phenomenon of catabolite repression in Yarrowia lipolytica yeast is
poorly studied.
The aim of this work was to study the metabolism of Yarrowia lipolytica yeast in
media containing two different carbon sources: glycerol/oleic acid; glucose/oleic
acid; glucose/hexadecane and its regulation.
It is evident from the Figure that when Yarrowia lipolytica was cultivated on the
mixture of glycerol and oleic acid, the concentration of these substrates started to
decrease just from the first hours of cultivation. Moreover, the utilization of these
two substrates went concurrently, although glycerol was utilized at a higher rate
than oleic acid. Glycerol kinase (the key enzyme of glycerol metabolism) and
two key enzymes of the glyoxylate cycle responsible for the metabolism of fatty
acids (isocitrate lyase and malate synthase) were induced from the fist hours of
cultivation and remained active to the end of the cultivation period. These results
suggest that glycerol is more easily utilizable substrate than oleic acid and probably
other fatty acids; however, glycerol does not suppress the metabolism of fatty acids.
In contrast, upon the cultivation of Yarrowia lipolytica on the mixture of glucose
1136
and oleic acid, the latter substrate began to be utilized only when the concentration
of glucose decreased from 10 to less than 2.5 g/L (on the 12th hour of cultivation).
The glycolytic enzymes pyruvate dehydrogenase and pyruvate carboxylase were
induced from the first hours of cultivation and remained at high levels until the
exhaustion of glucose in the medium. At the same time, the activities of isocitrate
lyase and malate synthase were very low during the metabolism of glucose, but
were rapidly induced after the exhaustion of glucose in the medium. These data
can be interpreted in such a manner that glucose at rather high concentrations suppresses enzymes involved in the metabolism of fatty acids.
When Yarrowia lipolytica was grown on the mixture of glucose and hexadecane,
the dynamics of growth and substrate consumption was typical of the diauxie phenomenon. Indeed, the utilization of hexadecane began only in several hours after
the time when glucose was completely exhausted in the cultivation medium. In this
case, the exhaustion of glucose arrested growth and the culture resumed growth
only after a lag period. The assay of enzymes showed that the glycolytic enzymes
pyruvate dehydrogenase and pyruvate carboxylase were active during the phase of
growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase
and malate synthase, were active during the phase of growth on hexadecane.
Thus, experiments on the cultivation of Yarrowia lipolytica on two substrate pairs:
glycerol/oleic acid and glucose/oleic acid and glucose/hexadecane indicated that
glycerol does not suppress the assimilation of oleic acid, whereas glucose suppresses it in such a manner that oleic acid begins to be consumed only after the
concentration of glucose in the medium falls to about zero.
References
1. K. D. Entian, H. J. Shuller. Glucose Repression (Catabolite Repression) in Yeast. In: Yeast
Sugar Metabolism. Biochemistry, Genetics, Biotechnology and Applications, F. K. Zimmerman,
K. D. Entian (Eds.), Technomoc Publishing, Basel, Switzerland, pp. 409-434 (1997).
2. J. M. Gancedo. Microbiol Mol Biol Rev 62, 334-361 (1998).
3. F. B. Guo and Y. Lin. J Biomol Struct Dyn 26, 413-420 (2009).
4. I. H. Cho, Z. R. Lu, J. R. Yu, Y. D. Park, J. M. Yang, M. J. Hahn, and F. Zou. J Biomol Struct
Dyn 27, 331-345 (2009).
1137
New Insights into Protein-DNA Electrostatic
Interactions: Beyond Promoters to Transcription
Factors Binding Sites
Electrostatic properties of genome DNA are well recognized to influence its interactions with different proteins such as histones (1, 2), and in particular the primary
recognition and regulation of transcription by RNA-polymerase. This enzyme may
identify promoters and evaluate their strength due to the peculiarity of their electrostatic profiles (3-7). The same problem of recognition of a limited number of
specific sequences in the long DNA molecule faces also transcription factors. To
reveal the role of electrostatic properties here we studied binding sites of different
families of these proteins.
174
Eugenia A. Krutinina
Gleb G. Krutinin
Svetlana G. Kamzolova
Alexander A. Osypov*
The analysis of the profiles using DEPPDB – DNA Electrostatic Potential Properties
Database – showed a number of common features, which can be illustrated with the
cAMP-CRP complex binding sites in the genome DNA in E.coli K12 (Figure 1).
Institute of Cell Biophysics of RAS,
The averaged profiles of the DNA electrostatic potential aligned around the CRP
dimer binding sites centers exhibit the pronounced rise in the negative potential value with the characteristic W-like profile in the concensus area of 16 bp
(TGTGA-N6-TCACA palindrome). The extensive (around 150-300 bp long),
symmetrical overall potential rise can not be explained by the influence of the
concensus itself and reflects the sequence organization of the flanking regions,
contributing to the high potential area formation. Apparently this sequence organization was selected evolutionary to support the binding site recognition by the
regulation protein molecule and its retention.
*[email protected]
Pushchino Moscow Region,
142290, Russia
It is worth noting that this high potential area is relatively AT-enriched though
doesn’t possess any textual consensus properties. Such enrichment is commonly
accepted as facilitating the DNA melting in the promoter regions. However, it is
clearly not the case in the present system, as the promoter core (and the initially
Figure1: Averaged electrostatic profile of the cAMP-CRP complex binding sites in the genome DNA in E. coli K12 (above) and the GC
content (below). The binding site consensus region is highlighted with gray.
1138
DNA melting point) lies downstream of the CRP binding sites, sometimes more
than 100 bp apart. So the function of this enrichment is rather the formation of the
high electrostatic potential trap for the CRP complex, especially given the fact of
the symmetry of this electrostatic valley, which wouldn’t be the case if due to the
promoter core influence.
The same overall properties, though vary in particular details, are typical to binding
sites of other families of transcription factors in a diverged range of bacterial taxa.
The data obtained reveal the role of electrostatic properties of DNA in the recognition of the transcription regulation proteins binding sites, further confirming their
universal importance in the protein-DNA interactions beyond the classical promoterRNA polymerase recognition and regulation. They demonstrate the necessity of the
studies of the electrostatic properties of genome DNA in addition to the traditional
textual analysis of its sequence.
References
3. R. V. Polozov, T. R. Dzhelyadin, A. A. Sorokin, N. N. Ivanova, V. S. Sivozhelezov,
S. G. Kamzolova. J Biomol Struct Dyn 16(6), 1135-43 (1999).
4. S. G. Kamzolova, A. A. Sorokin, T. D. Dzhelyadin, P. M. Beskaravainy, and A.A. Osypov.
J Biomol Struct Dyn 23(3), 341-346 (2005).
5. S. G. Kamzolova, V. S. Sivozhelezov, A. A. Sorokin, T. R Dzhelyadin, N. N. Ivanova, and
R. V. Polozov. J Biomol Struct Dyn 18(3), 325-334 (2000).
J Bioinform Comput Biol 4(2), 455-467 (2006).
7. A. A. Osypov, G. G. Krutinin, and S. G. Kamzolova. J Bioinform Comput Biol 8(3),
413-425 (2010).
1139
The Role of Electrostatics in Protein-DNA
Interactions in Phage Lambda
Physical properties of DNA are known to be essential for RNA polymerase-promoter
recognition. Especially electrostatic interactions between promoter DNA and RNA
polymerase is of considerable importance in regulating promoter function (1-5),
just like the electrostatic interactions between histones and DNA determine the
positioning of the nucleosomes and the expression of the genome (6, 7).
One of the elements that may play a crucial role in the promoter strength regulation
is the so-called “up-element”, which interacts with the alpha-subunit of the RNA
polymerase and thus facilitates its binding to the promoter. It was shown earlier that
the T4 phage strong promoters with pronounced “up-element” have high levels of
the electrostatic potential within it (3).
Using DEPPDB – DNA Electrostatic Potential Properties Database (5) – we studied
electrostatic properties of the lambda phage genome DNA. We observed that the
strong lambda phage promoters have pronounced “up-element” compared to the
absence of it in weak promoters. Promoters with intermediate strength possess
weak “up-element” (Figure 1).
Strong promoters also have the characteristic heterogeneity of the electrostatic profile,
known to differentiate promoters and coding regions. Pseudopromoters are located
in the region of high potential value with a prominent electrostatic trap. It is reported
that RNA polymerase binds them frequently and rests there for a long time (8).
Not only promoters are marked with peculiarities in the electrostatic properties.
Regulator proteins binding sites within the operators also have electrostatic features that correlate with binding ability of the corresponding regulatory proteins.
Apparently the sequence text itself shows weaker correlations. Another region that
shows a considerable increase in the electrostatic potential value is the attachment
site where the initial integration to the host genome DNA occurs.
Figure1: Electrostatic profile of a strong (3) weak (2) and intermediate-strength (1) promoters of the
phage lambda. The “up-element” area is highlighted with gray.
175
Gleb G. Krutinin
Eugenia A. Krutinina
Svetlana G. Kamzolova
Alexander A. Osypov*
Institute of Cell Biophysics of RAS,
Pushchino Moscow Region,
142290, Russia
*[email protected]
1140
The role of electrostatics in the DNA-protein interaction is directly confirmed by
the rigorous experiment evidence described in ref. 8. Using the visualization of the
individual molecules of RNA polymerase and phage DNA observed is the spatial
non-homogeneity of their interactions. The frequency of binding correlates with the
absolute value of the electrostatic potential along the DNA molecule.
These data highlight the universal role of electrostatics in the protein interactions
with the genome DNA.
References
S. G. Kamzolova. J Biomol Struct Dyn 16(6), 1135-43 (1999).
2. S. G. Kamzolova, A. A. Sorokin, T. D. Dzhelyadin P. M., Beskaravainy, and A. A. Osypov,
J Biomol Struct Dyn 23(3), 341-346 (2005).
3. S. G. Kamzolova, V. S. Sivozhelezov, A. A. Sorokin, T. R Dzhelyadin, N. N. Ivanova, and
R. V. Polozov. J Biomol Struct Dyn 18(3), 325-334 (2000).
J Bioinform Comput Biol 4(2), 455-467 (2006).
5. A. A. Osypov, G. G. Krutinin, and S. G. Kamzolova. J Bioinform Comput Biol 8(3),
413-425 (2010).
8. Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama, and T. Yanagida. Biophys J
76, 709-715 (1999).
176
Victor I. Danilov*
Vladimir V. Dailidonis
Tanja van Mourik
Herbert A. Früchtl
Institute of Molecular Biology and
Genetics, National Academy of Sciences
of Ukraine, 150 Zabolotnoho St., Kyiv,
The Study of the Nucleic Acid Base-Stacking by
Monte Carlo Method: Extended Cluster Approach
In the last two years we have seen publications in this Journal on nucleic acids based
on density functional theory and correlated ab initio calculations which examined
sugar-phosphate backbone, base stacking, hydrogen bonding, even ring expansion
and metal interactions. (1-3). Here a Metropolis Monte Carlo method based on the
extended cluster approach is used to investigate adenine-thymine (A/T), adenineuracil (A/U) and guanine-cytosine (G/C) base associates in a cluster containing
400 and 800 water molecules. It is shown that during the simulation each WatsonCrick base pair is transformed into a more favorable stacked configuration.
The results obtained allow to observe the whole process of convergence for the
first time (for more information, visit the Web site http://vd.bitp.kiev.ua).
Ukraine 03680
*[email protected] Table:
Energetic characteristics of the transformation of hydrogen-bonded base pairs to the stacked associates and of the base stacking reaction in water cluster (in kcal mol).
Transformation
ΔUtot
ΔUww
ΔUwb
Ubb
AT base pair > A/T stacked dimer
AU base pair > A/U stacked dimer
GC base pair > G/C stacked dimer
A 1 T > A/T stacked dimer
A 1 U > A/U stacked dimer
G 1 C > G/C stacked dimer
–15.9
–1.4
–32.3
–15.8
–8.8
–12.4
–3.4
12.6
20.3
–13.0
–3.2
–6.7
–18.8
–21.8
–50.4
–1.5
–1.9
–3.5
6.2
7.8
19.7
–4.3
–3.6
–2.3
It follows from this Table that all stacked associates in the water cluster are energetically more preferable to the corresponding Watson-Crick base pairs. The
changes in the interaction energies show that the water–base interaction (ΔUwb)
is the determining factor in favoring the stacked species over the base pair in an
aqueous cluster. This may be due to the smaller hydrophobic surface of the stacks.
The study showed that stacked dimers hydrate better than the hydrogen-bonded
associates. The formation of the A-T, A-U and G-C base pairs in the water clusters
was found to be energetically unfavorable, primarily due to the destabilizing contribution of the base-water interactions. The data allow us to calculate the transformation energy (ΔUtot) and its various contributions for all the three stacked associates
investigated in the water cluster. These results are given in the Table.
As can be seen, the formation of all stacked dimers was found to be favorable,
with transformation energies ranging from −8.8 to −15.8 kcal/mol. The preference
for the formation of these stacks results mainly from the favourable change in the
water-water interaction (Uww) and partly from the base-base interaction (Ubb)
during the base association reaction.
In contrast to the Watson-Crick base pairs, the formation of all stacked associates is
highly favorable. The water energy change, associated with the structural rearrangement of the water molecules around the bases during their association, contributes
most to the stabilization of the stacks. The stacked associates are significantly less
stabilized by the base-base interaction in comparison with the H-bonded base pairs.
Yet to a lesser extent this applies to the water-base interactions. Thus, the water–
water interaction is one of the main factors promoting stacked dimer formation, and
the data are a direct confirmation of the crucial role of the water-water interaction
in base stacking reported earlier in Ref. (4-6).
References
1. D. Vasilescu, M. Adrian-Scotto, A. Fadiel, and A. Hamza. J Biomol Struct Dyn 27, 465-476
(2010).
2. P. Sharma, S. Sharma, A. Mitra, and H. Singh. 19. J Biomol Struct Dyn 27, 65-81 (2009).
3. G. V. Palamarchuk, O. V. Shishkin, L. Gorb, and J. Leszczynski. J Biomol Struct Dyn 26,
653-661 (2009).
4. V. I. Danilov, I. S. Tolokh, Nature of the stacking of nucleic acid bases in water: a Monte
Carlo simulation. J Biomol Struct Dyn, 2, 119-130 (1984).
5. V. I. Danilov and T. van Mourik. Molecular Physics 106, 1487-1494 (2008).
6. V. I. Danilov, V. V. Dailidonis, T. van Mourik, and H. Fruchtl. (in preparation).
1141
1142
Coarse-Grained Model of Nucleic Acids
177
Maciej Maciejczyk1*
Aleksandar Spasic2
Adam Liwo3
Harold A. Scheraga4
1Department
of Physics and Biophysics
University of Warmia and Mazury
Olsztyn 10718, Poland
2Department
of Biochemistry and
Biophysics, University of Rochester
Medical Center, Rochester,
NY 14642, USA
3Faculty
of Chemistry University of
Gdańsk, Gdansk 80216, Poland
4Baker
Laboratory of Chemistry and
Atomistic simulations of nucleic acids are prohibitively expensive and, consequently, reduced models of these compounds are of great interest in the field. Most
coarse-grained models developed so far implemented harmonic (1, 2) or Go-like
potentials (3). Here we present physics-based coarse-grained model of nucleic
acids based on our recently developed method of coarse-graining (4). The model is
built of six types of rigid bodies connected by virtual bonds. Bases are represented
by several (three to five) centers of van der Waals and electrostatic interactions
(dipolar beads), deoyribose is approximated by single van der Waals interaction
center (neutral bead) and phosphate group is represented by charged van der Waals
sphere (charged bead). Recently developed method of coarse-graining was used
for parametrization of two-body long-range interactions between coarse-grained
objects (4). Bonded part of interaction energy was determined by fitting analytical
expression to potentials of mean force computed for the model system. Umbrella
sampling and WHAM methods were applied to the model three-nucleotide watersolvated system. Rigid-body equations of motion were integrated with simplectic
rotation matrix constraint method (5). Good numerical stability of microcanonical
simulation was achieved for timesteps up to 25 fs for the model DNA duplex.
References
1. W. R. Rudnicki, G. Bakalarski, and B. Lesyng. J Biomol Struct Dyn 17, 1097-1108 (2000).
2. M. Maciejczyk, W. R. Rudnicki, and B. Lesyng. J Biomol Struct Dyn 17, 1109-1116 (2000).
3. E. J. Sambirski, V. Oritz, and J. J. de Pablo. J Phys: Condens Matter, 21, 034105-034118
(2009).
4. M. Maciejczyk, A. Spasic, A. Liwo, and H. A. Scheraga. J Comp Chem 31, 1644-1655 (2010).
5. A. Kol, B. B. Laird, and B. J. Leimkuhler. J Chem Phys 107, 2580-2588 (1997).
Chemical Biology, Cornell University,
Ithaca, NY 14853, USA
*[email protected]
178
Garima Khandelwal1,2
Rebecca A. Lee2,4
B. Jayaram1,2,3
David L. Beveridge4
1Department
of Chemistry
2Supercomputing
Facility for
Bioinformatics and Computational Biology
3School
of Biological Sciences,
Indian Institute of Technology Delhi,
4Department
of Chemistry and Molecular
Biophysics Program, Wesleyan
University, Middletown, CT-06459, USA
[email protected]
[email protected]
[email protected]
[email protected]
Statistical Thermodynamics of DNA Sequences Based
on a Two State Model for the Base Pair Steps
A statistical thermodynamic framework is constructed for investigating the stability
of DNA sequences and their potential for interaction with ligands and proteins, on
the lines of structure based thermodynamics of Hilser and coworkers (1). Each base
pair step (the dinucletotide) is considered to be either closed (as in the intact double
helical DNA where in the Watson-Crick pairing and inter-strand and intra-strand
stacking is intact; or open ( as in the locally molten state in which the WatsonCrick pairing and inter-strand stacking is completely lost and intra-strand stacking
is partially disrupted). A sequence of n bases is characterized by n-1 base pair steps,
leading to a total of 2(n-1) microstates which can be enumerated explicitly. The bp
step free energies are adapted from experiment (2). This facilitates construction of
the partition function and development of thermodynamic parameters as statistical
mechanical ensemble averages over all the microstates. As an internal consistency
check, the computed average free energies are plotted against experimental melting
temperatures of 44 oligonucleotide sequences all at ~1M salt concentration, which
gives a correlation coefficient of 0.89. The methodology has the potential to address
questions concerning, sequence and stability, cooperativity in DNA-ligand/protein
binding etc.
References
1. V. J. Hilser, B. Garca-Moreno E., T. G. Oas, G. Kapp, and S. T. Whitten. Chemical Reviews
106(5), 1545-1558 (2006).
2. J. SanataLucia Jr. PNA, 95, 1460-1465 (1998).
3. G. Khandelwal, R. A. Lee, B. Jayaram, and D. L. Beveridge. (Manuscript in Preparation).
1143
In Silico Analysis of Structural Properties of Fungal
DNA Sequences and Promoter Prediction
Promoters play a central role in transcription initiation and gene regulation, so it is
necessary for these DNA regions to be differentiated from non-promoter sequences. Sequence motif based computational methods have not been able to identify
these regions with high degree of sensitivity and precision. On the other hand
several experimental and computational studies have shown that promoter sequences possess some special properties, which are common across all organisms. In
the current in silico study, we examined genomic regions upstream of genes of S.
cerevisiae with respect to their transcription start sites as well as the upstream
regions in all 7 yeast species with respect to their translation start sites, to see the
differences in their predicted structural features (stability, bendability and
nucleosome positioning preference, curvature, 2, 3). The core promoter regions
show different structural properties like lower stability, lpwer bendability and
slightly higher curvature compared to other neighbouring regions (1). Moreover
we also observed that TATA-containing promoters are less stable, more flexible and
more curved compared to TATA-less promoters, but both have similar nucleosome
positioning preferences. The variability of structural properties of TATA-less and
TATA-containing promoters may be due to differences in their role in regulation
of gene expression. Detailed analysis of these promoters, comparison with results
for prokaryotic promoters (4, 5) as well as quantitative prediction ability of our
in-house software ‘PromPredict’ will be presented.
179
Manju Bansal*
Venkata Rajesh Yella
Molecular Biophysics Unit,
Indian Institute of Science,
Bangalore 560012, India
*[email protected]
References
1.
2.
3.
4.
5.
A. Kanhere and M. Bansal. Nucleic Acids Res 33, 3165-3175 (2005).
F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010).
V. Rangannan and M. Bansal. Mol BioSyst 5, 1758-1769 (2009).
V. Rangannan and M. Bansal. Bioinformatics 26, 3043-3050 (2010).
Temperature-Induced Unfolding Thermodynamics of
DNA Hairpins Containing Internal Loops and Their
Targeting with Complementary Strands
The main focus of our research is to further our understanding of the physicochemical properties of nucleic acid structures. In particular, to investigate the melting behavior of unusual DNA structures, by determining their complete unfolding
thermodynamic profiles. In this work, we use a set of DNA hairpins as a model
to mimic a common motif present in the secondary structures of mRNA, i.e., a
stem-loop motif with an internal loop in the stem. Specifically, we used a combination of UV spectroscopy and differential scanning calorimetry (DSC) melting
techniques to determine the unfolding thermodynamics of a set of hairpins with
sequence: d(GCGCTnGTAACT5GTTACTnGCGC, where Tn corresponds to internal loops with n = 1, 3 or 5 and “T5” is an end loop of 5 thymines, as shown in the
figure. The UV melting curves of each hairpin show monophasic transition with
TMs that are independent of strand concentration, confirming their intramolecular
formation. Analysis of the DSC profiles indicates that the unfolding of each hairpin results from the typical compensation of a unfavorable enthalpy (breaking of
base-pair stacks) and favorable entropy contributions (release of ion and water molecules). The increase in the size of the internal loop from 2 to 10 thymines yielded:
a) lower TMs and similar enthalpy contributions; b) lower heat capacity values that
correlated with the lower releases of structural water molecules; and c) higher
ion releases. Furthermore, we used isothermal titration calorimetry and DSC to
Luis A. Marky
Iztok Prislan
Cynthia Lee
Hui-Ting Lee
180
Department of Pharmaceutical Sciences,
University of Nebraska Medical
Center, 986025 Nebraska Medical
Center, Omaha, NE 68198-6025
1144
181
Nicholas Taylor
Gary Male
Christoph W. Müller*
Structural and Computational Biology
Unit, European Molecular Biology
Laboratory (EMBL), 69117 Heidelberg,
Germany
*[email protected]
investigate directly and indirectly, respectively, the thermodynamics of the reaction
of each hairpin with their partially complementary strands. The results showed that
all three targeting reactions yielded favorable free energy contributions that were
enthalpy driven. Overall, this approach works because of the favorable heat contributions resulting from the formation of base-pair stacks involving the unpaired
bases of the loops. Supported by Grant MCB-0315746 from the NSF.
DNA Recognition in RNA Polymerase III Transcription
Eukaryotic transcription requires the coordinated activities of enhancers that recognize specific DNA target sites, co-activators and general transcription factors
to recruit RNA polymerases to their transcription start sites (1). Among the three
eukaryotic RNA polymerases, recruitment of RNA polymerase II is by far the
most complex process requiring a large number of different factors. In comparison,
recruitment of RNA polymerase I and III is simpler and therefore more amenable for
detailed structure-function analyses. RNA polymerase I and III are responsible for
producing non-coding RNAs such as pre-RNA and tRNA. Together, they contribute
up to 80% to the total transcriptional activity in growing cells and need to be tightly
regulated. In the last years there has been increasing awareness that mis-regulation
of RNA polymerase I and III is associated with different types of cancer (2).
Recruitment of RNA polymerase III to the transcription start site requires binding of
the general transcription factors TFIIIB and TFIIIC. TFIIIC is a large DNA-binding
complex with a total molecular weight of 0.6 MDa composed of six polypeptides.
TFIIIC can be subdivided into two subcomplexes, that initiate RNA polymerase III
transcription by binding to two internal promoter sites in tRNA genes named A-and
B-box. We are using a combination of X-ray crystallography, electron microscopy
and biochemistry to gain insights into the structural organization of the RNA polymerase III enzyme and its general transcription factors (3-5). During the last years
our group has determined crystal structures corresponding to ~2/3 of the entire
TFIIIC complex and obtained a low-resolution EM reconstruction of TFIIIC that
serves as a starting point to assemble the entire complex. We will present insights
into DNA recognition by the RNA polymerase III-specific general transcription
factor complex TFIIIC resulting from this studies.
References
1. R. D. Kornberg. Proc Natl Acad Sci 104, 12955-61 (2007).
2. R. J. White. Trends Genet 27, 622-629 (2008).
3. A. Mylona, C. Fernandez-Tornero, P. Legrand, M. Haupt, et al. Mol Cell 24, 221-232
(2006).
4. C. Fernandez-Tornero, B. Bottcher, M. Riva, C. Carles, et al. Mol Cell 25, 813-23 (2007).
5. C. Fernandez-Tornero, B. Bottcher, U. J. Rashid, et al. EMBO J. 29, 3762-72 (2010).
182
Yaakov (Koby) Levy
Weizmann Institute of Science, Israel
Protein Sliding and Jumping Along DNA
Interactions between proteins and nucleic acids are ubiquitous and central to the
life of cells. The remarkable efficiency and specificity of protein-DNA recognition presents a major theoretical puzzle given the size of the genome, the largea
number of molecular species in vivo at a given time, and the crowded environment
they inhabit. Our research is motivated at quantitatively advancing our understanding of the kinetics and mechanisms of protein-DNA recognition, the molecular
and physical principles of fast association, and protein recruitment by DNA. For
the first time, we have visualized protein sliding along DNA where the protein
binds DNA nonspecifically and performs a helical motion when it is placed in the
major groove. Using coarse-grained models we found that the spiral motion along
1145
the sugar-phosphate rail is typical to various DNA-binding motifs. This stochastic
dynamics that is governed by electrostatic forces has similar structural features to
the specific binding mode of the protein with the DNA. In our study, we address
the question of the linkage between the molecular architecture of DNA-binding
proteins and the search mechanism. We have explored the interplay between the
molecular characteristics of the proteins (e.g., DNA recognition motifs, degree of
flexibility, and oligomeric states) and the nature of sliding, intersegment transfer
events and the overall efficiency of the DNA search.
Indirect Readout by the P22 Repressor Protein:
How Charge and Solvent Organization Aid
Binding Site Discrimination
To form a lysogen, the repressor of bacteriophage P22 (P22R) must bind and discriminate between its six DNA binding sites on the P22 chromosome. Comparison
of the six naturally occurring binding site sequences shows that the sequences of the
symmetrically-arrayed outermost base pairs in these sites are highly conserved, but
the sequence of the innermost bases are not (Figure 1). Crystallographic studies show
the conserved bases are directly contacted by bound protein, but the non-conserved
central bases are not (1). Nonetheless through a sequence recognition strategy called
“indirect readout”, the affinity of P22R for DNA varies with the non-contacted base
sequence (2). We wish to understand the mechanism of indirect readout.
P22R DNA binding causes the non-contacted region to assume a B’-DNA configuration (1). The minor groove of B’-DNA is very narrow and contains a spine of highly
ordered solvent molecules (1). We hypothesize that indirect readout depends on P22R’s
ability to either sense and/or impose B’ structure on the bases in the non-contacted of
its binding site. Thus the ease with which the non-contacted bases can assume B’ configuration by P22R determines the protein’s affinity for the binding site.
We showed that P22R binding sites lacking a minor groove N2-NH2 group on the
base pairs at the non-contacted positions bind P22R ~12-fold better than those that
do not contain this group at these positions. This finding identifies the N2-NH2
group as the mediator of indirect readout by P22R. We also found that changing
either E44 or E48, which are directly juxtaposed to the DNA phosphate backbone
near the non-contacted bases (1) (Figure 2), to uncharged residues eliminates the
ability of P22R to recognize non-contacted base sequence. This finding suggests
that these residues function to force the P22R-bound DNA into the B’ configuration. An N46A mutation completely blocks P22R’s ability to bind to sites bearing
sequences in the non-contacted region, which cannot normally assume the B’ configuration. This finding suggests that N46 is responsible for inducing and/or stabilizing B’ conformation in the non-contacted bases in P22R-DNA complexes.
Figure 1: DNA sequences of naturally occuring operators in the P22 chromosome. The contacted and
conserved bases are boxed and in bold. The central non-contacted bases are positions 8-11.
183
Lydia-Ann Harris
Gerald Koudelka
University at Buffalo (SUNY),
109 Cooke Hall, Buffalo, New York
14260
[email protected]
[email protected]
1146
Figure 2: Positions of E44 and E48 relative to the DNA backbone. The P22R protein is above and the
DNA is below. E44 and E48 are spacefilled (yellow) the nearest phosphates are spacefilled (cyan) (2).
We have found P22R sensitivity to non-contacted base sequence, directly affects
repressor’s ability to sense sequence changes in the contacted bases. We provide
evidence that this effect may be due to the spine of hydration which may run along
the minor groove from one end of the binding site to the next. Together these results
provide a fascinating picture of how P22R uses indirect readout in binding site
discrimination.
References
1. D. Watkins. Biochemistry 47, 2325-2338 (2008).
2. L. Wu, A. Vertino, and G. B. Koudelka. J Biol Chem 267, 9134-9135 (1992).
184
Joseph Racca*
Yen-Shan Chen1
Nelson Phillips1
Agnes Jansco-Radek1
Michael Weiss1
Elisha Haas2*
1Department
of Biochemistry,
Case Western Reserve University,
Cleveland, OH 44106
2Faculty
of Life Sciences, Bar Ilan
University, Ramat Gan, Israel 52900
*[email protected]
A Kinetic Code for SRY-Dependent Transcriptional
Activation Based on DNA Intercalation
The HMG (High-Mobility-Group) box defines a superfamily of DNA-bending
proteins remarkable for partial DNA intercalation by “cantilever” side chains. Here,
we describe the role of the cantilever residue of SRY, the human male-determining
factor encoded by the Y chromosome. Mutations at this and related sites abutting
the sharp DNA bend cause gonadal dysgenesis leading to chromosome sex reversal. A yeast-one-hybrid model was designed to enable rapid and efficient screening of mutational libraries in relation to the database of clinical substitutions and
evolutionary relationships among HMG boxes. Our results demonstrate (a) that the
only allowed cantilevers are those observed among mammalian SRY and related
SOX sequences (Ile, Met, Leu, and Phe); (b) that mutations associated with de novo
sex reversal in vivo markedly impair DNA binding; and (c) that variant cantilevers
are associated with a range of kinetic on- and off-rates. To correlate the biophysical properties of the bent protein-DNA complex with gene-regulatory activity,
a transfection assay was developed in an immortalized rodent embryogenic cell
line derived from the differentiating gonadal ridge at the time of onset of SRY
expression. A biological read-out was provided by real-time quantitative PCR
analysis of the endogenous Sox9 gene, a key downstream target of SRY in the
male transcriptional program. Remarkably, transcriptional potency was observed
to correlate with the kinetic life time of the bent DNA complex and not its thermodynamic stability. Whereas a variant Val cantilever (too short to fully insert
within the DNA double helix) suffices in vitro to direct formation of a wellorganized but short-lived SRY-DNA complex of near-native DNA bend angle, no
gene activation was observed in the rodent cell culture model. We propose that
the active cantilever side chains act as a kinetic anchor to provide a long-lived
DNA bend, in turn enabling formation of a functional male-specific transcriptional
pre-initiation complex.
1147
The Mode of DNA Recognition and Dimerization
Specificity of MITF Revealed by
Three Crystal Structures
The Microphthalmia-associated Transcription Factor (MITF) regulates the expression of pigment-cell specific genes in melanocytes, the mature pigment producing
cells of the skin and hair follicles. In addition, MITF function is of major interest in
the investigation of the mechanisms leading to melanoma.
MITF belongs to the superfamily of basic Helix-Loop-Helix leucine zipper transcription factors (b-HLH-Zip). Like other b-HLH-Zip factors, MITF can bind a
subset of the canonical palindromic E-box sequence (CANNTG) as well as related
asymmetric motives like M-box (TCATNTG); nevertheless the exact mechanism in
which MITF recognizes the correct promoters of target genes is not yet fully understood. Within the b-HLH-Zip family, MITF associates with the Tfe factors, but no
heterodimeric complex containing MITF and the related Myc, MAX or USF-1 have
been observed, raising the question how this discrimination is achieved.
We determined the crystal structure of MITF in its apo form and of two different
complexes of MITF with DNA containing two different target motives (E-box
and M-box). We pursued the study measuring interactions between these DNA
motives and several MITF mutants with known phenotype in mice, using different
technique such as using Isothermal Titration Calorimetry, Fluorescence Anisotropy
or EMSA. Comparing structural, biophysical and biological data, this study reveals
a particular mechanism of DNA recognition by MITF and how MITF discriminates
between the E and M boxes. In addition, our data demonstrate an unusual mode of
dimerization that might explain how MITF select its heterodimerization partners.
185
Vivian Pogenberg1*
Viktor Deineko1
Morlin Milewski1
Alexander Schepsky2
Eirikur Steingrimsson2
Matthias Wilmanns1
1EMBL-Hamburg,
Notkestrasse 85, 22603 Hamburg,
Germany
2Department
of Biochemistry and
Molecular Biology, Faculty of Medecine,
University of Iceland, 101 Reykjavik,
Iceland
*[email protected]
186
A Convenient Fluorescence Assay of
Polyamide-DNA Interactions
Polyamides (PA) are distamycin-type ligands of DNA that bind the minor groove
and are capable of sequence selective recognition (1). This capability provides
a viable route to their development as therapeutics. PAs have limited intrinsic
fluorescence behavior, making this signal poorly suited for binding assays, and
the introduction of a dye to this structure requires additional synthetic steps and
can change uptake for cell-based assays (2). The most commonly performed
assays of DNA binding by PAs involve complex techniques like calorimetry (3),
surface plasmon resonance (3), and footprinting (4). Presented here is a simple and
convenient fluorescence assay for polyamide-DNA binding. PAs are titrated into
a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU a
few nt away from the PA binding site. In a study of 12 polyamides, PA binding
leads to a steady, reproducible decrease in fluorescence intensity that can be used
to generate binding isotherms. The assays works equally well with both short
(6-8 ring) and long (14 ring) polyamides, and Kd values ranging from 0.5 to at
least 300 nM (depending on PA composition and target sequence), were readily
obtained using a simple monochrometer configuration. Stronger Kd values were
confirmed with a PCR fragment and quantitative footprinting analysis. Competition assays indicate that the effects of the dye are typically negligible when the
dye-labeled dU residue is outside the binding site. When the dye is placed within
the predicted binding site, binding experiments show disturbances in the classical
binding trend. This effect provides a means to confirm the binding site in DNAs
with more than one potential binding site.
c/o DESY,
Cynthia M. Dupureur*
James K. Bashkin
Karl Aston
Kevin J. Koeller
Kimberly R. Gaston
Gaofei He
Department of Chemistry and the Center
for Nanoscience, University of Missouri
St. Louis, St. Louis, MO 63121, USA
*[email protected]
1148
This research is supported by NIH R01 AI083803-02.
References
1. P. B. Dervan and B. S. Edelson. Curr Opin Struct Biol 13, 284-299 (2003).
2. K. S. Crowley, D. P. Phillion, S. S. Woodard, B. A. Schweitzer, M. Singh, H. Shabany,
B. Burnette, P. Hippenmeyer, M. Heitmeier, and J. K. Bashkin. Biorg Med Chem Lett 13,
1565-1570 (2003).
3. H. Mackay, T. Brown, P. B. Uthe, L. Westrate, A. Sielaff, J. Jones, J. P. Lajiness, J. Kluza,
C. O’Hare, B. Nguyen, Z. Davis, C. Bruce, W. D. Wilson, J. A. Hartley, and M. Lee. Bioorg
Med Chem 16, 9145-9153 (2008).
4. J. W Trauger and P. B. Dervan. Methods Enzymol 340, 450-466 (2001).
187
Balasubramanian Harish
Jannette Carey
Chemistry Department, Princeton
University, Princeton,
NJ 08544-1009, USA
[email protected]
[email protected]
188
Pinak Chakrabarti1,2*
Sucharita Dey1
Arumay Pal2
Mainak Guharoy2
1Bioinformatics
2Department
Centre
of Biochemistry, Bose
Institute, P-1/12 CIT Scheme VIIM,
Kolkata 700054, India
*[email protected]
*[email protected]
A Mechanism to Understand the Dynamics
of Tryptophan Repressor
Trp repressor was an early paradigm for understanding DNA binding, but presented
many still-unsolved mysteries, including the non-cooperative binding of L-tryptophan, the presence of a buried water layer at the DNA interface, and extensive
local dynamics in the DNA-binding domains. The crystal structure of a temperature-sensitive mutant of E. coli TrpR now suggests a mechanism for the dynamics
of the wildtype protein. One subunit of the mutant crystal structure shows an extensive rearrangement in the DNA-binding region, whereas the other subunit is in the
wildtype conformation. Comparison with isomorphous wildtype crystals reveals
that the distorted conformation is not an artifact of crystallization. Reinvestigation
of NMR data for the mutant shows that this conformation can explain NOEs that
are not accounted for by a wildtype-like structure. A survey of the extensive NMR
data on the wildtype protein reveals that many puzzling features in the dynamics
of the protein could also be explained by the presence of this alternative conformation. This hypothesis is presently being tested by NMR and molecular dynamics
simulations.
Physicochemical Features of Protein-DNA
Interactions and the Identification of Interface
Region in DNA-Binding Proteins
The use of physicochemical features of protein-protein and protein-DNA interfaces
can act as useful constraints in docking studies (1-3). Amino acid residues important
for structure and function are evolutionary conserved, and tend to cluster together
at the binding site (4, 5). Even though recently, there is substantial progress on our
understanding of the interactions between DNA and proteins (6-8), still we lack
quantitative or semi quantitative description of the surfaces involved. Following
the protocol used in protein-protein interfaces (5), here we analyze the clustering
of conserved residues in protein-DNA interfaces and show how this and other features, such as the propensity of residues to occur in the interfaces, can be used for
the discrimination of the real interface from other random surface patches.
In 129 non-redundant interfaces from 126 protein-DNA complexes, 81% have the
conserved positions clustered within the overall interface region – indicated by rho
(ratio of parameters representing the degree of clustering of conserved residues
relative to the overall interface) being greater than 1. The use of rho can identify
the interface (with rank 1) from other randomly generated surface patches in ~46%
of the cases. The incorporation of the Euclidean distance of the composition of
a surface patch from the average value in all protein-DNA interfaces improves
1149
the efficiency by another 6%. While the effectiveness of the clustering in the discrimination of the real interface is rather mediocre, the use of Rp (9) (the number
of a residue type in a patch multiplied by its propensity to occur in the interface,
summed over all the residues in the patch) can identify 81% of the interfaces with
rank 1. Another parameter Dp, the number of potential hydrogen bond donors in the
patch gives an accuracy of ~65%.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
S. Ahmad, M. M. Gromiha, and A. Sarai. Bioinformatics 20, 477-486 (2004).
Y. Mandel-Gutfrend and H. Margalit. Nucleic Acids Res 26, 2306-2312 (1998).
S. Biswas, M. Guharoy, and P. Chakrabarti. Proteins 74, 643-654 (2009).
S. Ahmad, O. Keskin, A. Sarai, and R. Nussinov. Nucleic Acids Res 36, 5922-5932 (2008).
M. Guharoy and P. Chakrabarti. BMC Bioinformatics 11, 286 (2010).
C. Carra and F. A. Cucinotta. J Biomol Struct Dyn 27, 407-427 (2010).
R. P. Bahadur, P. Chakrabarti, F. Rodier, and J. Janin. J Mol Biol 336, 943-955 (2004).
Getting a Firm Grip on DNA: Understanding DNA
Recognition at the Finger-finger Interface of Zinc
Finger Proteins
Zinc finger nucleases hold tremendous potential for site-specifically
editing genomes in a variety of organisms (1). However, their utility is predicated on the ability to efficiently create sequence-specific C2H2 Zinc
Finger Proteins (ZFPs) for a wide variety of target sequences. Each zinc finger
module typically binds to a 3 bp core DNA element (DNA triplet). Zinc finger
modules have been characterized that can specify most of the sixty-four possible
DNA triplets. These modules can be assembled into multi-finger zinc finger proteins (modular assembly) that bind extended target sites (9-12 bp), where typically
three to four zinc fingers are employed for efficient DNA recognition. However,
modularly-assembled ZFPs often show poor specificity presumably due to incompatible specificity determinants at the finger-finger interface (2). To understand the
influence of these interactions on DNA recognition, we employed bacterial onehybrid selections (3, 4) to identify groups of amino acid residues at the interface of
two-finger modules that specify all sixteen 2 bp interfaces between the two DNA
triplets. In total, we could identify two-finger modules that specify >90% of these
targets. Further analysis of the selected modules suggests the presence of complex interactions at the finger-finger interface that contribute to context-dependent
effects on specificity. These selected finger pairs are functional in vivo, as zinc
finger nucleases employing these modules generate targeted lesions in zebrafish.
Ultimately, understanding finger-finger interactions will allow the rational design
of multi-finger ZFPs for their use as artificial proteins and aid the assignment of
specificities for naturally-occurring zinc finger proteins.
References
1. F. D. Urnov, E. J. Rebar, M. C. Holmes, H. S. Zhang, and P. D. Gregory. Nat Rev Genet 11,
636-646 (2010).
2. C. L. Ramirez, J. E. Foley, D. A. Wright, F. Muller-Lerch, S. H. Rahman, T. I. Cornu,
R. J. Winfrey, J. D. Sander, F. Fu, J. A. Townsend, T. Cathomen, D. F. Voytas, and
J. K. Joung. Nature methods 5, 374-375 (2008).
3. X. Meng, M. B. Noyes, L. J. Zhu, N. D. Lawson, and S. A. Wolfe. Nat Biotechnol 26,
695-701 (2008).
4. M. B. Noyes, X. Meng, A. Wakabayashi, S. Sinha, M. H. Brodsky, and S. A. Wolfe. Nucleic
Acids Res 36, 2547-2560 (2008).
189
Ankit Gupta1,2
Amy Rayla1
Ryan G. Christiansen3
Nathan D. Lawson1
Gary D. Stormo3
Scot A. Wolfe1,2,*
1Program
in Gene Function and
Expression
2Department
of Biochemistry and
Molecular Pharmacology, University
of Massachusetts Medical School,
Worcester, MA 01605, USA
3Department
of Genetics, Washington
University School of Medicine,
St. Louis, MO 63108, USA
*[email protected]
1150
190
Tianyin Zhou
Ana Carolina Dantas Machado
Remo Rohs*
Molecular and Computational Biology,
University of Southern California, 1050
Childs Way, RRI 404C, Los Angeles,
CA 90089, USA
*[email protected]
Intrinsic DNA Shape of Fis-DNA Binding Sites
Determines Binding Affinity
The bacterial nucleoid-associated protein Fis (factor for inversion stimulation)
binds to DNA of various DNA nucleotide sequences at binding affinities varying
in three orders of magnitude. Johnson and coworkers recently reported 11 crystal
structures of the Fis protein bound to high- and low-affinity sites (1). A common
feature of these Fis-DNA complexes is the narrow minor groove in the central
region of the DNA targets. Without the input of any structural data derived from
experimental studies, we predicted the shape of the naked Fis-DNA binding sites
based on our Monte Carlo algorithm (2). For this purpose, we generated an ideal
B-DNA double helix of a given sequence without any structural identity of dinucleotide steps and sampled the three-dimensional structure of high- and low-affinity
Fis-DNA binding sites. In agreement with the hypothesis presented by Johnson
and coworkers (1), we find that the shape of the naked binding site with the highest
Fis-DNA binding affinity already assumes a shape with a minor groove narrowing in its central region similar to the one observed in the crystal structure of the
complex. In contrast, the Fis-DNA site with the lowest binding affinity exhibits in
its unbound state a shape that is essentially the one of average B-DNA. Although
the Fis protein binds DNA largely non-specifically, the finding that DNA shape
determines binding affinity indicates a similar mechanism as the one that we previously described for the sequence-specific DNA recognition by the papillomavirus
E2 protein. The E2 protein binds to DNA that is intrinsically bent as observed in
the protein-DNA complex with high affinity and to DNA that is essentially straight
with low affinity (2). The data presented for the larger set of Fis-DNA binding sites
further validates our structure prediction method and emphasizes the important role
of DNA shape in protein-DNA recognition (3, 4). Moreover, protein-DNA binding
affinity was shown to be correlated with gene regulatory control (5), thus suggesting
that structural data that explains binding affinity leads to a better understanding
of protein-DNA recognition, both for largely non-specific DNA interactions with
architectural proteins and highly specific DNA readout by transcription factors.
References
1. S. Stella, D. Cascio, and R. C. Johnson. Genes Dev 24, 814-26
2. R. Rohs, H. Sklenar, and Z. Shakked. Structure 13, 1499-509 (2005).
3. R. Rohs, S. M. West, A. Sosinsky, P. Liu, R. S. Mann, and B. Honig. Nature 461, 1248-53
(2009).
4. R. Rohs, X. Jin, S. M. West, R. Joshi, B. Honig, and R. S. Mann. Annu Rev Biochem 79,
233-69 (2010).
5. S. V. Nuzhdin, A. Rychkova, and M. W. Hahn. Trends Genet 26, 51-3 (2010).
1151
Structural Studies of Nucleic Acid-Binding Proteins
Involved in Regulating Cell Differentiation
and Pluripotency
Transcription factors play a central role in regulating cell differentiation and
de-differentiation. The use of “cocktails” of transcription factors to promote the
reprogramming of adult fibroblasts into induced pluripotent stem cells (iPS) has
generated tremendous interest in biology and medicine. The originally reported sets
of iPS generating factors contained Oct4, Sox2, Klf4 and c-Myc (1) or Oct4, Sox2,
Nanog and Lin28 (2). Here we report on structural and biochemical studies of two
of these proteins, Klf4 and Lin28.
Klf4 (Krueppel-like factor 4) is a zinc-finger transcription factor required for the
maturation of epithelial tissues. Crystal structure analyses of two different zincfinger fragments of Klf4 reveal that the two C-terminal C2H2 zinc-finger motifs
of Klf4 are required for DNA site specificity and the induction of macrophage
differentiation (3). The N-terminal zinc finger, conversely, inhibits the otherwise
cryptic self-renewal capacity of Klf4. A Klf4 zinc-finger domain mutant induces
self-renewal and block of cell maturation.
Lin28 is a highly conserved RNA-binding protein and was described to modulate
the processing of let-7 microRNA precursors (4). The small protein contains a coldshock domain (CSD) and a tandem array of retroviral-type CCHC zinc fingers. Both
protein motifs are presumably involved in RNA binding. Crystal structure analysis
reveals that the Lin28 CSD resembles the bacterial cold shock proteins (5-8). The
presence of conserved nucleotide-binding subsites of the surface of Lin28 CSD
suggests a common mode of DNA or RNA single-strand binding of Lin28 and
bacterial cold shock proteins (9, 10).
References
1. K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichikasa, K. Tomoda, and S. Yamanaka.
Cell 131, 861-872 (2007).
2. J. Yu, M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J. L. Frane, S. Tian,
J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, I. I. Slukvin, and J. A. Thomson. Science
318, 1917-1920 (2007).
3. A. Schuetz, D. Nana, C. Rose, G. Zocher, M. Milanovic, J. Koenigsmann, R. Blasig,
U. Heinemann, and D. Carstanjen. Cell Mol Life Sci DOI 10.1007/s00018-010-0618-x
(2011).
4. S. R. Viswanathan and G. O. Daley Cell 140, 445-449 (2010).
5. H. Schindelin, M. Herrler, G. Willimsky, M. A. Marahiel, and U. Heinemann. Proteins:
Struct Funct Genet 14, 120-124 (1992).
6. H. Schindelin, M. A. Marahiel, and U. Heinemann. Nature 364, 164-168 (1993).
7. H. Schindelin, W. Jiang, M. Inouye, and U. Heinemann. Proc Natl Acad Sci USA 91,
5119-5123 (1994).
8. U. Mueller, D. Perl, F. X. Schmid, and U. Heinemann. J Mol Biol 297, 975-988 (2000).
9. K. E. A. Max, M. Zeeb, R. Bienert, J. Balbach, and Heinemann. J Mol Biol 360, 702-714
(2006).
10. K. E. A. Max, M. Zeeb, R. Bienert, J. Balbach, and U. Heinemann. FEBS J 274, 1265-1279
(2007).
191
Udo Heinemann*
Florian Mayr
Anja Schuetz
Macromolecular Structure and
Interaction Group, Max-Delbrück Center
for Molecular Medicine,
Robert-Rössle-Str. 10, 13125 Berlin,
Germany
*[email protected]
1152
192
Remo Rohs
Molecular and Computational Biology,
University of Southern California, 1050
Childs Way, RRI 404C, Los Angeles,
CA 90089, USA
[email protected]
The Role of DNA Shape on Protein-DNA Binding
Affinity and Specificity
Gene regulation requires highly specific interactions between proteins and their
DNA binding sites. This high level of binding specificity in protein-DNA readout is
achieved through the recognition of both linear sequence (base readout) and threedimensional structure (shape readout) (1). DNA shape is specifically recognized by
a variety of protein families, and we have identified two different ways of modulating DNA shape. One widely observed phenomenon is the variation of the shape
of the double helix as a function of nucleotide sequence. This mode of sequencedependent DNA shape was first found to affect the DNA binding specificity of the
Hox protein Scr (Sex combs reduced) (2). A general study showed the critical role
of DNA shape on the binding of other transcription factors to their target sites and
on the genome packaging through nucleosome formation (3).
Whereas the findings on the readout of sequence-dependent DNA shape are based
on the analysis of crystal structures, we now expanded the description of DNA
shape as binding affinity and specificity determinant including systems for which
no structural data is available. We used our Monte Carlo algorithm (4) for predicting the shape of various DNA binding sites. In one study we relate binding affinity
of the architectural protein Fis (factor for inversion stimulation) to the shape of
high- and low-affinity Fis-DNA binding sites (see poster abstract by Zhou et al.).
In another study the DNA structure predictions of multiple binding sites of the
eight Drosophila Hox proteins and their cofactors Extradenticle and Homothorax,
as identified in SELEX-seq experiments, reveal intrinsic DNA shape as a selection
criterion contributing to binding specificity (see poster abstract by Liu et al.).
In addition to the sequence dependence of DNA shape, we identified a different
mode for varying the shape of DNA binding sites. In the case of DNA recognition
by the tumor suppressor p53, the base pairing geometry at specific positions of the
p53 response element deviates from standard-Watson-Crick geometry (5). Certain
base pairs assume Hoogsteen geometry, thus affecting local DNA shape and in turn
the interaction with arginine residues that were found to be frequently mutated in
human tumors. If applicable to other systems, non-Watson-Crick base pairs would
effectively extend the four letter genomic alphabet, demonstrating the importance
of considering three-dimensional structure (6).
References
1. R. Rohs, X. Jin, S. M. West, R. Joshi, B. Honig, and R. S. Mann. Annu Rev Biochem 79,
233-69 (2010).
2. R. Joshi, J. M. Passner, R. Rohs, R. Jain, A. Sosinsky, M. A. Crickmore, V. Jacob,
A. K. Aggarwal, B. Honig, and R. S. Mann. Cell 131, 530-43 (2007).
(2009).
5. M. Kitayner, H. Rozenberg, R. Rohs, O. Suad, D. Rabinovich, B. Honig, and Z. Shakked.
Nat Struct Mol Biol 17, 423-9 (2010).
6. B. Honig and R. Rohs. Nature 470, 472-3.
1153
Exploring the DNA Binding Specificities of
Homeodomain Complexes using SELEX-sequencing
Hox genes encode an evolutionarily conserved set of homeodomain-containing
transcriptional regulators that play critical roles in the development of all metazoans. Although first discovered in Drosophila because of their role in anterior
(A)-posterior (P) axial patterning, these genes are now known to assign morphological identities along the AP axes in both vertebrates and invertebrates (1). Hox
proteins typically bind to degenerate AT-rich DNA sequences. This low degree of
sequence specificity in vitro contrasts with the highly gene-specific regulation Hox
proteins carry out in vivo. Complicating the Hox specificity problem is that this
family of proteins, which are encoded by eight Hox paralogs in Drosophila and
39 Hox genes in vertebrates, all bind to very similar DNA sequences via identical
DNA-contacting residues in their homeodomains (2). One way in which Hox proteins achieve a higher degree of DNA binding specificity is to bind cooperatively
with cofactors. One such cofactor is a heterodimer composed of Extradenticle (Exd;
Pbx in vertebrates) and its binding partner Homothorax (Hth; Meis in vertebrates),
both homeodomain proteins (2). Together, Exd-Hth bind cooperatively with Hox
proteins, allowing them to recognize features of the DNA that cannot be read in the
absence of these cofactors.
To explore the DNA binding specificities of the Hox transcription factors and their
cofactor Exd, we have combined the traditional method of SELEX (Systematic
Evolution of Ligands by Exponential Enrichment), which can be used to select
DNA binding sites, with next generation sequencing technology (e.g. Illumina
sequencing). This combination, termed SELEX-seq, allows the identification of
the complete repertoire of binding sites for any Hox-Exd combination after only a
small number of SELEX rounds. Subtle differences in DNA binding preferences
among the Hox-Exd complexes can be clearly observed using this technology. In
general Hox-Exd complexes seem to use a modular binding site architecture, in
which an octomer core sequence determines the DNA signature motif for various
Hox-Exd dimers while flanking nucleotides adjust the absolute affinity of a signature motif. Importantly, these motifs can explain certain aspects of in vivo Hox
function, and our findings suggest that signature motif selectivity is achieved in part
by Hox-Exd recognition of structural features of the DNA.
References
1. T. Iimura and O. Pourquie. Dev Growth Differ 49, 265-275 (2007).
2. R. S. Mann, K. M. Lelli, and R. Joshi. Curr Top Dev Biol 88, 63-101 (2009).
193
Matthew Slattery1,4,*
Todd R. Riley2,3
Namiko Abe1
Harmen J. Bussemaker2,3
Richard S. Mann1
1Department
of Biochemistry and
Molecular Biophysics,
Columbia University, 701 West 168th
Street, HHSC 1104, New York,
NY 10032, USA
2Columbia
University, Department
of Biological Sciences, 1212 Amsterdam
Avenue, New York, NY 10027
3Columbia
University, Center for
Computational Biology and
Bioinformatics, 1130 St Nicholas
4 Current
address: Institute for Genomics
and Systems Biology, University
of Chicago, 900 East 57th Street KCBD
10115, Chicago, IL 60637, USA
*[email protected]
1154
194
Todd R. Riley1,2,*
Matthew Slattery3,4
Namiko Abe3
Harmen J. Bussemaker1,2
Richard S. Mann3
1Columbia
University, Department of
Biological Sciences, 1212 Amsterdam
2Columbia
University, Center for
Computational Biology and
Bioinformatics, 1130 St Nicholas
Dimerization with Extradenticle Unlocks Latent DNA
Binding Specificities that Discriminate Hox Proteins
in Drosophila
Hox proteins constitute a subclass of the large homeodomain family of transcription
factors. They play a crucial role in body plan and tissue specification throughout
the animal kingdom (1). In the transcriptional network of the cell, each Hox protein
targets a distinct set of genes. Little is currently understood about the differences
in DNA binding specificity that underlie these functional differences. In particular,
Hox monomers have been shown to bind DNA with similar sequence preferences
(2, 3). We developed a method (“SELEX-seq”) that couples in vitro selection of
pools of random DNA with massively parallel sequencing. In combination with a
novel computational method for analyzing the data, based on a biophysical model,
it allows us to quantify relative binding affinities at unprecedented resolution for all
12-mer sequences. Application of SELEX-seq to dimers of each of the eight Drosophila Hox proteins with the co-factor Extradenticle (Exd) revealed the DNA binding specificities for this family of homeodomain proteins. Only in the context of
the Exd-Hox complex is the identity of the Hox protein manifested, through major
differences in how base identities in the core of the binding site are recognized. Our
analysis reveals that most Hox-Exd dimers have a unique DNA recognition signature. These differences in binding specificity in vitro may go a long way towards
explaining the functional differences between the Hox proteins observed in vivo.
3Department
of Biochemistry and
Molecular Biophysics, Columbia
University, 701 West 168th Street HHSC
1104, New York, NY 10032, USA
4Current
address: Institute for Genomics
and Systems Biology, University of
Chicago, 900 East 57th Street KCBD
10115, Chicago, IL 60637, USA
We present a novel method, based on a biophysical model of the SELEX procedure, which is capable of estimating binding affinities between a protein or protein
complex of interest and all oligonucleotide sequences of a given length with great
accuracy. Relative affinities are obtained by comparing the sequence composition
of later rounds to that of the initial round. In this comparison our method takes into
account the significant sequence biases in the initial pool of dsDNA molecules.
While the higher sequence counts associated with the later rounds of SELEX may
be preferable in terms of statistical accuracy, the accumulation of PCR-amplification noise and binding-site saturation leads to systematic error in the estimated
affinities. We developed a procedure that uses the earlier and later rounds together,
and thereby obtains affinities that are both accurate and precise.
*[email protected]
The unprecedented depth of the sequencing data allowed us to systematically determine the effective length of the Exd-Hox dimer. While a 12nt degenerate consensus
binding motif emerged for all Hox identities, we found that there exist striking differences in how different Exd-Hox dimers interact with the central hexanucleotide
of the binding site. By quantifying the differences in affinity for each class of binding motif, we were able to identify a unique DNA recognition signature for each of
the Exd-Hox dimers.
References
1. S. Banerjee-Basu and A. D. Baxevanis. Nucleic Acids Res 29, 3258-3269 (2001).
2. M. F. Berger, G. Badis, A. R. Gehrke, S. Talukder, A. A. Philippakis, L. Pena-Castillo,
T. M. Alleyne, S. Mnaimneh, and O. B. Botvinnik, et al. Cell 133, 1266-1276 (2008).
3. M. B. Noyes, R. G. Christensen, A. Wakabayashi, G. D. Stormo, M. H. Brodsky, and
S. A. Wolfe. Cell 133, 1277-1289 (2008).
1155
Structural Determinants of DNA-binding Specificity
for Drosophila Hox Proteins
Hox proteins are homeodomain transcription factors that help to define cellular
and tissue identities, and thus diversify body patterning on the anterior-posterior
axis (1). Understanding the DNA-binding specificities of Hox proteins will therefore provide insight into the regulation of Hox target genes and animal morphogenesis. Advanced by the SELEX-seq method, the complete repertoire of DNA
sequences that bind to Drosophila Hox proteins with their cofactors Extradenticle
(Exd) and Homothorax (Hth) has been obtained. A range of unique DNA-binding
specificities has been observed among these Hox proteins, raising the question of
how such diverse DNA-binding specificities are generated.
Using multiple sequence alignments of Hox proteins, we identified partially conserved residues that correlate with DNA-binding specificity. Possible functions
of these residues were inferred by mapping them onto available 3D structures. In
parallel, using Monte Carlo simulations (2), we predicted the width of DNA minor
groove for different Hox binding sites identified by SELEX-seq. We find that
DNA sequences preferred by Hox proteins that control posterior patterning have
similar minor groove shape, and this shape is different than that of DNA sequences
preferred by Hox proteins that define anterior morphology. In particular, within
the Exd-Hox binding site, high-affinity DNA sequences for Hox protein Scr have
two narrow regions while sequences preferred by Hox protein Ubx have only one.
As revealed by previous studies (3, 4), the additional narrow minor groove of Scr
induces enhanced negative electrostatic potential, which attracts Arg3 of Scr. Our
work leads to a new understanding of the structural basis of specific DNA-binding
for Hox proteins in Drosophila, linking DNA binding site preferences to common
DNA shapes.
References
1. R. S. Mann, K. M. Lelli, and R. Joshi. Curr Top Dev Biol 88, 63-101 (2009).
3. R. Joshi, J. M. Passner, R. Rohs, R. Jain, A. Sosinsky, M. A. Crickmore, V. Jacob, A. K.
Aggarwal, B. Honig, and R. S. Mann. Cell 131, 530-543 (2007).
(2009).
Peng Liu1,*
Remo Rohs2
Richard Mann3
Barry Honig1,**
195
1Howard Hughes Medical
Institute, Center for Computational
Biology and Bioinformatics, Department
of Biochemistry and Molecular
Biophysics, Columbia University, 1130
St. Nicholas Avenue, New York,
NY 10032, USA
2Molecular
and Computational Biology
Program, Department of Biological
Sciences, University of Southern
California, Los Angeles, California
90089, USA
3Department
of Biochemistry and
Molecular Biophysics, Columbia
University, 701 West 168th Street, HHSC
1104, New York, NY 10032, USA
* [email protected]
** [email protected]
1156
196
Dana R. Holcomb*
Thomas D. Tullius
Boston University,
Boston, MA 02215
*[email protected]
DNA Shape Patterns and Binding Specificities for
Drosophila Hox Proteins
The Hox family of transcription factors is essential for the patterning of the anterior-posterior axis, stem cell maintenance, and motor neuron specification in Drosophila (1-3). This family of transcription factors carries out highly specific gene
regulation; however, the source of specificity for binding of Hox proteins to DNA
is poorly understood. Hox proteins, such as Ultrabithorax (Ubx) and Sex combs
reduced (Scr), bind as monomers to degenerate DNA sequences with little specificity. In the presence of the homeodomain-containing cofactors Extradenticle (Exd;
Pbx in vertebrates) and Homothorax (Hth; Meis in vertebrates), Hox proteins bind
DNA sequences with a high degree of specificity, in part by recognizing sequencedependent DNA structural features, such as minor groove shape and electrostatic
potential (4).
In order to determine DNA structural features that are associated with specific
binding to Hox proteins, we are employing hydroxyl radical cleavage chemistry
to probe the structures of DNA sequences that have been identified as high-affinity
binding sites by the Mann and Bussemaker labs using the SELEX-seq technique.
The hydroxyl radical cleavage pattern depends on the local solvent accessibility of
each nucleotide in a duplex DNA molecule, thereby yielding a representation of
the sequence-dependent shape of a DNA molecule. We compare our results with
Monte Carlo-based computational predictions of DNA structure made by the Rohs
and Honig laboratories (5). We also use the ORChID (OH Radical Cleavage Intensity Database) and ORChID2 algorithms developed by our laboratory (6) to compute the sequence-dependent structure of SELEX-seq-identified binding sites.
In previous work our laboratory used ORChID to show that similar DNA structures
can arise from different DNA sequences (6). This result suggests that nucleotide
sequence similarity alone may not be enough to recognize all classes of Hox binding sites. By experimentally determining the shapes of DNA sequences that bind
to Hox proteins, and then comparing our experimental structural results to Monte
Carlo- and ORChID-based computational predictions of structure, we can identify
DNA shape-based binding preferences for Hox transcription factors.
References
1. J. S. Dasen and T. M. Jessel. Curr Topics Devel Biol 88, 169-200 (2009).
2. S. D. Hueber and I. Lohmann. Bioessays 30, 965-979 (2008).
3. S. Tumpel, L. M. Wiedemann, and R. Krumlauf. Curr Topics Devel Biol 88, 103-137
(2009).
4. R. S. Mann, K. M. Lelli, and R. Joshi. Curr Topics Devel Biol 88, 63-101 (2009).
5. R. Rohs, S. M. West, P. Liu, and B. Honig. Curr Opin Struct Biol 19, 171-177 (2009).
6. J. A. Greenbaum, B. Pang, and T. D. Tullius. Genome Res 17, 947-953 (2007).
Protein-DNA Interactions in the p53/DNA System
1157
The tumor suppressor protein p53 is a transcription factor (TF) that, in response
to various types of cellular stress, regulates the expression of a variety of genes
involved in cell-cycle control, apoptosis, DNA repair, and cell differentiation (1),
by first binding sequence-specifically to defined DNA targets (2). Abrogation of p53
sequence-dependent binding is implicated in ~50% of all known cancers (1). p53
molecules consist of four major functional domains (3). The N-terminus contains a
transactivation domain; the core domain contains the sequence-specific DNA binding domain (DBD); and the C-terminal domain (CTD) includes a tetramerization
domain (TD) and a regulatory domain that contain a separate sequence non-specific
DNA binding activity. The core domain of p53 contains 95% of the missense mutations identified in human tumors (4). This highlights the importance of sequencespecific DNA binding by p53 in maintaining genomic integrity and preventing
tumor formation. The consensus DNA response element (RE) consists of two decameric half-sites with the general form RRRCWWGYYY (R=A,G;W=A,T;Y=C,T),
separated by a variable number of base pairs (2). The WW doublet in the CWWG
center of p53 half-sites is uncontacted with the protein in the crystal structure of
p53DBD/DNA complexes (5, 6). Nonetheless, this position is highly conserved in
p53 binding sites.
197
We will present our recent study (7) demonstrating that p53DBD bind consensus
sequences differing in the CWWG center with different binding affinities and especially binding cooperativity. The binding cooperativity spans five orders of magnitude and is encoded in the structural properties of this region and in particular in the
torsional flexibility of the CWWG motif, as determined experimentally by us. The
torsionally flexible CATG motif, connected with binding sites related to cell-cycle
arrest genes, is bound with high affinity and low cooperativity by p53DBD. The
torsionally rigid CAAG and CTAG motifs are bound with lower affinity and high
cooperativity. These motifs are abundant in binding sites associated with low affinity apoptosis-related genes. Our results provide a molecular and structural basis
to recent findings that DNA binding cooperativity of p53 modulate the decision
between cell-cycle arrest and apoptosis (8).
p53 sits at a hub of cellular network controlling many genes, mostly by its function
as a TF that binds sequence-specifically to p53 REs. Knowledge of the full spectrum of p53 cellular connectivity is a prerequisite for a true understanding of what
can go wrong when p53 function is disrupted. We have determined, using proteinbinding microarrays, the binding affinity of p53DBD-TD to a large fraction of all
possible combinations of p53 REs, and will present these results.
References
1. B. Vogelstein, D. Lane, and A. J. Levine. Nature 408, 307-310 (2000).
2. W. S. El-Deiry, S. E. Kern, J. A. Pietenpol, K. W. Kinzler, and B. Vogelstein. Nature Gen
1, 45-49 (1992).
3. A. C. Joerger and A. R. Fersht. Annu Rev Biochem 77, 557-582 (2008).
4. M. Olivier, R. Eeles, M. Hollstein, M. A. Khan, C. C. Harris, and P. Hainaut. Hum Mutat
19, 607-14 (2002).
5. Y. Cho, S. Gorina, P. D. Jeffrey, and N. P. Pavletich. Science 265, 346-355 (1994).
6. M. Kitayner, H. Rozenberg, N. Kessler, D. Rabinovich, L. Shaulov, T. E. Haran, and
Z. Shakked. Mol Cell 22 741-753 (2006).
7. I. Beno, K. Rosenthal, M. Levitine, L. Shaulov, and T. E. Haran. Nuc Acids Res In Press
(2011).
8. K. Schlereth, R. Beinoraviciute-Kellner, M. K. Zeitlinger, A. C. Bretz, M. Sauer,
J. P. Charles, F. Vogiatzi, E. Leich, B. Samans, M. Eilers, C. Kisker, A. Rosenwald and
T. Stiewe. Mol Cell 38, 356-368 (2010).
Itai Beno
Karin Rosenthal
Michael Levitine
Lihi Shaulov
Tali E. Haran*
Department of Biology, Technion,
Technion City, Haifa 32000, Israel
*[email protected]
1158
198
H. Rozenberg
A. Eldar
M. Kitayner
Y. Diskin-Posner
A. Kapitkovsky
O. Suad
Z. Shakked*
Modulating DNA Binding Activity of P53 by Base
Sequence Effects and Amino-Acid Mutations
Our structural studies of the DNA binding domain of the tumor suppressor protein p53 and its complexes with various DNA targets demonstrate that four p53
molecules bind to two decameric DNA half-sites to form a dimer of dimers stabilized by protein-DNA and protein-protein interactions. The 3-D architecture of the
complex and its stability are dependent on the identity of the DNA half-sites and
on the DNA spacer between them. Several hot-spot mutations in the DNA binding domain of p53 abolish the protein’s DNA binding activity and its function as
a tumor suppressor. In certain cases, such activities can be restored by second-site
suppressor mutations. Our high-resolution crystal structures of wild-type p53, its
tumor-derived p53 mutants and the restored proteins in their free and DNA-bound
states provide a framework for understanding the molecular and structural basis of
p53 disfunction as a result of oncogenic mutations and its restoration by suppressor
mutations and potential drug molecules.
Weizmann Institute of Science,
Rehovot 76100, Israel
*[email protected]
199
Victor B. Zhurkin*
Feng Cui
Difei Wang
Laboratory of Cell Biology, NCI, NIH
Bethesda, MD 20892
*[email protected]
DNA Mechanics, Nucleosome Positioning and
p53-DNA Recognition
For many years, it has been assumed that the nucleosome positioning is defined
entirely by the energy of DNA deformation (bending and twisting) when it is
wrapped around the histone core [see the retrospective review by Trifonov (1) and
the accompanying commentaries (2, 3); many of the relevant detailed papers are in
(4-17)]. However, when the high-resolution NCP crystal structure was solved (18),
it became clear that bending of the nucleosomal DNA differs drastically from that
in the other protein-DNA complexes. The DNA bending in nucleosome is accompanied by strong lateral displacements of the DNA axis (Slide) that are critical for
formation of the DNA superhelical path in nucleosome (19). These severe DNA
deformations (denoted Kink-and-Slide), occur as a result of interactions with histone arginines which penetrate the DNA minor groove asymmetrically, so that their
side chains are closer to one DNA strand (13).
Recent computer simulations of oligonucleotide duplexes suggest that the sequencedependent deformability of DNA depends on the imposed constraints that mimic
the presence of bound protein. In particular, when the Slide constraints observed in
the nucleosome were used, the energy of the Kink-and-Slide deformation increased
in the order TA  CA:TG  CG (13). Bending into the minor groove brings the
highest increase in the deformation energy of DNA (19). Therefore, selection of
the ‘correct’ sequences for the minor-groove bending is likely to be the most critical part in the process of finding the optimal position of nucleosomes on DNA.
According to our current understanding (12, 13), the optimal minor-groove bending patterns contain the TA and CA:TG dimers in the pyrimidine-purine context
YYRR. Importantly, this sequence preference explains the exceptionally high stability of nucleosomes formed by the “TG repeat” (20) and the “601 sequence” (21)
containing numerous TTAG and TTAA fragments, respectively.
In addition to folding in nucleosomes, the DNA Slide is implicated in the sequencespecific recognition of DNA by the tumor suppressor protein p53 (22). The shearing deformation of the DNA axis caused by p53 binding (23-25) is consistent with
the Kink-and-Slide conformation described above. Therefore, structural organization of a p53 binding site in chromatin can regulate its affinity to p53 – for example,
exposure of the DNA site on the nucleosomal surface would facilitate p53 binding
to the response element (26). Our results indicate that there is a complex interplay
between the structural codes encrypted in eukaryotic genomes – one code for DNA
packaging in chromatin, and the other code for DNA recognition by transcription
factors (TFs). The two codes appear to be generally consistent with each other. At
least in some cases, such as p53 (26) and the glucocorticoid receptor (27), the DNA
wrapping in nucleosomes can facilitate the binding of a TF to its cognate sequence,
provided that the latter is properly exposed in chromatin.
References
1.
2.
3.
4.
5.
6.
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27.
E. N. Trifonov. Phys Life Rev 8, 39-50 (2011).
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V. B. Zhurkin. Phys Life Rev 8, 64-66 (2011).
A. Travers, E. Hiriart, M. Churcher, M. Caserta, and E. Di Mauro. J Biomol Struct Dyn 27,
713-724 (2010).
F. Xu and W. K. Olson. J Biomol Struct Dyn 27, 725-739 (2010).
E. N. Trifonov. J Biomol Struct Dyn 27, 741-746 (2010).
P. De Santis, S. Morosetti, and A. Scipioni. J Biomol Struct Dyn 27, 747-764 (2010).
G. A. Babbitt, M. Y. Tolstorukov, and Y. Kim. J Biomol Struct Dyn 27, 765-780 (2010).
D. J. Clark. J Biomol Struct Dyn 27, 781-793 (2010).
S. M. Johnson. J Biomol Struct Dyn 27, 795-802 (2010).
G. Arya, A. Maitra, and S. A. Grigoryev. J Biomol Struct Dyn 27, 803-820 (2010).
F. Cui and V. B. Zhurkin. J Biomol Struct Dyn 27, 821-841 (2010).
Y. V. Sereda and T. C. Bishop. J Biomol Struct Dyn 27, 867-887 (2010).
I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 26, 403-411 (2009)
I. Gabdank, D. Barash, and E. N. Trifonov. J Biomol Struct Dyn 28, 107-121 (2010).
C. A. Davey, D. F. Sargent, K. Luger, A. W. Mäder, and T. J. Richmond. J Mol Biol 319,
1097-1113 (2002).
M. Y. Tolstorukov, A. V. Colasanti, D. M. McCandlish, W. K. Olson, and V. B. Zhurkin.
J Mol Biol 371, 725-738 (2007).
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P. T. Lowary and J. Widom. J Mol Biol 276, 19-42 (1998).
S. R. Durell, R. L. Jernigan, E. Appella, A. K. Nagaich, R. E. Harrington, and V. B. Zhurkin.
In Sarma, R. H. and Sarma, M. H. (Eds.): Structure, Motion, Interaction and Expression
of Biological Macromolecules. Proceedings of the Tenth Conversation, 1997. New York,
Adenine Press, 1998, (2) pp. 277-296.
K. A. Malecka, W. C. Ho, and R. Marmorstein. Oncogene 28, 325-33 (2009).
Y. Chen, R. Dey, and L. Chen. Structure 18, 246-56 (2010).
M. Kitayner, H. Rozenberg, R. Rohs, O. Suad, D. Rabinovich, B. Honig, and Z. Shakked.
Nat Struct Mol Biol 17, 423-9 (2010).
G. Sahu, D. Wang, C. B. Chen, V. B. Zhurkin, R. E. Harrington, E. Appella, G. H. Hager and
A. K. Nagaich. J Biol Chem 285, 1321-1332 (2010).
M. Becker, C. Baumann, S. John, D. A. Walker, M. Vigneron, J. G. McNally, and
G. L. Hager. EMBO Rep 3, 1188-94 (2002).
1159
1160
200
Feng Cui1,*
Hope A. Cole2
David J. Clark2
Victor B. Zhurkin1
1Laboratory
2Program
of Cell Biology, NCI, NIH
in Genomics of Differentiation,
NICHD, NIH, Bethesda, MD 20892
*[email protected]
Yeast Nucleosomes Mapped with High Resolution
by Paired-end Sequencing Exhibit Strong
Positioning Patterns
Despite intense efforts, recently summarized in a series of publications (1-14),
understanding nucleosome positioning continues to be challenging. Recent
advances in massively parallel shotgun sequencing techniques allowed detecting
nucleosome positions across the genomes of yeast (15, 16), nematode (17) and
human (18). Millions of DNA fragments derived in the course of micrococcal
nuclease cleavage were partially sequenced at the 5’ end. As a result, the ‘properly’
trimmed nucleosome core particles have been mixed up with the over-digested or
incompletely trimmed nucleosomes. To resolve this ambiguity, we used the pairedend sequencing technique and obtained ~15 million paired reads that are uniquely
mapped to the yeast genome (19). The lengths of DNA fragments obtained in
this way span the interval from 120 bp (over-digested core particles) to 180 bp
(poorly trimmed nucleosomes), with the highest occurrence at 150 bp. The data for
147-152 bp-long fragments (~5 million) were compared with the two published
datasets, one with the same length interval, 147-152 bp (~72,000) (16), and the
other with the estimated length 147 bp (~50,000) (20).
The high-resolution nucleosomes in our new dataset are depleted in the promoter
regions and positioned regularly downstream of gene starts, consistent with the
earlier studies (16, 20). This regularity was further supported by the start-to-start
distance correlation function having periodicity of 160-170-bp, close to the yeast
nucleosome repeat ~165 bp. A detailed analysis of this distance correlation rendered a clear 10-bp periodicity, more pronounced than in the two published datasets (16, 20). Moreover, a stronger variation of occurrence of the AT-containing
fragments (21) was found in the paired-end nucleosomes (19). This new dataset
is being used to analyze the occurrence of the DNA dimers, trimers and tetramers
in various positions in the nucleosome core particles, and to further develop our
scheme for prediction of the nucleosome positioning (9).
References
713-724 (2010).
15. I. Albert, T. N. Mavrich, L. P. Tomsho, J. Qi, S. J. Zanton, S. C. Schuster, and B. F. Pugh.
Nature 446, 572-576 (2007).
16. T. N. Mavrich, I. P. Ioshikhes, B. J. Venters, C. Jiang, L. P. Tomsho, J. Qi, S. C. Schuster,
I. Albert, and B. F. Pugh. Genome Res 18, 1073-1083 (2008).
17. A. Valouev, J. Ichikawa, T. Tonthat, J. Stuart, S. Ranade, H. Peckham, K. Zeng, J. A. Malek,
G. Costa, K. McKernan, A. Sidow, A. Fire, and S. M. Johnson. Genome Res 18, 1051-1063
(2008).
18. D. E. Schones, K. Cui, S. Cuddapah, T-Y. Roh, A. Barski, Z. Wang, G. Wei, and K. Zhao.
Cell 132, 887-898 (2008).
19. H. A. Cole, B. H. Howard, and D. J. Clark. Submitted for publication (2011).
20. Y. Field, N. Kaplan, Y. Fondufe-Mittendorf, I. K. Moore, E. Sharon, Y. Lubling,
J. Widom, and E. Segal. PLoS Comp Biol 4, e1000216 (2008).
21. S. C. Satchwell, H. R. Drew, and A. A. Travers. J Mol Biol 191, 659-675 (1986).
1161
DNA Shape Patterns and Evolutionary Constraint in
Nucleosome Positioning Sequences
The process of wrapping DNA around a nucleosome core particle affects the functions encoded in the underlying sequence. Therefore, it is important to understand the
biological signals responsible for nucleosome positioning. Nucleosome positioning
is one of the current ‘hot’ issues and the various approaches and hypotheses on what
positions nucleosomes are discussed extensively in recent publications, particularly
in an issue of this journal devoted exclusively to this subject (1-14). One of the
major factors affecting the nucleosome positioning is DNA sequence. Despite the
observation that nucleosome positioning is sequence-directed, the core particle
makes no base-specific contacts with DNA. These circumstances suggest that
nucleosome positioning may be encoded through DNA structural properties and
not necessarily the primary sequence. To study this, we introduce a new algorithm
named ORChID2 that uses hydroxyl radical cleavage patterns to predict the singlenucleotide resolution shape of a DNA molecule. Analysis of ORChID2 DNA shape
profiles in well-positioned nucleosome sequences reveals a periodic and symmetric
pattern. Additionally, there is more information encoded in the DNA shape than the
primary sequence. Information content is consistently highest at regions where the
minor groove is narrow—suggesting a fundamental property of protein-DNA shape
recognition in nucleosome positioning. Finally, we present a new algorithm named
Chai2 that measures the single-nucleotide resolution evolutionary conservation of
DNA shape. Overlaying Chai2 constraint results on ORChID2 shape profiles in
nucleosome positioning sequences reveals interesting evolutionary conservation
patterns. We suggest that selective pressure operating on DNA shape can influence nucleosome positioning dynamics, and therefore be a major force in genome
evolution.
References
713-724 (2010).
11. S. M. West, R. Rohs, R. S. Mann, and B. Honing. J Biomol Struct Dyn 27, 861-866 (2010).
13. I. Gabdank, D. Barash and E. N. Trifonov. J Biomol Struct Dyn 26, 403-411 (2009).
201
Stephen C. J. Parker1,*
Sean M. West2,3
Eric Bishop4
Remo Rohs5
Barry Honig2
Thomas D. Tullius4,6
Elliott H. Margulies1
1National
Human Genome Research
Institute, National Institutes of Health,
Bethesda, Maryland, USA
2Howard
Hughes Medical Institute,
Center for Computational Biology
and Bioinformatics, Department of
Biochemistry and Molecular Biophysics,
Columbia University, New York, USA
3Center
for Genomics and Systems
Biology, Department of Biology,
New York University, New York, USA
4Program
in Bioinformatics, Boston
University, Boston, Massachusetts, USA
5Molecular
and Computational Biology
Program, Department of Biological
Sciences, University of Southern
California, Los Angeles, California, USA
6Department
of Chemistry, Boston
University, Boston, Massachusetts, USA
*[email protected]
1162
Molecular Dynamics Simulations on DNA Y10K
202
David L. Beveridge
Recent progress in the area molecular dynamics (MD) simulations on DNA over
the last 10 years will be presented, with an emphasis on force field developments,
convergence and stability at the nano- to microsecond level, and the state of validation based on comparisons with diverse experimental physical methods. Results
on the sequence effects on dynamical structure based on MD on all tetranucleotide
steps by the ABC consortium will be described, and current research on DNA axis
bending and flexibility will be included.
Dept of Chemistry and
Molecular Biophysics Program
Middletown, CT
[email protected]
203
Edward N. Trifonov1,2
1Genome
Diversity Center, Institute
of Evolution, University of Haifa,
Mount Carmel, Haifa 31905, Israel
2Department
of Functional Genomics and
Proteomics, Faculty of Science,
Masaryk University, Kotlarska 2,
CZ-61137 Brno, Czech Republic
[email protected]
Six Sensations: Six Second Genetic Codes
Human mind can accept the idea that in addition to classical triplet code some
other, “second genetic code” could exist. And its discovery, of course, would be
a great scientific sensation. But the human mind can not possibly imagine that
there could be several different codes. This is why every newly discovered code
(and there are, actually, many) is called by the same generic name “second genetic
code”. The second codes, indeed, all different, have been solemnly announced six
times (1-6), in 1988, 2001, 2001, 2006, 2008 and 2010.
Another, well known feature of human mind is fading memory. This is illustrated
by the above time series. The sensational discoveries are honored by the traditional
title of the “second genetic code”, and only the amnesia keeps the discoverers from
making grimaces.
What is good about the amnesia, however, it encourages to make yet new sensational discoveries (as by the short memory perception none are made before).
Will the sevenths Second Genetic Code be discovered? (Chicken would give all
previous codes different numbers, as it counts till seven).
References
1. G. Kolata. New York Times May 13 (1988).
2. Second genetic code could provide clues to schizophrenia, bipolar disorder. CBCNews
March 12 (2008).
3. E. Young. New scientist August 9 (2001).
4. N. Wade. New York Times July 25 (2006).
5. T. Hughes. The FASEB Journal 22, 262-268. (2008).
6. J. R. Tejedor and J. Valcárcel. Nature May 6 (2010).
1163
The UvrA•UvrB DNA Damage Sensor: Structure
and Mechanism
During nucleotide excision repair (NER), the UvrA•UvrB (AB) damage sensor identifies lesion-deformed DNA within a sea of undamaged DNA. We report the first
structure of the AB sensor and of a novel UvrA conformer. In the structure of the AB
sensor, a central UvrA dimer is flanked by two UvrB molecules, all linearly arrayed
along a DNA path predicted by biochemical studies. DNA is predicted to bind to
UvrA in the complex within a narrow and deep groove that is compatible with native
duplex DNA only. In contrast, the shape of the corresponding surface in all other
UvrA structures is wide and shallow and appears compatible with various types of
lesion-deformed DNA. These differences point to conformation switching between
the two forms as a component of the genome-scanning phase of damage sensing.
We also show that the highly conserved signature domain II of UvrA, which is adjacent to the proximal nucleotide-binding site, mediates a critical nexus of contacts to
UvrB and to DNA. Moreover, in the novel UvrA conformer, the disposition of this
domain is altered such that association with either UvrB or DNA is precluded. Concomitantly, nucleotide is uniquely absent from the proximal binding site. Thus, the
signature domain II is implicated in an ATP-hydrolysis-dependent conformational
change that detaches UvrA from both UvrB and DNA after initial damage recognition. Finally, the disposition and number of UvrB molecules in the AB complex,
both unanticipated, suggest that once UvrA departs, UvrB localizes to the site of
damage by helicase-mediated tracking along the DNA. Together these results permit
a high-resolution model for the dynamics of early stages in NER (1-6).
Research in the Jeruzalmi lab is supported by the NSF (MCB-0918161) and NIH
(GM084162).
References
1.
2.
3.
4.
5.
6.
S. Thiagalingam and L. Grossman, J Biol Chem 266, 11395-403 (1991).
S. Thiagalingam and L. Grossman, J Biol Chem 268, 18382-9 (1993).
E. Bertrand-Burggraf, C. P. Selby, J. E. Hearst, and A. Sancar, J Mol Biol 219, 27-36 (1991).
N. Goosen and G. F. Moolenaar, DNA Repair 7, 353-79 (2008).
D. Pakotiprapha, Y. Liu, G. L. Verdine, and D. Jeruzalmi, J Biol Chem 284, 12837-44 (2009).
D. Pakotiprapha, Y. Inuzuka, B. R. Bowman, G. F. Moolenaar, N. Goosen, D. Jeruzalmi,
and G. L. Verdine, Mol Cell 29, 122-33 (2008).
204
David Jeruzalmi*
Danaya Pakotiprapha
Martin A. Samuels
Koning Shen
Johnny Hu
Biology, Harvard University,
52 Oxford Street, Cambridge,
MA 02138, USA.
*[email protected]
Index to Authors
Abaturova, A.
977, 978
Abe, N.
1153, 1154
Adams, C.
1099
Agris, P. F.
1052,1072,1073
Agutter, P. S.
643
Ahmad, F.
331, 929
Ahmad, N.
929
Akella, S.
611
Alakent, B.
675
Albinsson, B.
1054, 1086
Aldersley, M. F.
1062
Alijanvand, H. H.
919
Aman, M. J.
1
Ampt, K. A. M.
1007
Anand, S.
1039
Andrianov, A. M.
1080, 1081
Anishchenko, I. V.
1080, 1081
Ansari, Z. A.
331
Antonyan, A. P.
1132, 134
Arakelyan, V. B.
1133
Arora, P.
1050
Artamonova, I. I.
1068
Aston, K.
1147
Atri, M. S.
919
Attaluri, S.
1103
Austen, K.
1108
Autexier, C.
1090
Avakyan, N.
1090
Ávila-Figueroa, A.
1106
Avvedimento, E. V.
1096
Azad, R.
1073
Bagchi, A.
653
Bahar, I.
1029, 1030, 1031, 1032, 1033
Bahrami, H.
211
Bairagya, H. R.
503, 637, 1008,1027
Bakan, A.
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Balayan, G.
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Banappagari, S.
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Banáš, P.
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Banerjee, A.
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Banerjee, P. R.
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Bansal. M.
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Bao, Z.-Q.
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Barvik, I.
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Bashkin, J. K.
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Basu, G.
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Bawa, T. S.
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Bean, H. D.
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Beichmanis, S.
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Dynamics, ISSN 0739-1102
Volume 28, Issue Number 6, ( 2011)
©Adenine Press (2011)
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Belotserkovskii, B. P.
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Beno, I.
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Bera, A. K.
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Besseova, I.
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Bishop, E.
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Bishop, T. C.
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Bocharova, T. N.
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Bolia, A.
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Bongiovanni, M.
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Borcherds, W.
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Börjesson, K.
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Boubakri, H.
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Bowden, M. G.
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Bredehöft, J. H.
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Breslauer, K. J.
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Bryce, R. A.
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Caetano, M. S.
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Canary, J.
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Canary, R. W.
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Cantara, W. A.
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Cao, J.
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Carvalho, I.
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Carvalho, K. T. G.
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Chaitanya, P. K.
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Chakraborty, B.
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Chalovich, J. M.
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Chamani, J.
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Chan, H. S.
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Chang, G.
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Chang, K.-W.
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Chang, K.-L.
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Chang, L.-K.
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Chattopadhyaya, R.
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Chavushyan, A.
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Cheguri, S.
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23, 187, 309,471, 773, 895
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Chen, H.-Y.
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39
Chen, L.
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Chen, W.
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Chen, X.
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Chiang, C.
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Cho, J.-E.
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Chong, L. T.
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Chou, K.-C.
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Chuang, C.-K.
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Clark, D. J.
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Cole, H. A.
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Colón, W.
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Commandeur, J. N. M.
1007
Corcho, F.
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Cornett, E.
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Costanzo, G.
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Cowburn, D.
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Cremer, C.
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Cremer, M.
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Cremer, T.
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Cui, F.
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Cunha, E. F. F.
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Dailidonis, V. V.
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Dalyan, Y.
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1165
1166
Dang, L.
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Danilov, V. I.
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Das, P.
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Das, S.
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Dastidar, S. G.
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Daughdrill, G. W.
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de Andrade, P.
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de los Santos, C.
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Deineko, V.
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Delaney, S.
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Deng, S.
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Derevyanko, A. G.
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Dey, R.
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Dey, S.
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Dhar. S.
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Dima, R. I.
617, 975,976, 979, 997
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Divsalar, A.
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Drevko, B. I.
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Duan, L.
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Dupureur, C. M.
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Dutta, A.
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Dutta, K.
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Dziak, D.
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Ebrahim-Damavandi, S.
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Efferth, T.
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El-Sagheer, A. F.
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Endutkin, A. V.
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Eremin, V. F.
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Esguerra, M.
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Estiu, G.
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Fakhoury, J.
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Fedorova, O. S.
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Finkelstein, A. V.
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Gaston, K. R.
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Gromova, E. S.
Gruebele, M.
Guharoy, M.
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Gunkel, M.
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Gur, M.
Haas, E.
Hakker, L.
Hamblin, G. D.
Hammond, N. B.
Han, S. W.
Hanawalt, P. C.
Hannestad, J. K.
Haran, T. E.
Harish, B.
Harris, K. A.
Harris, L.-A.
Harris, R. S.
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Hashimoto, K.
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He, J.
He, Z.
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Hellman, L.
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Ho, Y.
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Honig, B.
Honing, M.
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Horovitz, A.
323
996
1113
1077
983
107
1124
595
989
1147
881
1112
1120
1053
1034
1017, 1018
627
999
1093
653
1068
13
919
1065
431, 815
1057
1050
1131
615
1148
1082
1049
455
1108
1149
1032
1146
1052
1056
1114
421
1118
1054
1157
1148
1052, 1073
1145
1096
1124
1065
1087
1147
797
833
1151
1097
1117
1101, 1120
39, 743
1087
1060
1156
871
1155, 1161
1007
1087
1035
Horowitz, E. D.
1063
Hough, L. E.
1012
Hovhannisyan, G. G.
1133
Hsiao, C.
1071
Hsu, C.-H.
23
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Huang, G.
535
Huang, H.-J.
23, 187, 309, 895
Hübner, B.
1108
Hud, N. V.
1063, 64
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Ingle, S.
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Irth, H.
1007
Isaeva, M. P.
517
Isin, B.
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Ivanov, A. A.
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Jacobsen, D. M.
1028
Jain, S. S.
1079
James, T. L.
1082, 845
Jani, V.
845, 984, 985
Jansco-Radek, A.
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Jarem, D. A.
1095, 1106
Jatana, N.
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Jayaram, B.
133, 443, 669, 998, 1003, 1142
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Jeruzalmi, D.
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Jha, S.
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Ji, L.
871
Ji, L.-N.
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Ji, X.-L.
995, 621
Jia, J.
535
Jimenez-Cruz, C.
989
Jin, B.
421
Jinks-Robertson. S.
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Johnson, F.
1103
Jones, N. C.
1060
Jones, V.
1073
Josa, D.
907
Joshi, P. C.
1062
Joshi, R.
667, 845, 984, 985, 1017, 1018
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Kabanov, A.
1021
Kahlon, A. K.
201
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Kaluzhny, D. N.
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Kamzolova, S. G.
1121, 1122, 1137, 1139
Kamzolova, S. V.
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Kandaswamy, K. K.
405
Kapitkovsky, A.
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Kar, I.
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Karapetian, A. T.
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Karauzum, H.
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Kaufmann, R.
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Kaus, J. W.
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Kellner, R.
13
Khalo, I. V.
1130
Khan, S. H.
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Khandelwal, G.
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Khechinashvili, N.
1021
Kholodov, Y.
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Khomenko, V. A.
517
Khrameeva, E. E.
1112
Khrushchev, S.
977
Kieltyka, R.
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Kilså, K.
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Kim, D.-S.
277
Kim, J.
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1167
Kim, N.
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Kim, N. Y.
517
Kim, S. K.
421
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Kisel, M. A.
1080
Kitayner, M.
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Knaggs, M. H.
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Knyazeva, O.
978
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1147
Kolatkar, P. R.
405
Kolkman, A. J.
1007
Kolpashchikov, D. M.
1053
Komarov, V.
1021
Komatsoulis, G. A.
1018
Kondratyev, M.
1021
Koshy, C.
71
Koudelka, G.
1145
Koulgi, S.
984
Kovalenko, I.
977, 978
Kříž, Z.
393
Krasilnikova, M. M.
1118
Kravats, A.
1036, 1040
Krimer, D. B.
1117
Krutinin, G. G.
1122, 1137, 1139
Krutinina, E. A.
1122, 1137, 1139
Kuča, K.
393
Kulakovskiy, I. V.
1126, 1127
Kumar, R.
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Kuznetsov, N. A.
1099
Kwon, A.-H.
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LaBean, T. H.
1052
Langlois de Septenville, A.
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Latha, N.
1006
Lawson, N. D.
1149
Lee, C.
1143
Lee, H. M.
421
Lee, H.-T.
1143
Lee, K.-J.
23, 187, 309
Lee, R. A.
1142
Leonhardt, H.
1108
Leontis, N. B.
1070
Levengood, J.
1083
Levitine, M.
1157
Li, D.
1047
Li, L.
833
Li, L.-Y.
1089
Li, Y.
159, 1058
Liang, L.-M.
994
Liévin, J.
949
Likhatskaya, G. N.
517
Lin, C-H.
471
Lin, J.-G.
773, 895
Lincoln, P.
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Litke, J.
1058
Litvinov, R. I.
975, 976
Liu, D. D. W.
861
Liu, H.-L.
39, 743
Liu, K.-T.
39, 743
Liu, L.
343
Liu, P.
1155
Liu, R.
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Liu, S.-Q.
143, 621, 994, 995, 996
Liu, S.-X.
996
Liu, W.
1051
Liu, X.
535, 833
Liu, Y.
1049
Liu, Z.
Livshits, M. A.
Liwo, A.
Lobanov, M. Y.
Lomzov, A. A.
Lopez, M.
López, V.
Lowenhaupt. K.
Lu, L.
Lü, Z.-R.
Lukin, M.
Lundberg, E. P.
Luz, G. P.
Ma, B.-G.
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Machado, A. C. D.
Macickova-Cahova, H.
Maciejczyk, M.
Madduri, R.
Madej. T.
Maeshima, K.
Mahalakshmi, A.
Makeev, V. J.
Malac, K.
Malathi, R.
Male, G.
Malkiewicz, A.
Mallik, P.
Mamajanov, I.
Mancini, J.
Mangels, C.
Mann, R. S.
Mann, R.
Mansouri-Torshizi, H.
Mao, C.
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Margulies, E. H.
Markaki, Y.
Marky, L. A.
Martadinata, H.
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Martínez-Robles, M.-L.
Masulis, I. S.
Matthews, B. W.
Mauro, E. D.
Mayr, F.
McEachon, C.
McFarland, C. W.
McLaughlin, E.
Mechetin, G. V.
Medvedeva, Y. A.
Meierhenrich, U. J.
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Meng, Z.-H.
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97
939
1142
595
1130
1043
1117
1088
797
259
1103
1055
907
415, 619
1029
1150
1087
1142
1018
1005
1109
363
1126, 1127
991
1069
1144
1072
503, 1008
1064
1090
13
1153, 1154
1155
805, 1002
1048
1005
1113, 1161
1108
1143
1092
405
1117
1128
589
1061
1151
1066
1062
1079
1095
1126
1060
1060
1097, 1099
1111
996
1050
625, 993
1116
1147
1103
939
1115, 1118
1062, 1113
1112
1083
649
Mitra, C. K.
611
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239, 827
Mittal, A.
133, 443, 669, 998
Mittermaier, A.
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Mitternacht, S.
607
Mladek, A.
1077
Mlýnský, J.
1078
Mohanty, D.
1039
Moitessier, N.
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Monturus de Carandini, E.
1117
Moosavi-Movahedi, A. A.
211, 919
Morais, P. A. B.
787
Morgunov, I. G.
1134
Morozov, I.
977
Movahedi, A. A. M.
331
Mukerji, I.
1058
Mukherjee, G.
1003
Mukherjee, R.
983
Mukhopadhyay, B. P.
503, 637, 1008, 1027
Müller. C. W.
1144
Murthy, D. K.
379
Nagaraj, R.
639
Nahon, L.
1060
Nandi, T. K.
1027
Nasiri, R.
211
Nekrasov, A. N.
85
Neupane, R.
1079
Nguyen, K. V.
1104
Nguyen, T. L.
1, 641
Niasari-Naslaji, A.
919
Nielsen, P. E.
1084
Nikitina, V. E.
969
Nikolayevich, V. A.
1080
Nordén, B.
1055
Novikova, O. D.
517
Nurminski, E. A.
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O’Donnell, M.
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O’Neill, E.
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Ohayon, Y.
1046
Oliveira, A. A.
455
Oliveira, L. C. A.
227
Olmez, E. O.
675
Olson, W. K.
1075
Oltvai, Z. N.
1023
Orsag, P.
1087
Osypov, A. A.
1122, 1137,1139
Otyepka, M.
633, 1078
Ozkan, S. B.
1034, 1068
Ozoline, O. N.
1128
Pakotiprapha, D.
1163
Pal, A.
1148
Panchenko, A. R.
1005, 1065
Panchin, A. Y.
1068
Pande, J.
1001
Pankratov, A. N.
969
Park, Y.-D.
259
Parker, S. C. J.
1113, 1161
Parthiban, M.
71
Perez, A.
1029
Perez, J. J.
657, 986
Peters, J.
1071
Petrov, V. V.
1024, 1025
Phan, A. T.
1092
Phillips, N.
1146
Pieniazek, S. N.
1101
Pieniazek, S.
1053, 1074
Pino, S.
1061
1168
Pirchi, M.
987
Pivonkova, H.
1087
Plesa, C.
1055
Poddar, N. K.
331
Pogenberg, V.
1147
Poole, L. B.
51
Portnyagina, O. Y.
517
Potenza, A.
1079
Pradhan, S. K.
1093
PreetyPriya.
1016
Preus, S.
1086
Prislan, I.
1143
Pugalenthi, G.
405
Punetha, A.
759
Pyshnyi, D. V.
1130
Qian, G.-Y.
259
Racca, J.
1146
Rackovsky, S.
593
Rahmanpour, R.
575
Raindlova, V.
1087
Rajan, P.
1083
Ramalho, T. C.
227, 455, 645, 907
Ramanathan, R.
661
Ramasree, D.
379
Rao, Z.-H.
143
Rapoport, A. E.
567
Ray, B. K.
1088
Ray. A.
1088
Rayla, A.
1149
Rea, V.
1007
Reblova, K.
1070, 1076
Remeta, D. P.
1103
Resende, J. A.
787
Reyes, V. M.
1013, 1014, 1015, 1016, 1017
Rich. A.
1088
Ried, J.
1087
Riley, T. R.
1153, 1154
Rittschof, C. C.
1114
Riznichenko, G.
977, 978
Rocha, M. V. J.
227
Rodriguez, A.
986
Rohs. R.
1150, 1152, 1155, 1161
Ronald, S.
289
Rooman, M.
949
Rösch, P.
13
Rosenthal, K.
1157
Rouquette, J.
1108
Rout, M.
1012
Roy, S.
201, 729
Rozenberg, H.
1158
Rubin, A.
977, 978
Ryazanova, A. S.
1134
Saberi, M. R.
483
Saboury, A. A.
805, 919, 1002
Saeidifar, M.
805
Sagi, I.
1007
Sahoo, S.
827
Saleh, S.
1118
Salsbury, F. R. Jr.
51
Salyanov, V. I.
1131
Samchenko, A.
1021
Samuels, M. A.
1163
Sarma, R. H.
587
Sattarahmady, N.
211
Satyanarayanajois, S. D.
289
Saxena, A.
1018
Schepsky, A.
1147
Scheraga, H. A.
Schermelleh, L.
Schermerhorn, K. M.
Schermerhorn, K.
Schmit, J.
Schneider, D. M.
Schuetz, A.
Schulten, K.
Schvartzman, J. B.
Schwarzinger, S.
Sebest, P.
Seeman, N. C.
Sefidbakht, Y.
Sela-Passwell, N.
Semighini, E. P.
Seo, E.
Sha, R.
Shahinyan, M. A.
Shakked, Z.
Shakya. A.
Shanmugam, K.
Sharifzadeh, A.
Sharma, A.
Shaulov, L.
Shavkunov, K. S.
Shen, H.-B.
Shen, K.
Shenbagarathai, R.
Shenoy, S.
Sherman, W. B.
Shi, F.
Shi, L.
Shi, Q.
Shoemaker, B. A.
Shrivastava, I.
Shustrova, E. N.
Si, Y.-X.
Silva, C. H. T. P.
Simmel, F. C.
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Singh, S. K.
Singh, T.
Sinha, P. K.
Slattery, M.
Sleiman, H. F.
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Smirnova, E. A.
Smolina, I.
Solov’eva’ T. F.
Sonavane, U. B.
Sonavane, U.
Song, Y.
Sorokin, A. A.
Sowdhamini, R.
Spacek, J.
Spackova, N.
Spasic, A.
Sponer, J. E.
Sponer, J.
Šponer, J.
Srinivasan, S. P.
Srivastava, A. K.
Srivastava, A.
Stan, G.
Steingrimsson, E.
593, 1142
1108
1095, 1106
1103
999
1071
1151
978
1117
13
1087
1044, 1145, 1146, 1147,
1148, 1149, 1150, 1151
919
1007
787
259
1045, 1146, 1147, 1148,
1149, 1150
1132
1158
1088
759
919
201, 1006
1157
1128
175
1163
363
133
1050
535
1011
881
1005
1031
1068
259
635, 787
1057
331
1000
1003
1018
1153, 1154
1056, 1090
1108
1124
1085
517
845, 1017
984, 985
663
1121
71, 405
1087
1076
1142
1076, 77
1070, 1076, 1077
1078
1043
1008
1018
1020, 1036, 1040
1147
Stenkova, A. M.
517
Stevenson, B.
1099
Stormo, G. D.
1149
Streltsov, S. A.
1131
Su, J.
717
Suad, O.
1158
Subramanian, H. K. K.
1045
Suganthan, P. N.
405
Sumpter, B. G.
1077
Sun, M-.F.
309, 471, 773, 895
Sundar, D.
759
Sunyaev, S.
1062
Susova, O. Y.
1131
Szostak, J. W.
1059
Taft, C. A.
635, 787
Tainer, J. A.
1094
Takahashi, J.-I.
1060
Takashima, A.
1042
Tao, Y.
143, 996
Taylor, N.
1144
Tessari, M.
1007
Tetenbaum-Novatt, J.
1012
Thakur, A. R.
729
Thangudu, R. R.
1005
Thirumalai, D.
987
Thota, A.
983
Tjhen. R.
1082
Tolbert, B. S.
1083
Tomashevsky, A. A.
1025
Tonddast-Navaei, S.
1020, 1036
Tornaletti, S.
1118
Torosyan, M. A.
1132
Traaseth, N. J.
1011
Trahtenhercts, A.
1007
Trifonov, E. N.
107, 517, 567, 1060,1162
Trifonov, S.
977
Tripathi, S. K.
1000
Tsai, F.-J.
23, 187, 309, 471, 773, 895
Tsai, W.-B.
39, 743
Tsivileva, O. M.
969
Tuân, P. A.
1091
Tullius, T. D.
1073, 1113, 1114, 1156, 1161
Tutukina, M. N.
1128
Tuzikov, A. V.
1080, 1081
Tyagi, M.
1005
Udgaonkar, J. B.
988
Udomprasert, A.
1050
Ulyanov, N. B.
1082
Uma, V.
379
Uversky, V.
1041
Vahedian-Movahed, H.
483
van Mourik, T.
1140
Vardanyan, I.
123
Vardapetyan, H. R.
1134
Vardevanyan, P. O.
1132, 1133
Varughese, J. F.
159
Vasavi, M.
379
Veglia, G.
1011
Ventura, S.
655
Verardi, R.
1011
Verma, A.
661
Vermeulen, N. P. E.
1007
Veytsman, B. A.
1095
Viguera, E.
1116
Vishveshwara, S.
1037
Vitoc, C. I.
1058
Volle, C. B.
1105
1169
Volodin, A. A.
Vorobjev, Y. N.
Vottero, E. V.
Vrabel, M.
Wai, L. K.
Walter, N. G.
Wang, C.
Wang, D.
Wang, J.
Wang, K.-Z.
Wang, R.
Wang, S.
Wang, T.
Wang, W.
Wang, Y.
Weiglmeier, P. R.
Weill, N.
Weisel, J. W.
Weiss, M.
Weng, L.-P.
West, S. M.
Wheatley, E. G.
Widom, J.
Wiesner, J.
Wiest, O.
Wijmenga, S. S.
Wilhelmsson, L. M.
Williams, L. D.
Wilmanns, M.
Wilson, N. R.
1124
991
1007
1087
1091
1078
717
1158
343, 629, 992
955
1049, 1051
881
247, 545, 1048
323
1043, 881
13
1090
975, 976
1146
1089
1161
1053
1111
393
1023
1007
1055, 1086
1071
1147
1095, 1106
Wintjens, R.
Wolfe, S. A.
Won, C.-I.
Wu, H.
Wu, J. W.
Wu, L.
Wu, N.
Xiao, Y.
Xu, S.
Xu, X.
Xu, Y.
Xue, B.
Yaakov (Koby) Levy
Yan, L.
Yang, G.
Yang, H.-X.
Yang, J.-M.
Yang, Z.
Yao, N. Y.
Ye, M.
Ye, Z.
Yella, V. R.
Yennie, C. J.
Yin, S.-J.
Young, M. A.
Yu, H. W.
Yu, J.
Yuan, Y.
Zahedi, M.
Zahra Bathaie, S.
949
1149
277
1041
39, 743
97
323
431, 815
881
717
97
1041
1144
259
323
955
259
323
1120
1049
1043
1143
1104
259
1028
23, 187, 309
629
51
211
575
Zeng, J.
Zhang, A.-G.
Zhang, D.
Zhang, H.-Y.
Zhang, J.
Zhang, K.-Q.
Zhang, S.
Zhang, X.
Zhang, Y.
Zhao, J.-H.
Zhao, L.
Zhao, Y.
Zharkov, D. O.
Zheng, J.
Zhmurov, A.
Zhong, H.
Zhong, L.
Zhou, R.
Zhou, T.
Zhou, Y.
Zhou, Z.-L.
Zhuohang, M.
Zhurkin, V. B.
Zhuze, A. L.
Zima, V.
Zinchenko, A. A.
Zomot, E.
Zu, Y.
Zwier, M. C.
535
955
1005
619
861
994, 996
97, 247
881
557, 1029
39, 743
992
431, 815
1095, 1101
1048
975, 976, 997
1051
355
981
1150
1120
743
871
1158, 1160
1131
991
85
1032
323
980
Albany 2011: The 17th Conversation, June 14-18, 2011
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