Here - Index of

Transcription

Here - Index of

The 5th Central European Conference
"Chemistry towards Biology"
Book of Abstracts
September 8–11, 2010
Primošten, Croatia
International Steering Committee
Dušan Berek, Slovakia
Ivano Bertini, Italy
Pavel Hobza, Czech Republic
Boris Kamenar, Croatia
Venčeslav Kaučič, Slovenia
Robert Konrat, Austria
Henryk Kozlowski, Poland
Vladimir Král, Czeh Republic
Christoph Kratky, Austria
Tadeusz M. Krygowski, Poland
Zvonimir Maksić, Croatia
Henriette Molinari, Italy
Gábor Náray-Szabó, Hungary
Botond Penke, Hungary
András Perczel, Hungary
Ladislav Petruš, Slovakia
Janez Plavec, Slovenia
Jaroslaw Polanski, Poland
Lucio Randaccio, Italy
Vilim Šimanek, Czech Republic
Ioan Silaghi-Dumitrescu, Romania
Grazyna Stochel, Poland
Sanja Tomić, Croatia
Local Organizing Committee
Marija Abramić
Zvonimir Maksić
Branka Salopek-Sondi
Sanja Tomić
Robert Vianello
The 5th Central European Conference – Chemistry towards Biology, Primošten, September 8–11, 2010
ISBN–13 978–953–6690–83–1
EAN 9789536690831
Editorial board:
Marija Abramić
Zvonimir Maksić
Branka Salopek-Sondi
Sanja Tomić
Robert Vianello
Publisher:
Ruđer Bošković Institute
Bijenička 54, P. O. Box 180
HR–10002 Zagreb
Croatia
Contact:
http: www.irb.hr
Tel: +385 1 456 1111
Fax: +385 1 468 084
Year of publishing: 2010
Supported by:
The Ministry of Science, Education and Sports of the Republic of Croatia
Croatian Academy of Science and Arts
Ruđer Bošković Institute, Zagreb, Croatia
Croatian Biophysical Society
Contents
Short history of the
"Chemistry towards Biology" series of conferences ....................
7
Program of the conference ......................................................
9
List of poster presentations .....................................................
15
Abstracts of lectures ...............................................................
23
Abstracts of posters ................................................................
59
List of participants ..................................................................
143
Index of contributions .............................................................
153
A Short History of the "Chemistry towards Biology" Symposia
In 1997 Zvonimir Maksić, Boris
Kamenar and Lucio Randaccio
discussed
the
possibilities
of
reviving scientific collaborations in
the
South
West
European
countries after the decay and fall
of Yugoslavia. Based on the
traditional ties between Ljubljana,
Trieste
and
Zagreb
it
was
concluded that the first of the
series of biannual meetings should
take place in Trieste. It was
organized by L. Randaccio at the
University of Trieste in 1998 as the
CIS chemistry meeting involving researchers from Croatia, Italy and Slovenia. The
next ACIS meeting was organized by Z. Maksić on Brijuni islands in 2000. In
addition to chemists from Austria, a number of leading guest–scientists from the
Central European countries, including Czech Republic, Hungary, Poland and
Slovakia, have been invited too. Several important decisions were made at Brijuni
meeting.
First,
the
symposium
should
become
Central
European
one
encompassing countries mentioned above. Building on the similar cultures,
educational systems and existing scientific contacts, it should contribute to efficient
collaboration, better use of large equipment, mobility of young researchers and last
but not the least to the joint projects. Secondly, the symposium should be focused
on chemistry related to molecular biology according to suggestion of Ivano Bertini.
This was accepted and the idea of the Central European CtB symposia was born!
Hence, the Brijuni meeting was a precursor of the whole Series and ignition for the
first one organized by Slavko Kavčić at Portorož (Slovenia) in 2002. Tradition is
cherished by Symposia held in Seggau (Cristoph Kratky) in 2004, Krakow
(Grazyna Stochel) in 2006 and Dobogoko (Andras Perczel) in 2008. It is hoped that
symposia will maintain to be melting pots for chemists and molecular biologists,
with possible extention to experts in the related fields, to mention only
biophysicists.
7
8
Program of the Conference
Wednesday, 08 September 2010
13:00 – 21:00
Registration
16:45 – 17:00
Opening ceremony
Chairperson: Sanja Tomić
17:00 – 17:25
INVITED LECTURE IL1
17:25 – 17:40
LECTURE L1
17:40 – 18:05
INVITED LECTURE IL2
18:05 – 18:20
Emil Palecek, Institute of Biophysics, Czech Republic
"Electrochemistry of biomacromolecules. New trends in
protein and polysaccharide electroanalysis"
Krzysztof Lewinski, Jagiellonian University, Poland
"High pressure protein crystallography for structural
biology"
Mariusz Jaskolski, A. Mickiewicz University, Poland
"Towards understanding Nod factor biosynthesis: crystal
structure of rhizobial NodS N–methyltransferase"
LECTURE L2
Bianka Szalaine Agoston, Eötvös Loránd University, Hungary
"Structural and functional characterization of the
intrinsically disordered plant dehydrin ERD14"
18:20 – 19:30
Welcome reception
Thursday, 09 September 2010
Chairperson: Marija Luić
09:00 – 09:50
PLENARY LECTURE PL1
09:50 – 10:15
INVITED LECTURE IL3
Nenad Ban, Swiss Federal Institute of Technology, Switzerland
"The next frontiers in ribosome research"
Ivana Weygand–Đurašević, University of Zagreb, Croatia
"The many faces of serine activating enzymes in protein
biosynthesis"
9
10:15 – 10:40
INVITED LECTURE IL4
Siniša Volarević, University of Rijeka, Croatia
"Activation of a p53-dependent ribosome checkpoint is
responsible for congenital malformations in mice"
10:40 – 11:00
Coffee Break
Chairperson: Marija Abramić
11:00 – 11:50
PLENARY LECTURE PL2
11:50 – 12:05
LECTURE L3
12:05 – 12:20
Rudolf Zechner, Karl Franzens University, Austria
"Lipolysis: More than just the catabolism of fat"
Veronika Stoka, Jožef Štefan Institute, Slovenia,
"Cross–talk of the kallikrein–kinin and the renin–angiotensin
systems as revealed by a structural network"
LECTURE L4
Tea Pavkov–Keller, MPI of Biophysics, Germany
"How do S-layers self-assemble?"
12:20 – 14:30
Lunch Break
Chairperson: Mariusz Jaskolski
14:30 – 15:20
PLENARY LECTURE PL3
15:20 – 15:45
INVITED LECTURE IL5
15:45 – 16:00
Anna Tramontano, University of Rome "La Sapienza", Italy
"Structural bioinformatics: From a luxury to a necessity"
Peter Macheroux, Graz University of Technology, Austria
"Novel alkyl sulfatases for biocatalysis"
LECTURE L5
Gyula Batta, University of Debrecen, Hungary
"Thermal unfolding of a highly stable antifungal protein at
extreme temperatures"
16:00 – 16:30
Coffee Break
Chairperson: Peter Macheroux
16:30 – 16:55
INVITED LECTURE IL6
16:55 – 17:10
LECTURE L6
Christian Obinger, BOKU University, Austria
"Chemistry and physiological role of heme peroxidase–
mediated halogenation and oxidation reactions"
Marcel Zámocký, BOKU University, Austria
"From chemistry of bifunctional catalase-peroxidases
towards molecular evolution of the peroxidase-catalase
superfamily"
10
17:10 – 17:25
LECTURE L7
17:25 – 17:40
LECTURE L8
17:40 – 17:55
LECTURE L9
17:55 – 18:20
Marija Luić, Ruđer Bošković Institute, Croatia
"Validation of the catalytic mechanism of E. coli purine
nucleoside phosphorylase"
Delia Picone, University of Naples Federico II, Italy
"Structure/toxicity relationships in seminal RNase, a protein
with multiple shapes"
Nadezhda Kudryasheva, Institute of Biophysics, Russia
"Pysico–chemical classification of toxic effects on bioassay
system"
INVITED LECTURE IL7
Barbara Mohar, National Institute of Chemistry, Slovenia
"Optimized ligands for Rh–catalyzed enantioselective
hydrogenation"
18:30 – 19:45
Poster Session
20:00
Dinner
21:00
Meeting of the International Steering Committee
Friday, 10 September 2010
Chairperson: Zvonimir Maksić
09:00 – 09:50
PLENARY LECTURE PL4
09:50 – 10:15
INVITED LECTURE IL8
10:15 – 10:30
Nicholas S. Bodor, Center for Drug Discovery, Florida, USA
"Retrometabolic drug design: Soft drugs and chemical
delivery systems"
Ema Žagar, National Institute of Chemistry, Slovenia
"Dendritic polymers for drug delivery applications"
LECTURE L10
Gábor Pál, Eötvös Loránd University, Hungary
"Selective blocking of the lectin pathway of the complement
system with phage display evolved peptide inhibitors"
10:30 – 10:55
Coffee Break
Chairperson: Jaroslaw Polanski
10:55 – 11:20
INVITED LECTURE IL9
Grażyna Stochel, Jagiellonian University, Poland
New photosensitizers for photodynamic therapy of cancer"
11
11:20 – 11:45
INVITED LECTURE IL10
11:45 – 12:00
LECTURE L11
12:30 – 19:00
Silvano Geremia, University of Trieste, Italy
"Structural basis for vitamin B12 delivery and the rational
design of bioconjugates"
Laszlo Nyitray, Eötvös Loránd University, Hungary
"Affinity enhancement of linear peptide motifs by in vitro
evolution: the case of dynein light chain (DYNLL) binding
peptides"
Excursion to the “Krka Lakes” National Park
Saturday, 11 September 2010
Chairperson: Botond Penke
09:00 – 09:50
PLENARY LECTURE PL5
09:50 – 10:15
András Perczel, Eötvös Loránd University, Hungary
"Foldamer stability in Trp cage miniproteins and type II
diabetes”
Linda Luck, State University of New York, USA
"19F NMR studies of receptor proteins"
David M. Smith, Ruđer Bošković Institute, Croatia
"Subtle Differences with Important Consequences in
Enzymatic Diol Dehydration"
10:40 – 11:00
Coffee Break
10:15 – 10:40
Chairperson: Piotr Cysewski
11:00 – 11:50
PLENARY LECTURE PL6
11:50 – 12:15
Janusz M. Bujnicki, Inst. of Mol. and Cell Biology, Poland
"RNA 3D structure prediction: From comparative to de novo
modeling"
Victor Viglasky, Safarik University, Slovakia
"Thermodynamic aspects of intra- and intermolecular G–
quadruplexes"
12:20 – 14:30
Lunch Break
12
Chairperson: Robert Vianello
14:30 – 14:55
14:55 – 15:10
LECTURE L12
15:10 – 15:25
Bela Gyurcsik, University of Szeged, Hungary
"Specificity of the zinc-finger DNA interaction. Calculations
and experiments"
Valery Andrushchenko, Academy of Sciences, Czech Republic
"Experimental and computational IR/VCD studies of d(G)8
structural forms"
LECTURE L13
Tadeusz Marek Krygowski, University of Warsaw, Poland
"Formamide as the Lewis acid/base amphotheric solvent
molecule: a computational approach"
15:25 – 15:40
Poster Award Ceremony
15:40 – 16:00
Closing Session
13
14
List of Posters
P–1
Maria A. Alexandrova, Gennady A. Badun, Galina A. Vydryakova, Nadezhda S. Kudryasheva
Effect of radionuclides on luminous bacteria
P–2
Dávid Árus, Tamás Gajda
New high affinity zinc binding site of human ZnT3 zinc transporter protein
P–3
Markus Auer, S. Alexander Teufer, Marcel Zamocky, Margit Bernroitner, Paul G. Furtmüller,
Christian Obinger
Heterologous expression and characterization of novel cyanobacterial heme peroxidases
P–4
Zsófia Balogh, Viola Bardóczy, Gergely Lautner, Beata Komorowska, Róbert E. Gyurcsányi, Tamás
Mészáros
Aptamers as virus detecting molecules
P–5
Lubos Bauer, Katarina Tluckova, Viktor Viglasky
Use of electrophoretic and spectroscopic methods in G–quadruplex research
P–6
Nadezda V. Belogurova, Nadezda S. Kudryasheva
Discharged photoprotein obelin: fluorescence peculiarities
P–7
2
Branimir Bertoša, Sanja Tomić, Maja Aleksić , Grace Karminski-Zamola
QSAR analysis and proposal of new heterocyclic compounds with potential antitumor activity
P–8
Gustavo A. Bezerra, Greg Wasney, Masoud Vedadi, Doug Cossar, Sirano Dhe-Paganon, Marija
Abramić, Peter Macheroux, Karl Gruber
Thermodynamic and structural studies of human dipeptidyl peptidase III with substrates and
inhibitors
P–9
Miroslava Bilecová – Rabajdová, Peter Urban, Jana Mašlanková, Alexander Ostró, Mária Mareková
Detection of vascular markers of patients with gynecological malignancies
P–10
Andrea Bodor, András Perczel, Á. Zotter, J. Ovádi
TPPP/p25: a new unstructured protein with GTPase activity
P–11
Ferenc Bogár, Zoltán Násztor, Balázs Leitgeb, Botond Penke
The influence of fluoride and iodide ions on the stability of Trp-cage miniprotein: a computational
study
P–12
Ana Brcko, Maja Brajlović, Saša Kazazić, Nina Jajčanin Jozić, Branka Salopek-Sondi
A possible interplay between two conserved Cys residues of auxin-amidohydrolase BrILL2 from
Brassica rapa L.
15
List of Posters
P–13
Sanja Tomić, Grit Straganz, Daniela Buongiorno, Michael Ramek, Hrvoje Brkić
Diketone Cleaving Dioxygenase 1 – Computational Study
P–14
Dorota Stępień, Michał K. Cyrański
Molecular complexes of phloroglucinol derivatives with pyridines
P–15
Piotr Cysewski
Post-SCF ab initio quantum chemistry characteristics of 8-oxogunine intermolecular interactions in
B-DNA
P–16
Anikó Czene, Béla Gyurcsik, Ida Noémi Jakab-Simon, Kyosuke Nagata,Hans Erik Mølager
Christensen
HNH motif as active centre of chimeric metallonucleases for gene therapy
P–17
Zsolt L. Datki, Blaine R. Roberts, Akos Hunya, Adam Gunn, Eva Kondorosi, Paul A. Adlard, Viktor
Szegedi, Gabor Juhasz , Dora Simon, Livia Fulop, Istvan Foldi, Zsolt Bozso, Katalin Soos, Gabor
Kozma, Akos Kukovecz, Colin L. Masters, Zoltan Konya, Ashley I. Bush
Alzheimer Risk Factors Age and Gender Induce Aβ Aggregation by Raising Extraneuronal Zinc
P–18
Zografia Boulsourani, Seyedehraziyeh Hosseinian, Evagellia Vassiliou, George Geromichalos,
Katia Repana, Efthalia Yiannaki, Catherine Raptopoulou, Dimitra Chatzipaulou-Litina, Catherine
Dendrinou-Samara
Synthesis, antiinflammatory and anticancer activity of binuclear and trinuclear copper (II) complexes
P–19
Ines Despotović, Zvonimir B. Maksić
Protonation of some Supramolecular Compounds Based on Pyridine Subunits
P–20
László Fábián, László Oroszi, Elmar K. Wolff, Pál Ormos, András Dér
Fast integrated optical switching by the protein bacteriorhodopsin
P–21
Etelka Farkas, Orsolya Szabó
2+/3+
and Fe2+/3+ Binding by Natural Siderophores and Model Hydroxamic Acids
Mn
P–22
Viktor Farkas, Petra Rovó, Gábor Tóth, András Perczel
Miniprotein structure: a chiroptical studies
P–23
Lívia Fülöp, Dóra Simon, Zsolt Bozsó, Tamás Janáky, Gábor Kozma, Ákos Kukovecz, Botond
Penke
Problems of controlling the aggregation of the synthetic beta amyloid peptide for use in biological
experiments
P–24
Paul. G. Furtmüller, Julius Kostan, Björn Sjöblom, Georg Mlynek, Stephanie Füreder, Michael
Wagner, Holger Daims, Christian Obinger, Kristina Djinović-Carugo
Structural and functional analyses of chlorite dismutases from two nitrite-oxidizing bacteria
16
List of Posters
P–25
Martina Glušič, Polona Ropret, Jože Grdadolnik
The binding of cadmium(II) to reduced form of glutathione: Vibrational study
P–26
Marina Grabar, Sanja Tomić, Lidija Tumir, Ivo Piantanida, Ivo Crnolatac
Molecular modeling of phosphonium cyanine dyes in complex with DNA
P–27
Sabine G. Gruber, Gustav Vaaje-Kolstad, Fabiola Matarese, Rubén López-Mondéjar, Christian P.
Kubicek, Verena Seidl-Seiboth
Regulation of fungal chitinases containing LysM motifs and adjacent LysM-proteins in Trichoderma
atroviride
P–28
Dávid Héja, Katalin Zboray, Dávid Szakács, András Szabó, Miklós Sahin-Tóth, Gábor Pál
Selective inhibitors of human chymotrypsin C developed by phage display
P–29
Balázs Leitgeb, Liza Hudoba, Gábor Janzsó, Gábor Rákhely
Structural investigation of palindromes of temporin antimicrobial peptides by molecular dynamics
methods
P–30
Liza Hudoba, Gábor Janzsó, Gábor Rákhely, Balázs Leitgeb
Characteristic structural features of palindrome sequences of indolicidin and tritrpticin
P–31
Imre Jákli, Dóra Menyhárd Karancsiné, András Perczel
Pair correlations in beta structures: from structural databases to the real life
P–32
Christa Jakopitsch, Jutta Vlasits, Christian Obinger
Mechanism of hydrogen peroxide oxidation in catalase-peroxidases
P–33
Veronika Jancsik, Emese É. Várkonyi
The multi-faceted melanin-concentrating hormone
P–34
Gábor Janzsó, Ferenc Bogár, Liza Hudoba, Botond Penke, Gábor Rákhely, Balázs Leitgeb
Folding processes of alanine-based peptides containing basic amino acids: helix and H-bond
formation
P–35
Gábor Janzsó, Ferenc Bogár, Liza Hudoba, Botond Penke, Gábor Rákhely, Balázs Leitgeb
Folding processes of alanine-based peptides containing basic amino acids: folding time, pathways
and stability
P–36
Gábor Janzsó, Balázs Leitgeb, Gábor Rákhely, Botond Penke, Ferenc Bogár
Replica exchange molecular dynamics simulations of Aβ1-42 and its isopeptide
17
List of Posters
P–37
Alexander A. Kamnev, Lev A. Dykman, Anna V. Tugarova
Novel possibilities of FTIR spectroscopy in bioscience: From interactions at nanobioconjugates to
bacterial cells
P–38
Alexander A. Kamnev, Roman L. Dykman, Krisztina Kovács, Ernő Kuzmann, Attila Vértes
Spectroscopic approaches for studying the effects of abiotic factors on microbial molecular signaling
P–39
Ágnes Kasza, Viktor Szegedi, Gábor Juhász, Zsuzsanna Frank, Zsuzsa Penke, Botond Penke
ICV injected Aβ-peptide induces dysfunctions in hippocampus and in spatial memory: model for
Alzheimer's disease
P–40
Galina A. Kudryasheva, Elena V. Nemtseva
Biological effect of halogenated anthracenes on biochemical processes
P–41
Saška Marczi, Marijana Radić Stojković, Ivo Piantanida, Ivan Mihaljević, Ljubica Glavaš-Obrovac
Antiproliferative activity, uptake and intracellular distribution of urea-phenanthridinium conjugates
P–42
Marijana Marković, Jasmina Sabolović
Molecular modeling of crystallization processes of trans and cis bis(L-valinato)copper(II) from
aqueous solution
P–43
Goran Mikleušević, Marta Narczyk, Lucyna Magnowska, Beata Wielgus-Kutrowska, Agnieszka
Bzowska, Marija Luić
Steady-state kinetic analysis of E. coli purine nucleoside phosphorylase active site mutants
P–44
Nađa Došlić, Milena Petković, Jurica Novak
Infrared spectrum of the acetic acid dimer in the O–H stretching region
P–45
Katarzyna M. Majerz-Maniecka, Robert Musioł, Agnieszka Skórska-Stania, Josef Jampilek,
Barbara J. Oleksyn, Jarosław Polański
Pyridine and quinoline as privileged moieties in drug design: potential AChE reactivators, antifungal
and HIV integrase inhibitors
P–46
Łukasz Orzeł, Dorota Rutkowska-Żbik, Leszek Fiedor, Grażyna Stochel
Structural and electronic effects on the reactions of metallochlorophylls formation
P–47
Michael I. Oshtrakh, Aron L. Berkovsky, Amit Kumar, Suman Kundu, Alexander V. Vinogradov,
Tatiana S. Konstantinova, Vladimir A. Semionkin
Relationship of the heme iron stereochemistry and Mössbauer hyperfine parameters in different
oxyhemoglobins
18
List of Posters
P–48
Michael I. Oshtrakh, Arina V. Alenkina, Nikolai V. Sadovnikov, Vladimir A. Semionkin
Comparative analysis of human liver ferritin, chicken liver and spleen, and pharmaceutically
important ferritin models using Mössbauer spectroscopy
P–49
Wojciech P. Oziminski, Piotr Garnuszek, Aleksander P. Mazurek
Theoretical modeling of Pt-histamine complex hydrolysis and interactions with guanine and adenine
P–50
Gábor Paragi, Lajos Kovács, Zoltán Kupihár, Célia Fonseca Guerra, F. Matthias Bickelhaupt
Neutral or positively charged new tetramer structures: a computational study of xanthine and uric
acid derivatives
P–51
Nena Peran, Zvonimir B. Maksić
Calculation of the Asparagine pKa Values in Water Using COSMO, COSMO–RS and Modified
Cluster–Continuum Models
P–52
Gordana Pirc, Jernej Stare, Janez Mavri
Car–Parrinello simulation of hydrogen bond dynamics in sodium hydrogen bissulfate
P–53
Primož Šket, Janez Plavec
Assessing the Role of Loops on Cation Movement within DNA G-quadruplexes
P–54
Agata Kurczyk, Pawel Mazur, Barbara Janik, Andrzej Bak, Tomasz Magdziarz, Jaroslaw Polanski
Mining databases for the analysis of catechol and azanaftalene polypharmacology
P–55
Mária Bučková, Jana Godočíková, Marcel Zámocký, Christian Obinger, Bystrík Polek
Cloning and molecular analysis of katG genes from the soil fungi Chaetomium globosum and
Chaetomium cochliodes
P–56
László Radnai, Péter Rapali, Alíz Tichy-Rács, Csaba Hetényi, Veronika Harmat, Weixiao Wahlgren,
Gergely Katona, László Nyitray
Interaction of the LC8 dynein light chain with myosin Va and EML3: structural studies
P–57
Peter Rapali, Laszlo Nyitray, Gabor Pal
In vitro evolution of dynein light chain (DYNLL) binding peptides via phage display
P–58
Katalin Revesz, Tamas Meszaros, Miklos Csala
A bacterial model for identification of putative transporters
P–59
Giovanni N. Roviello, Domenica Musumeci, Cristian D’Alessandro, Enrico M. Bucci, Carlo Pedone
Synthesis and DNA/RNA binding studies of cationic peptides
P–60
Petra Rovó, Viktor Farkas, Pál Stráner, Beáta Huszka, András Perczel
Structure and dynamic studies of exendin-4: A new model for ligand-receptor interaction
19
List of Posters
P–61
Joanna Równicka-Zubik, Anna Sułkowska, Iwona Zubik-Skupień, Agnieszka Szkudlarek,
Małgorzata Maciążek-Jurczyk, Barbara Bojko, Wiesław W. Sułkowski
A comparison of drug-binding ability of bovine and human serum albumin using fluorescence
spectroscopy
P–62
Małgorzata Maciążek-Jurczyk, Anna Sułkowska, Joanna Równicka-Zubik, Agnieszka Szkudlarek,
Barbara Bojko, Wiesław W. Sułkowski
1
Determination of the binding site of antirheumatoid drugs in serum albumin. Fluorescence and H
NMR studies
P–63
Agnieszka Broniec, Anna Pawlak, Marta Wrona-Król, Andrzej Żądło, David H. Thompson, Tadeusz
J. Sarna
Interaction of plasmalogens with singlet oxygen and oxidizing free radicals
P–64
Éva Sija, Annalisa Dean, Tamás Kiss
Hydroxypyridinecarboxylic acids as possible chelating agents in the therapy of neurodegenerative
disorders
P–65
Dóra Simon, Lívia Fülöp, Zsolt Bozsó, Róbert Rajkó, Zsolt László Datki, Tamás Janáky, Botond
Penke, Dezső Virók
Protein chip based interactome analysis of Aβ indicates an inhibition of the cellular translation
machinery
P–66
Zuzana Sochorová Vokáčová, Lukas Trantírek, Vladimir Sychrovský
Does nonplanarity of nucleic acid bases affect NMR parameters?
P–67
Ivan Sondi, Branka Salopek-Sondi
Influence of the Primary Structure of Enzymes on the Formation of CaCO3 Polymorphs: A
Comparison of Plant and Bacterial Ureases
P–68
Gerhard Stadlmayr, Gordana Wozniak-Knopp, Christoph Hasenhindl, Florian Rüker, Christian
Obinger
Stability engineering of the Fc fragment of human IgG1 by targeted mutagenesis
P–69
Pál Stráner, András Perczel
Enhancing solubility of proteins isolated from inclusion bodies: Expression and NMR study of the
GLP-1 receptor
P–70
Dávid Szakács, Dávid Héja, Gábor Pál
Phage Display of Single Chain Ecotin on the Surface of M13
P–71
Halina Szatyłowicz, Nina Sadlej-Sosnowska
Strength of individual hydrogen bonds in G-C and A-T Crick-Watson base pairs
20
List of Posters
P–72
Imola Cs. Szigyártó, László I. Simándi
Functional models for catechol oxidase and phenoxazinone synthase
P–73
Anna Sułkowska, Agnieszka Szkudlarek, Joanna Równicka-Zubik, Iwona Zubik-Skupień,
Małgorzata Maciążek-Jurczyk, Barbara Bojko, Wiesław W. Sułkowski
Binding of ibuprofen to bovine serum albumin
P–74
Jelena Veljković, Marina Šekutor, Kata Mlinarić-Majerski, Krešimir Molčanov, Biserka Kojić-Prodić
2,6 3,10 5,9
Synthesis and crystal structures of novel pentacyclo[5.4.0.0 .0 .0 ]undecane hydrazones
P–75
Miroslava Štefanišinová, Mária Kožurková, Vladimíra Tomečková, Mária Mareková
Determination of the binding affinities of DNA with chalcone derivates and their influence on
mitochondria
P–76
KatarinaTluckova, Lubos Bauer, Viktor Viglasky
Topological variability of G-quadruplexes in oncogenic promoters region
P–77
Lidija–Marija Tumir, Ivo Piantanida, Marina Grabar, Sanja Tomić
The interactions of bis-phenanthridinium–nucleobase conjugates with double stranded DNA
P–78
Peter Urban, Miroslava Bilecová - Rabajdová, Jana Mašlanková, Jarmila Veselá, Mária Mareková
Ischemic-reperfusion injury of the small intestine and changes in gene expression
P–79
Katalin Várnagy, Dóra Kiss, Zsuzsanna Kovács, Katalin Ősz, Daniele Sanna, Eugenio Garribba,
Giovanni Micera
Transition metal complexes of non-proteinogenic histidine analogue amino acids and their tripeptide
derivatives
P–80
Bojana Vukelić, Branka Salopek-Sondi, Igor Sabljić, Jasminka Špoljarić, Dejan Agić, Marija Abramić
Dipeptidyl peptidase III from human symbiont Bacteroides thetaiotaomicron: isolation and
characterization
P–81
Andrzej Wojtczak, Anna Kozakiewicz
Modeling studies of potato NTPDase1: an insight into the catalytic mechanism
21
List of Posters
22
LECTURES
23
Invited lecture
Electrochemistry of biomacromolecules.
New trends in protein and polysaccharide electroanalysis
Emil Palecek*, Veronika Ostatna, Mojmir Trefulka, Hana Cernocka,
Martin Bartosik
Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Kralovopolska
135, 612 65 Brno, CR, * [email protected]
Electroactivity of nucleic acids was discovered fifty years ago [1–2]. At present
electrochemistry of nucleic acids is a mature field, involving a number of
applications in biotechnologies, particularly in DNA hybridization sensors useful in
DNA sequencing and diagnostics [3]. Determination of specific sequences in
genomic DNA without PCR amplification still remains a challenge.
In recent decades electrochemistry of proteins focused on a relatively small group
of conjugated proteins containing non-protein redox centers. Using the constant
current chronopotentiometric stripping (CPS) and hanging mercury drop or solid
amalgam electrodes we observed a well-developed electrocatalytic peak (peak H)
produced by all tested proteins at nanomolar concentrations. Peak H differs from
the previously studied electrochemical signals of proteins particularly (i) by its
ability to detect proteins down to nanomolar and subnanomolar concentrations [3,4]
(regardless of the presence or absence of cysteines in the protein molecules) and
(ii) by its high sensitivity to local and global changes in protein structures [4–8] and
redox states [10]. Until very recently it was believed that proteins are denatured
when adsorbed at bare mercury electrode surfaces [3,4].
We have shown that proteins retain their native structures when adsorbed at Hg
electrode close to the potential of zero charge. Depending on ionic conditions and
speed of the electrode charging to negative potentials [4–8], proteins can either
retain their native structures or undergo surface denaturation at bare mercury
electrodes. Bare Hg electrodes have been used to study aggregation of alphasynuclein (AS) protein involved in Parkinson’s disease [6]. Using CPS peak H early
changes in AS structure, preceding the protein aggregation has been uncovered.
We showed that using thiol-modified Hg electrodes changes in protein structure at
the electrode surface could be well controlled. The chemically modified Hg
electrodes are particularly useful for recognition of changes in protein structures
resulting from single amino acid exchange in mutated proteins such as tumor
suppressor protein p53, which plays a pivotal role in the development of cancer.
Pico- or femtomoles of p53 proteins were sufficient for the analysis.
24
Invited lecture
Recent progress in elucidation of biological functions of oligosaccharides (OSs),
and polysaccharides (PSs), including their roles in cancer and some
neurodegenerative diseases, supported improvement of analytical methods. Due
the alleged electroinactivity of PSs, electrochemical methods lagged behind.
Recently we have shown that sulfated PSs produce the electrocatalytic peak H [11] .
Moreover, electroactive labels can be introduced in PSs and OSs using complexes
of six-valent osmium with different nitrogenous ligands [Os(VI)L] [12,13]. The PSOs(VI)L adducts produce three redox couples at carbon and Hg electrodes and an
electrocatalytic peak (only at Hg electrodes) enabling determination of PSs and
OSs at picomolar level. In this way OSs in glycoproteins can be determined.
[1] E. Palecek, E. Nature 1960, 188, 656–657.
[2] E. Palecek, E. Electroanalysis 2009, 21, 239–251.
[3] E. Palecek, F. Scheller, J. Wang (eds.) Electrochemistry of nucleic acids and proteins.
Towards electrochemical sensors for genomics and proteomics. 2005, Elsevier,
Amsterdam. 789 pp.
[4] E. Palecek, V. Ostatna, Electroanalysis 2007, 19, 2383–2403.
[5] V. Ostatná, B. Dogan, B. Uslu, S. Ozkan, E. Palecek, J. Electroanal. Chem. 2006,
593, 172–178; V. Dorcak, E. Palecek, Electroanalysis 2007, 19, 2405–2412; V. Ostatná,
E. Palecek, Electrochim. Acta 2008, 53, 4014–4021; V. Ostatná, F. Kuralay, L. Trnková, E.
Palecek, Electroanalysis 2008, 20, 1406–1413.
[6] E. Palecek, V. Ostatna, M. Masarik, et al. Analyst, 2008, 133, 76–84.
[7] E. Palecek, V. Ostatna, Chem. Commun. 2009, 1685–1687.
[8] E. Palecek, V. Ostatna, Analyst 2009, 124, 2076–2080.
[9] M. Zivanovic, M. Aleksic, V. Ostatna, et al. Electroanalysis, 2010, in press.
[10] V. Dorcak, E. Palecek Anal. Chem. 2009, 81,1543–1548.
[11] A. Strmečki, M. Plavšić, B. Ćosović, et al. Electrochem. Commun. 2009, 11, 2032–
2035.
[12] M. Trefulka, E. Palecek, Electroanalysis 2009, 21, 1763–1766.
[13] M. Trefulka, E. Palecek, Electroanalysis 2010, submitted.
25
Lecture
High pressure protein crystallography for structural
biology
Krzysztof Lewiński*, Katarzyna Kurpiewska
Faculty of Chemistry, Jagiellonian University, Kraków, Poland, [email protected].,
[email protected]
Pressure is an important environmental parameter that is widely used to study
physicochemical aspects of proteins. While only a few high pressure
macromolecular structures have been determined until now, the results of those
studies revealed the potential importance of this method for structural biology. A
protein in solution equilibrates among multiple ensembles including native
conformer and locally unfolded intermediates that differ in free energy and partial
molar volume. By displacing the equilibrium toward more compact ensembles,
pressure increase the population of higher energy conformers[1]. While standard
crystallographic experiments give the structure that reflects an average structure of
conformers present in the crystal, the crystallography at high pressure conditions
has the ability to determine structure of higher energy conformers preceding
unfolded states. The increasing number of high pressure studies on protein folding
is related to observation that partially folded intermediates may give rise to
misfolded proteins, aggregates and amyloids that are associated with many
neurogenerative diseases, such as spongiform encephalopathies, Alzheimer's
disease and Parkinson's disease[2]. The use of high pressure offers an opportunity
to understand the mechanism of these processes and may help to develop new
therapeutic approaches.
[1] K. Akasaka, Chem. Rev. 2006, 106, 1814–1835.
[2] J. L. Silva, Y. Cordeiro, D. Foguel, BBA 2006, 1764, 443–451.
26
Invited lecture
Towards understanding Nod Factor biosynthesis: crystal
structure of rhizobial NodS N-methyltransferase
Ozgur Cakici1, Michal Sikorski1, Tomasz Stepkowski1, Grzegorz
Bujacz1, Mariusz Jaskolski1,2,*
1
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland,
[email protected].
2
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan,
Poland, [email protected].
NodS is a SAM-dependent N-methyltransferase involved in the biosynthesis of Nod
Factor (NF) in nitrogen-assimilating rhizobia. NF is a modified chitooligosaccharide
signal molecule that must be recognized by the legume plant host before
productive bacterium-plant symbiosis is established. So far, there has been no
structural information about the NodS enzyme from any rhizobium. We have
undertaken X-ray crystallographic studies of recombinant NodS protein from
Bradyrhizobium japonicum, which infects lupine. Two crystal forms, of ligand-free
NodS and of NodS in complex with S-adenosyl-L-homocysteine (SAH), which is a
byproduct of the methylation reaction, were obtained and their structures were
refined to 2.43 Å and 1.85 Å resolution, respectively. Although the overall fold is
similar as in other SAM-dependent methyltransferases (a seven-stranded open βsheet, flanked by α-helices on each side), NodS has also specific features
connected with binding of its unique oligosaccharide substrate. In particular the Nterminal helix gets ordered on SAM binding, thus not only closing the methyl-donor
cavity, but also shaping a long canyon on the molecular surface that is evidently
the binding site for the acceptor molecule. By gaining insight about how NodS
binds its donor and acceptor substrates, we hope to better understand the
mechanism of NodS and the basis of its functional difference in various rhizobia.
NodS from Bradyrhizobium japonicum
in cartoon rainbow representation, with
the SAH ligand shown in ball-and-stick
mode.
27
Lecture
Structural and Functional Characterization of the
Intrinsically Disordered Plant Dehydrin ERD14
Bianka Szalainé Ágoston1,2, Dénes Kovács2, Péter Tompa2, András
Perczel1,3
1
Protein Modelling Group, Hungarian Academy of Sciences and Eötvös Loránd University,
H 1117 Pázmány P. s. 1/A., Budapest, Hungary
2
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences,
H 1113 Karolina út 29., Budapest, Hungary
3
Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd
University, H 1117 Pázmány P. s. 1/A., Budapest, Hungary
Dehydrins are a class of stress proteins that belong to the family of Late
Embryogenesis Abundant (LEA) proteins in plants, so named because they are
highly expressed in late stages of seed formation. In somatic cells, their expression
is very low under normal conditions, but increases critically upon dehydration
elicited by water stress, high salinity or cold. Dehydrins are thought to be
intrinsically disordered, which represents a challenge in understanding their
structure-function relationship. Herein we present the full NMR assignment of the
185 amino acid long ERD14 (Early Response to Dehydration 14), which is a K3Stype, typical dehydrin of A. thaliana. Secondary chemical shifts as well as NMR
relaxation data show that ERD14 is fully disordered under near native conditions.
However, a closer look at the data shows five distinct sequential regions of
somewhat restricted flexibility with a small but clear tendency towards helicity (on
average: 15 %). Three of these regions coincide with the three conserved Ksegments of ERD14, proposed already to form helices upon interaction with SDS
or lipid vesicles. These results suggest that ERD14 may have partially preformed
elements for functional interaction with its partner(s) and set the stage for further
detailed structural and functional studies of ERD14 both in vitro and in vivo.
28
Plenary lecture
The next frontiers in ribosome research
Nenad Ban
ETH Zurich, Institute of Molecular Biology and Biophysics, Swiss Federal Institute of
Technology
The main goal of the research in my laboratory is to study structures of prokaryotic
and eukaryotic ribosomes, ribosomal subunits and their complexes with various
factors involved in protein synthesis with an aim to better understand this process
[1–3]
. Recently, we have expanded our studies to investigate structure and function
of large eukaryotic multienzyme complexes such as fatty acid synthases, giant
multifunctional enzymes that contain seven catalytic domains and catalyze all
steps of fatty acid synthesis. We are using crystallography as the primary method
in combination with electron microscopy and biochemical experiments. Some of
the recently obtained mechanistic insights from our group into the coupling
between protein synthesis on the ribosome and various subsequent cellular
processes will be discussed.
[1] R. Bingel-Erlenmeyer, R. Kohler, G. Kramer, A. Sandikci, S. Antolić, T. Maier, C.
Schaffitzel, B. Wiedmann, B. Bukau, N. Ban, Nature 2008, 452, 108–11.
[2] C. Schaffitzel, M. Oswald, I. Berger, T. Ishikawa, J.P. Abrahams, H.K. Koerten, R.I.
Koning, N. Ban, Nature 2006, 444, 503–6.
[3] L. Ferbitz, T. Maier, H. Patzelt, B. Bukau, E. Deuerling, N. Ban, Nature 2004, 431,
590–6.
29
Invited lecture
The many faces of serine activating enzymes
in protein and peptide biosynthesis
Ivana Weygand-Đurašević
University of Zagreb, Faculty of Science, Department of Chemistry, Zagreb, Croatia,
[email protected]
Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved
enzymes that catalyze amino acid activation and their transfer to tRNA.
Aminoacyl-tRNAs are then used as substrates for ribosomal protein biosynthesis.
The specificity of aaRSs is thought to be established and fixed in the earliest
stages of evolution. In this context, it is notable that the two types of seryl-tRNA
synthetases (SerRSs), bacterial- and methanogenic-type, which both catalyze
seryl-tRNA formation, represent the only aaRSs with different mode of recognition
of the same amino acid substrate, reflecting their distinct evolutionary origin. In
addition to aaRS aminoacylation activities (canonical activity), there is increasing
evidence that these enzymes are also involved in processes not directly related to
the protein synthesis. Analysis of completed genomes reveals that many
organisms, which possess full length functional aaRSs genes for housekeepng
enzymes also posses duplicated aaRSs genes or display additional ORFs
encoding single domain aaRS-like proteins. Some of these "additional" aaRSs or
aaRS-like enzymes have developed unexpected functions. We have identified the
genes for putative seryl-tRNA synthetase homologs widespread in bacterial
genomes and characterized three of them functionally and structurally [1]. They
resemble the catalytic domain of highly diverged, atypical seryl-tRNA synthetases
(aSerRSs) found only in methanogenic archaea [2], but they are deprived of the
tRNA-binding domain and lack canonical tRNA aminoacylating activity. Instead,
they transfer activated amino acid to phosphopantetheine prosthetic group of
putative carrier proteins. Remarkably, in comparison to SerRSs, truncated aSerRS
homologs display different and relaxed amino acid specificity. Their enzymatic
activity is reminiscent of adenylation domains in nonribosomal peptide synthesis,
and thus they represent an intriguing link between programmable ribosomal protein
biosynthesis and template-independent nonribosomal peptide synthesis.
[1] M. Mocibob, N. Ivic, S. Bilokapic, T. Maier, M. Luic, N. Ban, I. Weygand-Durasevic,
Proc. Natl. Acad. Sci. USA, 2010, in press.
[2] S. Bilokapic, T. Maier, D. Ahel, I. Gruic-Sovulj, D. Söll, I. Weygand-Durasevic, N. Ban,
EMBO J. 2006, 25, 2498–2509.
30
Invited lecture
Activation of a p53-dependent ribosome checkpoint is
responsible for congenital malformations in mice
Martina Barkić, Slađana Crnomarković, Siniša Volarević
Department of Molecular Medicine and Biotechnology, Faculty of Medicine, University of
Rijeka, Croatia
The capacity to detect and appropriately respond to many different stresses that
interfere with functional homeostasis is essential for organismal survival. Recent
evidence suggests that the nucleolus, the site of ribosome biogenesis, plays a
critical role in sensing and responding to many external and internal stresses. To
understand these processes, we have used a genetically defined in vivo mouse
models in which ribosome biogenesis could be manipulated. In the Belly Spot and
Tail (Bst) mice, which suffer from defects of the eye, skeleton, and coat
pigmentation, ribosomal biogenesis is impaired by mutation in one allele of
ribosomal protein l24 gene (Rpl24). It has been hypothesized that these
pathological manifestations result exclusively from faulty protein synthesis. We
have recently demonstrated that up regulation of the p53 tumor suppressor during
the restricted period of embryonic development significantly contributes to the Bst
phenotype. Our results imply that activation of a p53-dependent checkpoint
mechanism in response to ribosomal protein deficiencies might also play a role in
the pathogenesis of congenital malformations and possibly other diseases in
humans.
31
Plenary lecture
Intracellular Lipolysis and the Regulation of Lipid and
Lipoprotein Metabolism
Rudolf Zechner
Institute of Molecular Biosciences, University of Graz, Austria
The process of lipolytic hydrolysis of cellular triglyceride (TG) depots is designated
lipolysis and is of central importance in lipid and energy homeostasis. Lipolysis
requires at least two TG hydrolases, adipose triglyceride lipase (ATGL) and
hormone-sensitive lipase (HSL). Although both enzymes act within the same
biochemical pathway, they exhibit different substrate specificities and are regulated
by completely different mechanisms. The generation and characterization of
induced mutant mice lacking or overexpressing ATGL or HSL in specific tissues
helped to elucidate the physiological function of these enzymes in adipose tissue,
muscle, liver and macrophages. Taken together our results demonstrate that TG
hydrolysis not only affects the availability of FA as substrates for energy
production, but also impacts many other cellular and vascular processes including
cell signaling and lipoprotein metabolism.
32
Lecture
Cross-Talk of the Kallikrein-Kinin and the ReninAngiotensin Systems As Revealed by a Structural
Network
Veronika Stoka*, Vito Turk
J. Stefan Institute, Department of Biochemistry and Molecular and Structural Biology,
Ljubljana, Slovenia, [email protected], [email protected]
The kallikrein-kinin and renin-angiotensin systems (KKS-RAS) are two highly
regulated proteolytic pathways which are involved in various physiological and
pathological processes, i.e. cardiovascular and renal homeostasis, inflammation,
growth and development, among others.
At the protein level, it is a challenging endeavor to differentiate between direct
physical interactions and functional associations, which do not involve direct atomic
contacts between macromolecules. However, this information can be assessed
from an atomic-resolution characterization of the protein interfaces, thus resulting
from the three-dimensional-protein-protein interactions.
To gain insight into the multilayered interaction of the KKS-RAS systems, we
present a protein network thus built up on three-dimensional domain-domain
interactions. The essential domains that connect these systems are: Cystatin,
Peptidase_C1, Thyroglobulin_1, Insulin, CIMR (Cation-independent mannose-6phosphate receptor repeat), fn2 (Fibronectin type II domain), fn1 (Fibronectin type I
domain), EGF, Trypsin, and Serpin. Surprisingly, we found that the CIMR domain
is located at the core of the network, thus linking both systems. From the latter, all
domain interactors up to level 4 were retrieved, thus showing a more
comprehensive representation of the KKS-RAS structural network.
V. Stoka, V. Turk, Biol. Chem. 2010, 391, 443–454.
33
Lecture
How do S-layers self-assemble?
Tea Pavkov-Keller* 1,2, Janet Vonck1, Eva M. Egelseer3, Uwe B. Sleytr3,
Werner Kühlbrandt1, Walter Keller2
1
Structural Biology, Max-Planck Institute of Biophysics, Frankfurt, Germany.
2
Institute of Molecular Biosciences, Karl-Franzens University, Graz, Austria.
3
Department of Nanobiotechnology, University of Natural Resources and Applied Life
Sciences, Vienna, Austria.
* corresponding author; [email protected]
Bacterial cell surface layer (S-layer) proteins are one of the most abundant cellular
proteins with the ability to form crystalline arrays on prokaryotic cells. Different
biological functions and promising nanobiotechnological applications have been
demonstrated. However, detailed structural information on S-layer proteins is very
scarce. For determining the structure-function relationship of SbsC, the S-layer
protein from Geobacillus stearothermophilus ATCC 12980, deletion mutants were
produced. It was shown that the N-terminal part is responsible for binding to a
secondary cell wall polymer (SCWP) and that the C-terminal part is essential for
self-assembly.
Combining X-ray crystallography and electron microscopy we could, for the first
time, describe how the S-layer self-assembles. We present three X-ray structures
of the different truncated forms of the S-layer protein SbsC[1,2]. The protein consists
of 9 domains: one coiled-coil domain and 8 Ig-like domains. The domains are
connected via short linkers forming an elongated molecule with a great flexibility.
These high resolution structures could be fit in an electron density map obtained by
3D-reconstruction of negatively stained 2D-crystals of a full length SbsC protein.
The thickness of the assembled S-layer could be determined by tomography. The
domains and the interaction sites responsible for self-assembly were identified.
[1] T. Pavkov, E.M. Egelseer, M. Tesarz, D.I. Svergun, U.B. Sleytr, W. Keller, Structure
2008, 16(8), 1226.
[2] M. Kroutil, T. Pavkov, R. Birner-Gruenberger, M. Tesarz, U.B. Sleytr, E.M. Egelseer, W.
Keller, Acta Crystallogr 2009, F65(10), 1042.
34
Plenary lecture
Structural bioinformatics: from a luxury to a necessity
Anna Tramontano
Department of Biochemical
[email protected].
Sciences,
Sapienza
University,
Rome,
Italy,
The proteins perform most of the functions of a living organism, for examples they
can catalyse chemical reactions, activate other proteins in response to external
factors or at specific times, identify and eliminate foreign molecules, maintain the
physical integrity of the cell, transmit signals, recognize and transport small
molecules. How can proteins perform so many and diverse functions so efficiently
and in such a coordinated fashion? The trivial answer is that they assume a threedimensional shape that allows them to position specific chemical groups in the
correct position to perform the desired function. Does this imply that, given the
three dimensional shape of a protein, we can deduce which function it performs in
a living cell and, perhaps, conceive ways to interfere with it? The answer is
unfortunately that, in general, this is not the case.
We can nevertheless devise a “periodic” table of protein structures and use it to
gain some clues about their functional properties. Our table is not as elegant as the
periodic table of elements because we need to take into account several
dimensions, sequence, structure and evolution at a minimum. Furthermore, the
emerging picture is much more complex and fuzzy than we would like so that its
interpretation has occupied many researchers for decades.
On the basis of what we learn from the analysis of these solved instances of the
problem, we cab predict the structure of a protein from its amino acid sequence in
many cases. The evolutionary mechanisms imply that proteins mostly evolved via
small sequence variation, usually single amino acid substitutions, insertions and
deletions. Therefore the sequences of proteins that are “sufficiently” closely
evolutionary related in evolution preserve detectable similarities.
The next, perhaps more important question is to which extent a relationship in
structural space mirrors a relationship in functional space. This is a much harder
problem that is attracting much interest because only by solving it, we can properly
interpret the genomic and post-genomic data and fully exploit their power in
shaping the life sciences of the future.
35
Invited lecture
Active site characterisation of the first sec-alkyl
sulfatase: towards the deracemisation of sec-alcohols
Tanja Knaus1, Markus Schober2, Kurt Faber2, Peter Macheroux1
1
Institute of biochemistry, Graz University of Technology, Petersgasse 12, A–8010 Graz,
Austria
2
Department of Chemistry, University of Graz, Heinrichstrasse 28, A–8010 Graz, Austria
Following the detection of sec-alkyl sulfatase activity in Pseudomonas sp.
DSM6611 [1] the gene encoding the enzyme was identified by a combined protein
analysis and whole-genome sequencing approach. Recombinant expression of the
gene enabled us to purify the first alkyl sulfatase specific for the hydrolysis of secalkylsulfates such as 2-octyl sulfate. The enzyme was also shown to be specific for
the hydrolysis of the (R)-stereoisomer of the substrate into the (S)-product (2octanol) by stereoinversion at the chiral carbon atom. Sequence comparison of the
sec-alkyl-sulfatase with other members of the metallo-β-lactamase family revealed
a close relationship to the previously characterized prim-alkyl sulfatase (SdsA1)
from Pseudomonas aeruginosa [2]. In fact, both enzymes share the canonical
topology of this protein family, including a dinuclear Zn-cluster in the active site.
However, close inspection of the amino acid residues in the active site of SdsA1
and the novel sec-alkyl sulfatase revealed obvious differences in the substrate
binding pocket. In order to understand the structural parameters underlying
substrate preference and stereoselective hydrolysis, we have carried out a
structure based site-directed mutagenesis study. Here, we will present first results
and discuss the potential for engineering of the active site to create enzymes with
altered substrate- and stereo-preference.
[1] P. Gadler, K. Faber, Trends Biotechnol. 2007, 25, 83–88.
[2] G. Hagelueken, T. M. Adams, L. Wiehlmann, U. Widow, H. Kolmar, B. Tümmler, D. W.
Heinz, W.-D. Schubert, Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7631–7636.
36
Lecture
Thermal unfolding of a higly stable antifungal protein at
extreme temperatures
Gyula Batta*1, Ádám Fizil1, Tibor Kurtán1, Zoltán Gáspári3, Teréz
Barna2, Katalin E. Kövér1, Éva Leiter2, István Pócsi2, Florentine Marx4
1
Institute of Chemistry, University of Debrecen, Debrecen, Hungary,
[email protected].
2
Institute of Biology, University of Debrecen, Debrecen, Hungary,
3
Institute of of Chemistry, Eötvös Loránd University, Budapest, Hungary,
4
Innsbruck Medical University, Division of Molecular Biology, Innsbruck, Austria,
The functional aspects of structure and dynamics of the small (55 aa) antifungal
protein PAF have recently been disclosed by NMR[1]. This protein is harmless for
mammalian cells and has many potential applications (e.g. against Aspergillosis).
At 4o C PAF is stable in solution for years. Reversible thermal unfolding/folding[2] of
PAF could be monitored in the –10 ... +80o C temperature range using the peak
integrals in 15N–1H HSQC spectra. Automatic peak assignment transfer was
achieved with the new lonelyness parameter concept. It seems that the
hydrophobic core of the protein is maintained with the three disulfide bonds, and
this core is the most stable entity that holds the greek-key supersecondary
structure together as long as possible. The exterior of PAF unfolds "earlier" and at
least two "independent" unfolding stages are suggested. CD studies and
thermodynamic interpretation of the results are in progress.
Acknowledgement:
This research was supported by the NKTH–OTKA CK–77515 Grant.
[1] G. Batta, T. Barna, Z. Gáspári, S. Sándor, K. E. Kövér, U. Binder, B. Sarg, L. Kaiserer,
A. Kumar-Chhillar, A. Eigentler, É.Leiter, N.Hegedüs, I. Pócsi, H.Lindner and F.Marx:
FEBS Journal., 2009, 276, 2875–2890.
[2] A. Pastore, S. R. Martin, A. Politou, K.C. Kondapali, T. Stemmler and P.A. Temussi: J.
Am. Chem. Soc. 2007, 129, 5374–5375.
37
Invited lecture
Chemistry and physiological role of heme peroxidasemediated halogenation and oxidation reactions
Christian Obinger1, Marzia Bellei2, Stampler Johanna1,
Paul G. Furtmüller1, Gianantonio Battistuzzi2
1
Metalloprotein Research Group, Department of Chemistry, Division of Biochemistry,
BOKU, Vienna, Austria, Email: [email protected]
2
Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy
Human heme peroxidases are able to catalyze the two-electron oxidation of
halides (Cl–, Br–, I–) and thiocyanate as well as the one-electron oxidation of
nitrite[1], thereby generating reactive species that participate in hormone synthesis,
host defence against foreign microorganisms, as well as in immunomodulation and
tissue degradation in inflammatory diseases. Various halogenated and/or oxidized
biomolecules serve as biomarkers of peroxidase activity and are known to
accumulate in certain pathologies. Typically, the heme in these enzymes is
covalently linked to the apoprotein by two ester bonds, and in one case
(myeloperoxidase) additionally by a unique sulfonium ion bond. As a consequence
the prosthetic group is distorted from planar conformation with small structural
differences being reflected by distinct spectral and redox properties as well as
reactivities towards substrates. The thermodynamics and kinetics of the involved
redox reactions
is
[3]
myeloperoxidase
demonstrated[1,2]. Based
on
the known
structure
of
and lactoperoxidase a mechanism for hypohalous acid
production and release is provided.
Recent phylogenetic analysis suggests that predecessor genes of chordata
peroxidases have segregated early in evolution. Before organisms developed an
acquired immunity, their antimicrobial defence seemed to depend on enzymes that
produce antimicrobial reaction products. These heme proteins (even found in
prokaryotes) evolved to important components in the innate immune defence
system.
[1] M. Zederbauer, P.G. Furtmüller, S. Brogioni, C. Jakopitsch, G. Smulevich, C. Obinger,
Nat. Prod. Rep. 2007, 24, 571–584.
[2] D.R. Ramos, M.V. Garcia, M.L. Canle, J.A. Santaballa, P.G. Furtmüller, C. Obinger, J.
Inorg. Biochem. 2008, 102, 1300–1311.
[3] X. Carpena, P. Vidossich, K. Schroettner, Calisto, S. Banerjee, J. Stampler, M. Soudi,
P.G. Furtmüller, C. Rovira, I. Fita, C. Obinger, J. Biol. Chem. 2009, 284, 25929–25937.
38
Lecture
From chemistry of bifunctional catalase-peroxidases
towards molecular evolution of the peroxidase-catalase
superfamily
Marcel Zámocký1, 2, Marcus Motz1, Paul G. Furtmüller1, Jutta Vlasits1,
Christa Jakopitsch1, Christian Obinger1
1
BOKU, Muthgasse 18, A–1190 Vienna, Austria, Email: [email protected]
2
Department of Microbiology, Institute of Molecular Biology, Slovak Academy of Sciences,
SK–84551 Bratislava, Slovakia
Catalase-peroxidases are heme b containing bifunctional oxidoreductases that
represent ancestral enzymes with obvious catalytic promiscuity but not certainly
known physiological function. In further evolution these two-domain proteins gave
rise to a very abundant peroxidase-catalase superfamily[1] of metalloproteins
encoded in genomes of both prokaryots and eukaryots with currently over 4,200
known genes. According to structural peculiarities all members of this superfamily
can be classified in three distinct classes [2]. The most variable Class I (focus of this
contribution) involves reaction specificities varying from H2O2 dismutase, ascorbate
peroxidase to cytochrome c peroxidase. The genome-database mining and
phylogenetic analysis revealed the presence of two further subfamilies of hybridtype peroxidases that are phylogenetically positioned between the families of
cytochrome c peroxidases and ascorbate peroxidases. Our experimental research
is currently focused on KatGs i.e. the bifunctional catalase-peroxidases. These
proteins are present not only in eubacteria from which they originate but
corresponding katG genes were transferred during the evolution also in archaeons
and in lower eukaryots by the means of horizontal gene transfers (HGT). Most
abundant eukaryotic KatGs are ascomycetous catalase-peroxidases. Among
phytopathogenic fungi they were proposed to be involved in the response to the
oxidative burst of the host. We have expressed heterologously three distinct fungal
katG genes and purified them to homogeneity. These highly purified recombinant
fungal KatGs allowed for the first time a detailed investigation of their physical and
chemical properties and comparison of their kinetics and spectroscopic behavior
with their bacterial counterparts. Directed evolution of fungal katG genes will
produce mutants in selected hot spot regions to address important aspects of
structure-function relationships within bifunctional catalase-peroxidases.
[1] M. Zámocký, P. G. Furtmüller, C. Obinger, Arch.
10.1016/j.abb.2010.03.024
[2] K.G. Welinder, Curr. Opin. Struct. Biol. 1992, 2, 388–393.
Bioch.
Biophys.
2010,
doi:
39
Lecture
Validation of the catalytic mechanism of E. coli purine
nucleoside phosphorylase
Marija Luić1*, Zoran Štefanić1, Goran Mikleušević1, Marta Narczyk2,
Beata Wielgus-Kutrowska2, Agnieszka Bzowska2
1
Ruđer Bošković Institute, Zagreb, Croatia, marija.luić@irb.hr
2
Department of Biophysics, Institute of Experimental Physics, University of Warsaw,
Warsaw, Poland, [email protected]
Purine nucleoside phosphorylase (PNP) is the key enzyme in the purine salvage
pathway[1]. It catalyses the reversible phosphorolytic cleavage of the glycosydic
bond of purine nucleosides and some analogues.
Biologically active form of the Escherichia coli purine nucleoside phosporylase
(PNP) is a homohexamer, whose structure could be described as a trimer of
dimers. In order to validate a catalytic mechanism proposed for this enzyme[2], five
active site mutants: Arg24Ala, Asp204Ala, Asp204Asn, Arg217Ala and
Asp204Ala/Arg217Ala were prepared. All mutated residues are very important for
the catalytic activity, since their change into alanine reduces activity of the enzyme
by at least 100-fold. Activity of the mutants vs natural substrates adenosine,
inosine and guanosine as well as 7-methylguanosine confirms that catalysis
involves protonation of the purine base at the position N7 by the side chain of the
Asp204. Kinetic studies as well as the crystal structures of wild type and Arg24Ala
mutant in complexes with phosphate are carried out and their results will be
presented. These results provide insight into the structure and catalytic mechanism
of E. coli PNP. Since E. coli PNP has shown to be a promising candidate for
tumour-directed gene therapy[3], this may help in design mutants useful for medical
use.
[1] A. Bzowska, E. Kulikowska, D. Shugar, Pharmacol. Ther. 2000, 88, 349–425.
[2] G. Koellner, A. Bzowska, B. Wielgus-Kutrowska, M. Luić, T. Steiner, W. Saenger, J.
Stẹpiński, J. Mol. Biol. 2002, 315, 351–371.
[3] Y. Zhang, W. B. Parker, E. J. Sorscher, S. E. Ealick, Curr. Top. Med. Chem. 2005,
5(13), 1259–74.
40
Lecture
New insights on the structure/toxicity relationships in
seminal RNase, a protein with multiple shapes
Delia Picone 1*, Carmine Ercole1, Gerardino D’Errico1, Marisa Lista1,
Roberta Spadaccini 2
1
Dipartimento di Chimica “Paolo Corradini”, Università Federico II di Napoli, 80126,
Napoli, Italy, [email protected]
2
Dipartimento di Scienze Biologiche ed Ambientali, Università del Sannio, 82100,
Benevento, Italy, [email protected]
Among the vertebrate ribonucleases. bovine seminal ribonuclease is the only
dimeric enzyme, which naturally exists as a mixture of swapped and un-swapped
forms [1]. Besides the hydrolytic activity, the protein displays additional biological
properties, including antitumor activity. In the cytosol, only the swapped form of
BS-RNase keeps a dimeric structure, in which the two subunits interchange the Nterminal ends. The quaternary structure allows this protein to evade the
ribonuclease inhibitor (RI), a protein that binds strongly most ribonucleases.
To understand the molecular basis of the swapping mechanism of BS-RNase, also
with the aim to increase the stability of the swapped form and possibly to improve
the basal anti-tumour activity, we have designed several mutants and evaluated
their structural and functional properties [2–3], but so far none of them displayed a
swapping propensity or an antitumor activity higher than the native protein. Using a
complementary approach, we have inserted key residues of BS-RNase into the
sequence of RNase A [4]. We have obtained a protein with the same swapping
propensity of BS-RNase, but its low cytotoxic activity indicated that the swapping is
not sufficient to elicit the cytotoxic activity, which is instead the result of a
complicated interplay of different effects, including a positive surface potential to
allow membrane interaction. Based on these considerations, here we report the
design and the characterization of the first BS-RNase variant with an improved
cytotoxicity.
Finally, EPR measurements acquired in the presence of spin-labelled synthetic
models of membrane, indicate that this effect can be related to a stronger
interaction with the phospholipid molecules of the cell membrane.
[1] R. Piccoli, M. Tamburrini, G. Piccialli, A. Di Donato, A. Parente, G. D’Alessio, Proc. Natl. Acad.
Sci. USA 1992, 89, 1870–1874.
[2] D. Picone, A. Di Fiore, C. Ercole, M. Franzese, F. Sica, et al, J Biol Chem 2005, 280, 13771–78.
[3] C. Ercole, R. Spadaccini, C. Alfano, T. Tancredi, D. Picone, Biochemistry 2007, 46, 2227– 32.
[4] C. Ercole, R. A. Colamarino, E. Pizzo, F. Fogolari, R. Spadaccini, D. Picone, Biopolymers
2009, 91, 1009–17.
41
Lecture
Physico-Chemical Classification of Toxic Effects on
Bioassay System
Nadezhda S. Kudryasheva
Institute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia, [email protected]
Bioluminescent (BL) assays based on luminous marine bacteria and their enzyme
reactions are widely used for toxicity monitoring in water media; BL intensity is a main
testing parameter in the assay procedure. Physico-chemical characteristics of
exogenous compounds (ECs) in water solutions determine their toxic effects, which
can be classified as physics, chemical and/or biochemical effects in the BL assay
systems. Basing on broad investigation of effects of model toxic ECs on BL assay
systems, classification of the effects on BL enzyme reaction is suggested. Five
mechanisms are discussed: (1) change of electron-excited states’ population and
energy transfer, (2) change of efficiency of S-T conversion in the presence of external
heavy atom, (3) change of rates of coupled reactions, (4) interactions with enzymes
and variation of enzymatic activity, (5) nonspecific effects of electron acceptors.
Effects of different groups of compounds were discussed according to the
classification suggested. Energy transfer processes (mechanism 1) contribute to BL
intensity change in the presence of ECs with electron-excited states energy lower than
that of bioluminescent emitter. Fluorescent ECs of this kind can provoke changes in BL
spectra. Iodine- and bromine-substituted ECs change efficiency of S-T conversion
(mechanism 2), but contribution of this mechanism is much lower than contribution of
mechanism (4). Organic and inorganic oxidizers (quinones, metals of variable valency,
etc.) produce specific changes in BL kinetics: BL induction period appears; its value
depends on concentration and redox potential of oxidizers. Competition of the
oxidizers with FMN for NADH in reaction of NADH:FMN-oxidoreductase is responsible
for these changes (mechanism 3). Such specific BL kinetics changes make BL
enzymatic assay specific to oxidizers: oxidative toxicity of solutions can be evaluated
by BL induction period, while general toxicity – by maximal BL intensity. Interactions
with enzymes (mechanism 4) is a prevalent mechanism for most of ESs; its efficiency
depend on hydrophobicity of organic ESs, atomic weight of haloid substituents, or
electron acceptation properties of metal ions. Interactions of ECs with enzymes were
studied using time-resolved fluorescent technique. Mechanism 5 is specific for
solutions of polar ESs, e.g., metal salts.
BL assays were found to be sensitive to alpha- and beta-radionuclides. Role of
peroxides (mechanism 3) and electron transfer (mechanism 5) in BL activation and
inhibition in the radionuclide solutions is discussed.
42
Invited lecture
Optimized ligands for Rh-catalyzed enantioselective
hydrogenation
Barbara Mohar*, Michel Stephan
Laboratory of Organic and Medicinal Chemistry, National Institute of Chemistry, Hajdrihova
19, 1001 Ljubljana, Slovenia; e–mail: [email protected]
Our new diversified series of P-stereogenic diphosphines[1] from Knowles’ DiPAMP
ligand exhibit highly enhanced efficiency in Rh-catalyzed hydrogenation of a widerange of olefins.
LIGANDS
R-SMS-Phos
RO
BigFUS
OR
P
Ph
RO
P
OR
P
Ph
Ph
(RO)n
P
Ph
(RO)n
R= branched/functionalized alkyl or aryl
n= 1 3
Switching DiPAMP’s o-anisyl groups to other aryls bearing branched or
functionalized RO-groups demonstrated that such modification was crucial for an
improved catalysis on the contrary to what was assumed earlier.[2] In addition, new
P-stereogenic polyalkoxyphenyl-based ligands were introduced which exhibited
exceptional activity and enantioselectivity for the reduction of top challenging
substrates. These ligands have been used in the synthesis of various chiral
building blocks such as α- / β-amino acids, α-amino phosphonates, amines,
alcohols, acids, diacids, hydroxyl acids, etc.
[1] a) B. Zupančič, B. Mohar, M. Stephan, Org. Lett. 2010, 12, 1296–1299; b) M. Stephan,
D. Šterk, B. Mohar, Adv. Synth. Catal. 2009, 351, 2779–2786; c) B. Zupančič, B.
Mohar, M. Stephan, Adv. Synth. Catal. 2008, 350, 2024–2032; d) M. Stephan, B.
Mohar (PhosPhoenix SARL, National Institute of Chemistry of Slovenia),
FR2005/2887253, WO2006/136695, US2010/099875.
[2] W. S. Knowles, Angew. Chem. Int. Ed. 2002, 41, 1998–2007.
43
Plenary lecture
Retrometabolic Drug Design: Soft Drugs and Chemical
Delivery Systems
Nicholas S. Bodor1
1
Center for Drug Discovery, University of Florida, Gainesville, USA, [email protected].
Despite considerable progress during the last decades, rational drug design is still
an elusive goal. It has, however, become clear that the development of effective
pharmaceutical agents with minimal side effects requires that targeting and
metabolism considerations form an integral part of the drug design process.
Retrometabolic approaches represent systematic drug design methodologies that
thoroughly integrate structure-activity and structure-metabolism relationships into
the drug design process and are aimed to design safe new drugs with an improved
therapeutic index. They incorporate two major design concepts : soft drugs (SD)
and chemical delivery systems (CDS), respectively.
Soft drugs are new, active molecules, often isosteric/isoelectronic analogues of a
lead compound, specifically designed to allow predictable and controllable
metabolism into inactive metabolite(s) after exerting the desired therapeutic effect.
A total of five major approaches have been identified, and two of them, the
“inactive metabolite-based” and the “soft analog” approaches proved to be the
most useful and successful strategies. In both cases, the usual oxidative metabolic
processes were replaced by design, with hydrolytic ones. Already, many ingenious
and successful soft drug examples have been described. Soft corticosteroids
represent one of the most successful areas, but there are a number of others, such
as soft β-blockers, soft anticholinergics, soft estrogens, soft antimicrobials, soft
calcineurin inhibitors and others. Loteprednol etabonate, a soft corticosteroid, is on
the US and world market in various different products. Since soft drug design is
based on well-defined, specific steps, these were incorporated into a general
computer program which generates virtual soft drug libraries and ranks them on
the basis of isosteric/isoelectronic analogy and metabolic conversion rates criteria.
The chemical delivery systems (CDS) are biologically inactive molecules designed
to enhance drug delivery to a particular organ or site. This is achieved by
sequential enzymatic conversion steps into the corresponding active drug and
exploiting differential influx-efflux processes through biological barriers. One such
system is used to target drugs to the brain. Here, the drug is chemically modified
44
Plenary lecture
by introduction of a 1,4-dihydrotrigonelline targetor moiety, which through
conversion to a hydrophilic quaternary form provides brain-targeting and sustained
release via a ‘lock-in’ mechanism behind the blood-brain-barrier. An extension of
this system also allowed brain targeting of peptides. Another basic approach takes
advantage of enzymes located in specific organs. A combination of ‘oxime
hydrolase’ and ‘ketone reductase’ enzymes allowed targeting to the eye of some
antiglaucoma agents. Clinical ‘proof of concept’ studies for betaxoxime, the oxime
analog of betaxolol, will be presented.
45
Invited lecture
Dendritic polymers for drug delivery applications
Ema Žagar*1, Sebastjan Reven2
1
National Institute of Chemistry, Ljubljana, Slovenia, [email protected].
1
EN-FIST Centre of Excellence, Dunajska 156, 1000 Ljubljana
2
Lek Pharmaceuticals d.d., Sandoz Development Center Slovenia, Ljubljana, Slovenia,
[email protected].
Aqueous solubility of drugs plays a decisive role in formulation development. The
number of solubilization techniques such as cosolvent addition, micellar
solubilization through surfactants, use of cyclodextrins, pH modification, solvent
recrystalization, spray drying and prodrug formulation have been developed for
drug solubilization. The effect of linear polymers on drug solubility has been widely
investigated in the case of solid dispersions, but only few products using this
technology are commercially successful. In the past decade the developed
dendritic polymers bring a new challenge in this field.
Dendritic polymers, i.e., dendrimers and hyperbranched polymers, possess unique
highly branched molecular architecture and a large number of functional groups.
Due to their distinctive mechanical, chemical and physical properties they have
attracted considerable and increasing interest in the field of drug delivery.
Dendrimers have well defined monodisperse perfectly branched structures, which
consist of fully branched, i.e., dendritic repeat units, and unreacted terminal repeat
units. They are synthesized tedious with many protection and deprotection
synthetic and purification steps, which make their large-scale production difficult
and expensive. On the other hand, hyperbranched polymers are simpler to
produce on a large scale via one-pot synthesis. However, this simplified procedure
yields fewer regular structures and broad molar mass distributions. Hyperbranched
polymers consist not only of dendritic and terminal repeat units but also of linear
ones, which are regarded as defects in their branched structures.
In this presentation the possibility of dendritic polymers as solubilization enhancers
affecting drug solubility and dissolution rate will be presented.
46
Lecture
Selective blocking of the lectin pathway of the
complement system with phage display evolved peptide
inhibitors
Andrea Kocsis1, József Dobó1, Adrienna K. Kékesi2,3, Róbert Szász4,
Péter Závodszky1, Péter Gál1, Gábor Pál 5 *
1
Institute of Enzymology of HAS, Budapest, Hungary [email protected].
Dept. of Physiology and Neurobiology of ELTE, Budapest, Hungary, kakekesi
dec001.geobio.elte.hu
3
Proteomics Group of the Biology Institute, ELTE, Budapest, Hungary
4
University of Debrecen, Health Science Center, 2nd Department of Internal
Medicine, Debrecen, Hungary, [email protected]
5
Department of Biochemistry of ELTE, Budapest, Hungary, [email protected].
2
The complement system, an essential part of the innate immune system, can be
activated through three distinct routes: the classical, the alternative and the lectin
pathways. The contribution of individual activation pathways to different biological
processes can be assessed by using pathway-selective inhibitors. Here we report
the first lectin pathway-specific peptide inhibitors developed by phage display
against mannose-binding lectin-associated serine proteases MASP-1 and MASP-2.
Two different serine protease inhibitor scaffolds were used as starting points for
library design. Based on the selected peptide sequences several peptides were
produced and characterized. All these peptides block the lectin pathway activation
completely while leaving the classical and the alternative routes intact and fully
functional. It indicates that from all complement proteases only MASP-1 and/or
MASP-2 are inhibited by these peptides. MASP-2 has been considered to be the
key enzyme of the lectin pathway as in vitro studies showed that it has all activities
required to activate the pathway. This key role was further confirmed by the fact
that our selective MASP-2 inhibitors were able to perfectly block the lectin pathway.
MASP-1 on the other hand is a more enigmatic protease believed to be only an
auxiliary component of the pathway. However, we demonstrated that the lectin
pathway can be perfectly blocked by selective MASP-1 inhibitors as well. Based on
comparative analysis of several functional assays we suggest that MASP-1 has a
crucial role in the initiation steps of lectin pathway activation possibly by activating
MASP-2. Since the lectin pathway has been implicated in several life threatening
pathological states including the extensive myocardial tissue damage after cardiac
attack, these inhibitors could be considered as lead compounds towards
developing lectin pathway blocking therapeutics.
47
Invited lecture
New photosensitizers for photodynamic therapy of cancer
Janusz M. Dabrowski1, Luis G. Arnaut2, Mariette M. Pereira2, Krystyna
Urbanska3, Grażyna Stochel1
1
Faculty of Chemistry, Jagiellonian University,Ingardena 3, 30060 Kraków, Poland
[email protected]
2
Department of Chemistry,University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal
3
Department of Biophysics,Jagiellonian University,Gronostajowa 7,30-387 Kraków, Poland
Photodynamic therapy (PDT) is a treatment modality for the selective
destruction of cancerous and non neoplastic pathologies based on the use of
photosensitizer, light and molecular oxygen to produce highly reactive oxygen
species resulting in necrosis and/or apoptosis of the treated cells, shutting down
the tumor microvasculature and stimulation of the host immune system.[1]
In this communication the usefulness of new class of halogenated
tetrapyrrolic photosensitizers in the photodynamic therapy of cancer is assessed
and recommendations are given for the design of more effective PDT protocols
employing these photosensitizers and near infrared radiation. The transparency of
tissues is optimal above 700 nm and the irreversible formation of singlet oxygen
requires sensitizers with singlet states below 800 nm. Thus, NIR photons are ideal
for PDT and the absorption peak of halogenated bacteriochlorins at 750 nm is
ideally suited to maximize both tissue penetration and efficient singlet oxygen
generation. With bacteriochlorins it will become possible to treat efficiently larger
tumours. [2] Prior to biological tests (cytotoxicity, cellular uptake, dose-dependent
phototoxicity), photochemical characterization (singlet and triplet lifetimes, quantum
yields of fluorescence, singlet oxygen and superoxide ion generation) of the
studied compound is presented.
[1] G. Stochel, M. Brindell, W. Macyk, Z. Stasicka, K. Szaciłowski, Bioinorganic
photochemistry, Wiley, Chichester, 2009.
[2] E. F. F. Silva, C. Serpa, J. M. Dabrowski, G. F. F. Sá, C. J. P. Monteiro, L. G. Arnaut,
S. J. Formosinho, G. Stochel, K. Urbanska, S. Simões, M. M. Pereira, Chem. Eur. J. 2010.
48
Invited lecture
Structural basis for vitamin B12 delivery and the rational
design of bioconjugates
Silvano Geremia1
1
University of Trieste, Department of Chemical Sciences, Centre of Excellence in
Biocrystallograpy, Trieste, Italy, [email protected].
Cobalamin (Cbl, vitamin B12) is an essential micronutrient for mammals, because
the corrin ligand of this cobalt-complex is synthesized only by bacteria and two
important enzymes: methylmalonyl-CoA mutase and methionine synthase have a
B12 derivative as cofactor [1]. Mammals have developed a complex system for
internalization of this vitamin from the diet. Three binding proteins (haptocorrin,
intrinsic factor, transcobalamin (TC)) and several specific cell surface receptors are
involved in the process of intestinal absorption, plasma transport and cellular
uptake [2]. Rapidly growing cells, as tumor cells, take up proportionally more vitamin
B12 than non-proliferating cells. Therefore, vitamin B12 is a potential vehicle that
can carry drugs to a specific cells and its bioconjugates have the potential
application as diagnostic for therapeutic antitumour agents [1,2]. The crystal
structures of B12 transport proteins reveal a two-domain architecture, with an Nterminal alpha(6)-alpha(6) barrel and a smaller C-terminal beta domain [3]. One Cbl
molecule is buried inside the domain interface. Structural information permits a
rational approach to the design of new B12-based bioconjugates. In particular, the
structures point to the 5’-hydroxyl group of the ribose moiety as the most promising
site for the covalent attachment of active ligands to cobalamin to preserve the
affinity of the resulting analogue for the transport proteins [4]. Recent results and
future prospects of B12 bioconjugates will be presented.
[1] L. Randaccio, S Geremia, N. Demitri, J. Wuerges, Molecules 2010, 15, 3228–3259; L.
Randaccio, S. Geremia, N. Demitri, J. Wuerges, Trends in Inorg. Chem. 2009, 11, 1–
19; L. Randaccio, S. Geremia, J. Wuerges, J. Organomet. Chem. 2007, 692, 1198–
1215.
[2] J. Wuerges, S. Geremia, L. Randaccio, Biochem. J. 2007, 403, 431–440.
[3] J. Wuerges, G. Garau, S. Geremia, S. N. Fedosov, T. E. Petersen, L. Randaccio, Proc.
Natl. Acad. Sci. U. S. A. 2006, 103, 4386–4391; J. Wuerges, S. Geremia, S. N.
Fedosov, L. Randaccio, IUBMB Life 2007, 1–8.
[4] P. Siega, J. Wuerges, F. Arena, E. Gianolio, S. N. Fedosov, R. Dreos, S. Geremia, S.
Aime, L. Randaccio, Chemistry 2009, 15, 7980–7989.
49
Lecture
Affinity enhancement of linear peptide motifs by in vitro
evolution:
the case of dynein light chain (DYNLL) binding peptides
Péter Rapali1, László Radnai1, Veronika Harmat2, Wixhiao Walgren3, Gergely
Katona3, Csaba Hetényi1, Gábor Pál1, László Nyitray1
1
Department of Biochemistry,
Hungary, [email protected]
3
2
Institute of Chemistry, Eötvös University Budapest,
Department of Chemistry, University of Gothenburg, Sweden
The highly conserved homodimer dynein light chain (DYNLL) is a eukaryotic hub
protein. It is a micromolar binder of linear epitopes having the loose consensus
sequence, [DS]KX[TVI]Q[TV][DE]. The binding motif is frequently located in
intrinsically disordered regions in the vicinity of coiled coil structures. DYNLL might
be a dimerization engine regulating its partners involved in cancer development,
transcription regulation and apoptosis. Gene knockout or knockdown of DYNLL
causes cell death through apoptosis suggesting that it should be a potential drug
target protein. In order to explore weather the binding motif of DYNLL is
thermodynamically optimal we applied an in vitro evolution approach, phage
display. A naive peptide library was displayed on M13 phage in a bivalent manner
using a Leu-zipper. The in vitro selected consensus sequence, VSRGTQTE is
similar to the natural one, but is extended by an additional binding determinant, a
Val, which increases the affinity tenfold. Dimerization through the Leu-zipper
further increases the affinity into the subnanomolar range. Interestingly, we
identified a human protein, EML3 that contains the phage-selected consensus
sequence located also in a disordered region. Structural details of the affinity
increase is explained based on x-ray crystallography and molecular modeling
studies. The results show that natural evolution of the DYNLL binding motif was
driven towards a biologically rather than thermodynamically optimal affinity. The
phage-selected high affinity peptide presented here could be used a competitive
inhibitor for therapeutic purposes.
50
Plenary lecture
Foldamer stability in Trp cage miniproteins and type II
Diabetes
Petra Rovó1, Viktor Farkas2, Pál Stráner1, András Láng1,
Gábor K. Tóth3, András Perczel1,2
1
University, Pázmány Péter sétány 1/A, H–1117 Budapest, Hungary,
2
Protein Modelling Group MTA-ELTE, Institute of Chemistry Eötvös Loránd University,
Pázmány Péter sétány 1/A, H–1117 Budapest, Hungary, and Department of Medical
3
Chemistry, Faculty of General Medicine, University of Szeged, Dóm tér 8., H–6720
Szeged, Hungary
We are studying the structural features of selected Exendin-4 related molecules.
They could serve as a highly potent GLP-1 receptor agonist, while its N-terminally
truncated analogs could be a high affinity antagonists, both types related to type II
diabetes. Aiming to find peptides of increased helicity, NMR and ECD
spectroscopic methods were applied to assess the role of each helix forming amino
acid residues in the stabilization of the overall fold. NMR restrain based solution
structures were obtained for the most folded analogues. We will present on how
the elongation and helix-stability alteration reflects to the cooperative nature of Trpcage miniprotein fold. The 20-residue long, Trp cage miniproetin, NLYIQWLKDG
GPSSGRPPPS, labelled as Tc5b by Neidigh and co-workers (Nat. Struct.
Biol.2002, 9, 425–430) starts at the N-terminus by an alpha-helix, continues by a
310-helix and concludes at its C-terminal by a poly-proline II helix. All of these
structural subunits are packed against the central Trp6. Various spectroscopic
methods (e.g. NMR, ECD) will be presented on how to analyses structure,
backbone dynamics, stability and foldamer interactions of both the natural and 15Nlabelled miniprotein variants (Hudáky, P. et. al. (2008) Biochemistry 47, 1007-1016,
Rovó P. 2009 unpublished).
Figure: Superposition and secondary structure or
foldamer distribution of several Trp cage miniprotein
variants.
51
Invited lecture
19
F NMR Studies of Receptor Proteins
Linda A. Luck
State University of New York at Plattsburgh, Plattsburgh, NY, USA,
[email protected].
19
F NMR has proven to be a powerful tool in the study of protein structure and
dynamics because the 19F nucleus is easily incorporated at specific labeling sites,
where it provides a relatively nonperturbing yet sensitive probe with no background
signals. Recent applications of 19F NMR from our laboratory in mapping out
structural and functional features of receptor proteins, including the glucose and
galactose-binding protein, the leucine-binding proteins, the human soluble tissue
factor, and the human estrogen receptor. These illustrate the utility of 19F NMR in
the analysis of protein conformational states even in molecules to large or unstable
for full NMR structure. These studies rely on the fact that the 19F nucleus is
sensitive to changes in the local conformational environment, which include van
der Waals packing interactions and local electrostatic fields. Additional information
can be obtained by the use of solvent-induced isotope shifts and paramagnetic
probes, which reveal solvent exposure of labeled residues. Also the use of
fluorinated ligands allows the determination of the role of the hydrogen bonding
interactions at specific sites. The studies will demonstrate the usefulness of this
nucleus in revealing important structural and kinetic features of protein
conformational changes, ligand-binding and protein-protein interactions.
52
Invited lecture
Subtle Differences with Important Consequences in
Enzymatic Diol Dehydration
David M. Smith1,*, Borislav Kovačević1, Danijela Barić1,
Gregory M. Sandala2, Leo Radom2
1
Rudjer Boskovic Institute, Zagreb, Croatia, [email protected].
2
University of Sydney, Sydney, Australia
The microbial dehydration of 1,2-diols is important for several reasons. On one
hand, this process enables certain types of bacteria to utilize these chemicals for
metabolic purposes. [1] On the other hand, chemical industry has found a novel way
to use this process to generate 1,3-propanediol, for environmentally friendly textile
production.[2] In both contexts, a better understanding of the mechanism is
desirable.
This contribution will outline recent computational investigations aimed at
understanding both the fundamental mechanism of action as well as the
inactivation of the enzymatic dehydration systems. Our approach combines
modern quantum chemical tools, to accurately model the chemical transformations,
with a classical molecular mechanical description of the surrounding protein.[3] The
calculations have revealed several subtle differences between closely related
enzymes that have important consequences on the final outcomes of the reactions.
[1] L. Macis, R. Daniel, G. Gottschalk, FEMS Microbiol. Lett. 1998, 164, 21.
[2] H. Miller, Int. Fiber J. 2000, 15, 14.
[3] K. Condic-Jurkic, H. Zipse, D. M. Smith, J. Comput. Chem. 2010, 31, 1024.
53
Plenary lecture
RNA 3D structure prediction: from comparative to de
novo modeling
Janusz M. Bujnicki1,2
1
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular
and Cell Biology, PL–02-109 Warsaw, Poland
2
Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology, Adam
Mickiewicz University, PL–61-614 Poznan, Poland
Email: [email protected]
WWW: http://iimcb.genesilico.pl
RNA is a large group of functionally important biomacromolecules. In striking
analogy to proteins, the function of RNA depends on its structure and dynamics,
which in turn is encoded in the linear sequence. There are numerous methods for
computational prediction of protein 3D structure from sequence, with comparative
modeling being the most reliable approach if a structure of a related molecule is
available. However, there are very few such methods for RNA. We have developed
tools for 3D modeling of RNA structure: ModeRNA for comparative modeling and
SimRNA for de novo modeling. As an input, ModeRNA requires a 3D structure of a
template RNA molecule, and a sequence alignment between the target to be
modeled and the template. ModeRNA can model posttranscriptional modifications,
a functionally important feature analogous to posttranslational modifications in
proteins. ModeRNA can also model DNA structures or use them as templates. It is
equipped with many functions for merging fragments of different nucleic acid
structures into a single model and analyzing its geometry, and can build models of
very big molecules, such as ribosomal RNAs (>1000 nucleotides). SimRNA is a
coarse-grained method for simulating RNA folding. It employs a reduced
representation (3 atoms per nucleotide), a statistical potential derived from a
database of known structures as an energy function, and a Monte Carlo searching
scheme. It can model RNA structures based solely on sequence information, but
the simulation can be also restricted by user-defined restraints. SimRNA-generated
structures of low energy are often very similar to the native structure. These tools
will be useful for studying sequence-function relationships in RNA molecules.
54
Invited lecture
Thermodynamic aspects of intra- and intermolecular Gquadruplexes
Viktor Víglaský, Ľuboš Bauer, Katarína Tlučková
Safarik University, Faculty of Siences, Institute of Chemistry, Department of Biochemistry,
Kosice, Slovakia, [email protected].
Guanine-rich sequences of nucleic acids may fold into secondary structural forms
called G-quadruplexes. G-quadruplexes are a rapidly growing theme of interest
with promising repercussions in our understanding of biology and practical
applications in medical fields, materials science, and biotechnology. Naturally
occurring oncogenic promoters (c-myc, c-kit, bcl-2, etc.) and telomeric repeats may
form G-quadruplexes in the guanine-rich strands, as well as a bimolecular Gquadruplex that targets HIV-1 integrase. These structures can be
thermodynamically more stable than DNA duplex. Various analytical techniques
are currently applied for the routine assessment of the stoichiometry that generally
includes conditions not representative of the environment in which the structural
studies are performed1. Although G-quadruplexes are extremely stable structures
their DNA strands arrangement shows a huge conformational variability which
depend on the condition and DNA sequence. In addition, details about driving
forces which govern topology and molecularity of G- rich sequences folding remain
still unknown. Except the type of ions, mainly potassium and sodium, also specific
ligands and crowding condition mimicking agents influence on the structural
variability of G- quadruplexes2.
[1] M. Webba da Silva, Methods 2007, 43, 264 –277.
[2] V. Viglasky, L. Bauer, K. Tluckova, Biochemistry 2010, 49, 2110–2120.
55
Invited lecture
Specificity of the zinc-finger DNA interaction.
Calculations and experiments
Béla Gyurcsik*1, Gábor Nagy1, Tamás Körtvélyesi2
1
Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H–
6720 Szeged, Hungary, E–mail: [email protected]
2
Department of Physical Chemistry and Material Sciences, University of Szeged, Aradi
Vértanuk tere 1, H–6720 Szeged, Hungary, E–mail: [email protected]
Zinc-finger (ZF) domains were first identified in transcription factors, highly specific
proteins that control gene activation. A ZF domain consists of two antiparallel βstrands and an α-helix. Two cysteines and two histidines coordinate the zinc(II)ion.
A ZF domain recognizes three subsequent bases in DNA sequence through
specific interactions with the bases exerted within the major groove. The amino
acids responsible for specific contacts are at positions –1, 1, 2, 3, 5, and 6
numbered relative to the start of the α-helix[1]. By the variation of these residues ZF
proteins can be designed to recognize practically any nucleotide sequence. ZF
proteins linked to a catalytically active agent form specific artificial enzymes such
as the ZF-FokI chimeric nucleases[2]. One of their most important applications
could be the gene therapy by specifically positioned chromosomal cleavage
increasing the frequency of homologous recombination[3]. The question of
specificity of the DNA binding is a critical requirement for such future applications.
To get an impression about the possibility of theoretical modelling of the specificity
of interactions, we performed calculations on a specific and nonspecific DNA
complexed with a three ZF-array protein based on semiempirical approaches. A
newly designed ZF array was positioned on the obtained specificity scale as to be
able to bind the DNA specifically. The synthesis, purification and gel shift
experiments have been performed to confirm the theoretical predictions. The
results of the above investigations will be presented.
Acknowledgments: This work has received support through the Hungarian
Science Foundation (OTKA-NKTH CK80850 and OTKA K72781 and K61577).
[1] J.G. Mandell, C.F. Barbas III, Nucl. Acids Res. 2006, 34, W516.
[2] Y.-G. Kim, J. Cha, S. Chandrasegaran, Proc. Natl. Acad. Sci. USA, 1996, 93, 1156.
[3].E.A. Moehle, J.M. Rock, Y.-L. Lee, Y. Jouvenot, R.C. DeKelver, P.D. Gregory, F.D.
Urnov, M.C. Holmes, Proc. Natl. Acad. Sci. USA 2007, 104, 3055.
56
Lecture
Experimental and computational IR/VCD studies of d(G)8
structural forms
Valery Andrushchenko1*, Dimiter Tsankov2, Maria Krasteva3,
Hal Wieser3, Petr Bouř 1
1
Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Prague, Czech
Republic, [email protected].
2
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria,
[email protected].
3
Department of Chemistry, University of Calgary, Calgary, Canada, [email protected].
Oligo- and polynucleotides rich in guanine bases, such as polyG, can form different
single- and multiple-stranded structures in solution. Infrared (IR) and vibrational
circular dichroism (VCD) spectroscopy have been widely used for characterization of
such systems.[1–4] However, due to a large variety of possible conformations,
interpretation of the spectroscopic data and subsequent structural assignments
remain ambiguous. Rather different IR and VCD spectra have been ascribed to fourstranded quadruplex structures.[1–4] The observed differences have been sometimes
attributed to metastable polyG quadruplex forms,[1,2] or to a quadruplex-duplex
transition.[2]
In an attempt to resolve this ambiguity we performed a combined experimental and
computational IR/VCD study of d(G)8 octamer. Experimental spectra were measured
at standard conditions used by other authors. Theoretical spectra were calculated for
single-, double- and quadruple-stranded d(G) 8 systems employing a multi-scale
approach. The computational methodology included initial molecular dynamics (MD)
simulations, followed by ab initio calculations of IR and VCD spectra for the whole
octamers using the Cartesian coordinate tensor transfer technique (CCT).[5] On the
basis of a comparison of the computed spectra with experiment the most probable
experimental structures could be determined.
[1] F. B. Howard, J. Frazier, H. T. Miles, Biopolymers 1977, 16, 791.
[2] A. G. Petrovic, P.L. Polavarapu, J. Phys. Chem. B 2008, 112, 2245.
[3] M. R. Guzman, J. Liquier, S. K. Brahmachari, E. Taillandier, Spectrochim. Acta, Part A
2006, 64, 495.
[4] J. Nový, S. Bohm, J. Králová, V. Král, M. Urbanová, Biopolymers 2008, 89, 144.
[5] P. Bouř, J. Sopková, L. Bednárová, P. Maloň, T. A. Keiderling, J. Comput. Chem. 1997,
18, 646.
57
Lecture
Formamide as the Lewis acid/base amphotheric solvent
molecule: a computational approach
Halina Szatyłowicz1, Tadeusz M. Krygowski2*
1
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00–664 Warsaw,
Poland, Email: [email protected]
2
Department of Chemistry, Warsaw University, Pasteura 1, 02–093 Warsaw, Poland, Email:
[email protected]
Solvent effects on chemical and biochemical processes as well as on
physicochemical properties have been subject of huge amount of works, which are
nicely reviewed in excellent monograph by Reichardt.[1] The idea of amphotheric
properties of aprotic solvents considered in terms of Lewis acid/base theory was
presented in mid 70-ties[2] and is also associated with the Kamlet and Taft approach
to the solvent effect in which solvents are classified into: solvent H–bond acceptor
basicities (HBA) [3] and H–bond donor acidities (HBD).[4] The H–bond formation is
possible for each non-carbon atoms of formamide.
Base
AH
H
O
C
H
Base
HA
N
H
Base
The dots show potential
localization of H-bonding
Application of the method based on the approaching of either the base (e.g. F–) or
acid (e.g. HF)[5] to the appropriate Lewis acidic or basic centers, respectively, allows
to estimate numerically their power (in acidity or basicity) by analyzing characteristics
of the H–bonding formed. Computation for the above presented modeling is carried
out at the B3LYP/6–311+G** level.
[1] Ch. Reichardt, Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH, Weinheim,
2003.
[2] T.M. Krygowski, R.W. Fawcett, J. Am. Chem. Soc. 1975, 97, 2143; R.W. Fawcett, T.M.
Krygowski, Austr. J. Chem. 1975, 28, 2125.
[3] M.J. Kamlet, R.W. Taft, J. Am. Chem. Soc. 1976, 98, 377–383.
[4] M.J. Kamlet, R.W. Taft, J. Am. Chem. Soc. 1976, 98, 2886–2894.
[5] T.M. Krygowski, J.E Zachara, H. Szatyłowicz, J. Phys. Org. Chem. 2005, 18, 110–114.
58
POSTERS
59
Poster
Effect of radionuclides on luminous bacteria
Maria A. Alexandrova1*, Gennady A. Badun3, Galina A. Vydryakova2,
Nadezhda S. Kudryasheva1,2
1
Siberian Federal University, Krasnoyarsk, Russia, [email protected]
2
Institute of Biophysics SB RAS, Krasnoyarsk, Russia,
3
Moscow State University, Chemistry Dept., Moscow, Russia.
Increase of radioactive contamination in environment is an important problem of
modern ecology. Luminous bacteria are perspective assay systems for monitoring
of radiation toxicity in solutions of radionuclides. Their luminescent intensity is a
useful physiological parameter for revealing effects of toxic media. Effects of
americium-241, high-radioactive alpha-emitting radionuclide, a product of
plutonium radioactive decay, and tritium, beta-emitting nuclide, on luminous
bacteria were monitored in our study.
Growth and luminescence of Рhotobacterium Phosphoreum 1883 from Collection
of IBP SB RAS, Krasnoyarsk were under study in the presence of americium-241
(0.2 - 6.7 kBq/L) and tritium (250–108 kBq/L) in nutrient media. Accumulation of
radionuclides in bacterial cells was evaluated.
Control (without radionuclides) and tested (with radionuclides) bacterial
suspensions were examined and compared. The suspensions were sampled at
two stages of growth - exponential and stationary, and bioluminescent intensity and
quantum yields were determined.
It was found that americium-241 suppressed bacterial growth; the effect became
more pronounced after 20-h of exposure. Tritium activated bacterial growth at all
times of exposure.
In the presence of americium-241, luminescent intensity and quantum yields were
increased in exponential-stage samples and decreased in stationary-stage
samples. In the presence of tritium, activation occurred simultaneously in both
types of samples.
We found that bacterial cells accumulate 70% of americium-241 and 25% of
tritium.
Role of peroxides (as secondary products of water solution radiolysis) and electron
transfer processes in effects of the radionuclides on luminous bacteria is
discussed.
60
Poster
New high affinity zinc binding site of human ZnT3 zinc
transporter protein
Dávid Árus1, Tamás Gajda1
1
Department of Inorganic and Analytical Chemistry, University of Szeged, H–6701 Szeged,
Hungary (E–mail: [email protected])
Dysfunction of zinc homeostasis was found to be associated with several chronic
diseases, e.g. asthma, diabetes and neurodegenerative disorders. ZnT3 is a brain specific
zinc transporter protein, which is located in the membranes of zinc-rich glutamatergic
presynaptic vesicles. The accumulation of zinc in these vesicles by ZnT3 followed by the
fast release of Zn2+ ions is related to the amyloid neuropathology of Alzheimer’s disease.
However, nothing is known about ZnT proteins at molecular level, especially concerning to
metal ion sensing, binding, translocating and sequestering properties. An intriguing
feature shared by ZnT proteins is an intracellular His-rich loop between the
transmembrane domains IV–V. This loop as a putative metal binding site (MBS) of these
proteins is widely accepted in the literature[1].
Although, this His-rich loop as putative MBS of
ZnT proteins (MBS1, Figure 1) may be attractive
for integrative aspects, both zinc binding and
transport mechanism of ZnTs may differ from
each other considerably. For example, the
number of His residues and hence the zinc ion
binding ability of these His-rich loops are Figure 1. Predicted membrane topology of human
protein. The locatin of the conserved,
considerably different within the ZnT family. On ZnT3
putative metal binding sites are indicated.
the other hand, the ZnT proteins contain
several further well conserved extramembranal
sequences with potentially high metal binding capacity: (i) the cytoplasmic C-terminal tail
of human, mouse and bovine ZnT3 have a metalloprotease-like metal binding motifHDXHX8H- (MBS2); (ii) the cytoplasmic N-terminal tail of human and some mammalian
ZnT2 and ZnT3 proteins possess a –HHCH- sequence (MBS3). To understand the
mechanism(s) of the of ZnT transporters first the zinc binding site(s) should be clarified.
Our earlier studies on metal-ion binding properties of His-rich peptides indicated, that the 3
separated His-residues in MBS1 and MBS2 cannot be regarded as a high affinity Zn2+binding site without the assistance of special tertiary structure of the protein. Since the 3D
structure of human ZnT3 is unknown, here we report solution chemical investigation of the
zinc(II) and nickel(II) complexes of Ac-PFHHCHRD-NH2 peptide, identical with the MBS3
sequence of human ZnT3 protein. Our results (based on pH-potentiomety, UV-Vis, CD and
NMR spectroscopy), indicate very high zinc binding affinity for this peptide, ~4.5 magnitude
higher than that of the analogous Ac-HHGH-OH peptide [2]. Moreover, the comparison
with nickel(II) suggests considerable zinc selectivity.
[1] D.J. Eide, Biochim. Biophys. Acta, 2006, 1763, 711–722.
[2] A. Jancsó, Z. Paksi, N. Jakab, B. Gyurcsik, A. Rockenbauer, T. Gajda, Dalton Trans.,
2005, 3187–3194.
61
Poster
Heterologous expression and characterization of novel
cyanobacterial heme peroxidases
Markus Auer1, S. Alexander Teufer1, Marcel Zamocky1,2,
Margit Bernroitner1, Paul G. Furtmüller1, Christian Obinger1
1
BOKU - University of Natural Resources and Applied Life Sciences, Vienna Institute of
BioTechnology, Department of Chemistry, Division of Biochemistry, Metalloprotein
Research Group, Vienna, Austria, Email: [email protected]
2
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
Heme peroxidases can be divided in two distinct superfamilies. One superfamily
(peroxidase-catalase superfamily) consists predominantly of catalase-peroxidases,
ascorbate peroxidases, cytochrome c peroxidases, manganese and lignin peroxidases
and plant secretory peroxidases. By contrast, mammalian peroxidases belong to the
second superfamily, entitled as peroxidase-cyclooxygenase superfamily (1). Going back to
the roots of this widespread superfamily we can find prostaglandin synthases
(cyclooxygenases), multi-domain proteins like peroxidasins, peroxinectins, peroxicins,
peroxidockerins and dual oxidases suggesting a complex evolutionary distribution.
Here, we describe two cyanobacterial peroxidases (2) of striking sequence homology with
human peroxidases that could be structurally related with the ancestors of the peroxidasecyclooxygenase superfamily. These prokaryotic metalloenzymes form a separate cluster
within the sixth subfamily. Sequence alignment suggests the presence of all essential
residues known to be relevant in catalysis and halide binding of human peroxidases (1,3)
including distal His-Arg-Gln and proximal His etc. Even Asp and Glu that are involved in
heme to protein linkages and heme distortion in mammalian peroxidases (3) are found.
Cloning of the corresponding proteins from Microcoleus chthonoplastes PCC 7420 and
Lyngbya sp. PCC 8106, heterologous expression in and purification from E. coli as well as
preliminary spectral and enzymatic characterization is described. Sequence, spectral and
enzymatic data are presented, compared and discussed with respect to known structurefunction relationships of the human oxidoreductases lactoperoxidase and
myeloperoxidase.
(1) M. Zamocky, C. Jakopitsch, P. G. Furtmüller, C. Dunand, C. Obinger, Proteins 2008,
72, 589–605.
(2) M. Bernroitner, M. Zamocky, P. G. Furtmüller, G. A. Peschek, C. Obinger, J. Exp. Bot.
2009,60, 423–440.
(3) M. Zederbauer, P. G. Furtmüller, S. Brogioni, C. Jakopitsch, G. Smulevich, C. Obinger,
Nat. Prod. Rep. 2007, 24, 571–584.
62
Poster
Aptamers as virus detecting molecules
Zsófia Balogh1, Viola Bardóczy2, Gergely Lautner3,
Beata Komorowska4, Róbert E. Gyurcsányi3, Tamás Mészáros*1
1
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry,
Semmelweis University, Budapest, Hungary
2
Department of Applied Biotechnology and Food Science, Budapest University of
Technology and Economics, Budapest, Hungary
3
Department of Inorganic and Analytical Chemistry, Budapest University of Technology
and Economics, Budapest, Hungary
4
Research Institute of Pomology and Floriculture, Mazovia, Poland
Aptamers are single-stranded oligonucleotides that effectively bind various target
molecules and rival antibodies in means of sensitivity, selectivity and costeffectiveness. Although the number of aptamer related publications continuously
increases, their diagnostic application is still in its infancy. We aimed to develop
apple stem pitting virus (ASPV) specific DNA aptamers and apply them as protein
specific sensor molecules in various bioanalytical assays. The applied SELEX
(Systematic Evolution of Ligands by Exponential Enrichment) protocol resulted in
identification of highly discriminative, ASPV coat protein specific aptamer
sequences. According to surface plasmon resonance analysis, the selected
aptamers bind their protein targets with Kd values in 10–9–10–8 M range. Next, we
used the selected aptamers for ASPV detection in complex matrices. First, we
developed a protocol for aptamer based detection of membrane immobilized virus
proteins and demonstrated the native and denaturated protein decorating capacity
of the selected aptamers. Second, we invented a novel Double Oligonucleotide
Sandwich-Enzyme Linked Oligonucleotide Assay (DOS-ELONA) method for
determination of virus coat protein concentration in complex matrix. The offered
DOS-ELONA completely relies on aptamers circumventing the antibody demand of
ELISA. Most importantly, we showed that DOS-ELONA is an apt approach for
sensitive and specific identification of ASPV infected plant samples. Collectively,
we selected the first plant virus specific aptamers, proved that they are more
sensitive than the available ASPV specific antibody and provided the very first
instance for measuring protein concentration and virus identification in complex
sample matrices by DOS-ELONA.
63
Poster
Use of electrophoretic and spectroscopic methods in Gquadruplex research
Lubos Bauer*, Katarina Tluckova, Viktor Viglasky
Department of Biochemistry, Institute of Chemistry, Faculty of Sciences, P. J. Safarik
University, Kosice, Slovakia, [email protected]
G-quadruplexes are built from the stacking of successive G–G–G–G tetrads and
stabilized by bound monovalent Na+ and K+ cations[1]. The conformation of
quadruplexes is endowed with a high degree of polymorphism in terms of strand
stoichiometry and polarity, glycosidic torsion angles, groove size, and connecting
loops. In nature, guanine-rich sequences are found in some important regions such
as telomeres, and in important proto-oncogenes promoter elements. Gquadruplexes formed within the promoter regions of these genes may play a
crucial role in transcriptional regulation, therefore emerging as promising targets for
the development of anticancer drugs[2,3]. We have analyzed different telomeric and
other nontelomeric G-quadruplex forming sequences in the presence of Na+ , K+
and PEG 200 by using of UV absorption spectroscopy, circular dichroism and
electrophoretic methods. The presence of multiple G-quadruplex conformations in
solution was observed, making structural elucidation difficult. Temperature gradient
gel electrophoresis (TGGE) is useful for the elucidation of structural variability and
thermal stability of quadruplex conformers. TGGE allows us to separate and
evaluate the most abundant conformers and to obtain relevant thermodynamic
parameters[4].
[1] M. Gellert, M. N. Lipsett, D. R. Davies, Proc. Natl. Acad. Sci. USA 1962, 48, 2013–
2018.
[2] D. J. Patel, A. T. Phan, V. Kuryavyi, Nucleic Acids Res. 2007, 35, 7429–7455.
[3] Y. Qin, L. H. Hurley, Biochimie 2008, 90, 1149–1171.
[4] V. Viglasky, L. Bauer, K. Tluckova, Biochemistry 2010, 49, 2110 –2120.
64
Poster
Discharged photoprotein obelin: fluorescence
peculiarities
Nadezda V. Belogurova1, Nadezda S. Kudryasheva1,2
1
Institute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia, [email protected].
2
Siberian Federal University, Krasnoyarsk, 660041, Russia, [email protected].
Photoprotein obelin, the enzyme-substrate complex of polypeptide with 2hydroperoxycoelenterazine, is responsible for bioluminescence of marine hydroid
Obelia longissima[1]. Addition of Ca2+ to the photoprotein triggers the
bioluminescent reaction with light emission. The product of the bioluminescent
reaction – enzyme-bound coelenteramide – is a fluorescent protein called
‘discharged’ obelin. It is stable and highly fluorescent. The work considers
dependence of its light-induced fluorescence on Ca2+ concentration. Increase of
Ca2+ concentration enhanced the fluorescence intensity of discharged obelin; the
dependence was found as linear in double logarithmic coordinates at Ca2+
concentration range 10–7–10–6 М, both in excitation and emission spectra.
Bioluminescence spectra of obelin and fluorescence spectra of discharged obelin
are known to be complex and variable[2]. The fluorescence spectra of discharged
obelin were divided into components. It has been found that contributions of the
components to experimental excitation and emission spectra depended on Ca2+
concentration. The data suggest enzymatic conformational transition in discharged
obelin at ~ 5·10–7 M of Ca2+ concentration. Spectra variations were attributed to
acidity changes of discharged obelin chromophore (coelenteramide) in its
fluorescent state S*1.
[1] Z. Liu, E. S. Vysotski, C. J. Chen, J. Rose, B. C. Wang, J. Lee, Protein Sci. 2000, 9,
2085–2093.
[2] N. V. Belogurova, N. S. Kudryasheva, R. R. Alieva, A. G. Sizykh, J. Photochem.
Photobiol. B. 2008, 92, 117–122.
65
Poster
QSAR analysis and proposal of new heterocyclic
compounds with potential antitumor activity
Branimir Bertoša1, Sanja Tomić1, Maja Aleksić 2,
Grace Karminski-Zamola2
Division of Physical Chemisty, Ruđer Bošković Institute, Bijenička cesta 54, 10 000
Zagreb, Croatia, [email protected]
1
Faculty of Chemical Engineering and Technology, Marulićev trg 19, 10 000 Zagreb,
Croatia, [email protected]
2
Quantitative Structure–Activity Relationship (QSAR) models for predicting
antitumor activity of heterocyclic amides and quinolones from benzo[b]thiophene-,
thieno[3,2-b]thiophene- and thieno[2,3-b], thiophene series were built. Dataset
consisted of 56 compounds with measured antitumor activity. For each compound
VolSurf descriptors were derived and correlated with the biological activity using
Partial Least Square analysis. Beside standard approaches for building QSAR
models, the approach based on a small dataset of 10 compounds selected
regarding the results of Principal Component Analysis (PCA), was tested. The
latter approach was shown as successful and it seems useful for planning
experiments in order to speed up and simplify the search for new drug candidates.
Results of PCA and QSAR analysis enabled identification of molecular properties
with the highest impact on antitumor activity. Volume, sum of the hydrophobic
surface areas and presence of the group that can be easily ionized in the pH range
from 4 to 9, were found to be highly important for successful antitumor activity of
the investigated compounds.[1] Using this knowledge, new compounds were
proposed for synthesis and biological testing. Their activities were predicted using
the derived QSAR models and the proposed compounds were shown as promising
antitumor candidates.
N
N
S
(CH2)3N(CH3)2
O
[1] B. Bertoša, M. Aleksić, G. Karminski-Zamola Int. J. Pharm. 2010, 294, 106–114.
66
Poster
Thermodynamic and structural studies of human
dipeptidyl peptidase III with substrates and inhibitors
Gustavo A. Bezerra1, Greg Wasney2, Masoud Vedadi2, Doug Cossar2,
Sirano Dhe-Paganon2,3, Marija Abramić4, Peter Macheroux5,
Karl Gruber1
1
Institute of Molecular Biosciences, University of Graz – Graz, Austria,
[email protected],
2
Structural Genomics Consortium and 3Department of Physiology – Toronto, Canada.
Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb,
Croatia.
4
5
Institute of Biochemistry – Graz University of Technology – Graz, Austria.
Dipeptidyl-peptidases III (DPP III), also known as enkephalinase B, are zinc-dependent
enzymes that specifically cleave dipeptides from the N-termini of its peptide substrates,
which include biologically active neuropeptides like angiotensins and endomorphins [1]. In
this way, DPP III from mammals plays important physiological functions, for instance, the
regulation in the pain modulatory system. DPP III enzymes from various sources have
been studied for decades through biochemical analyses but the three-dimensional
structures of the yeast and human DPPIII are only currently available and a catalytic
mechanism has been proposed based on related enzymes [2]. However, the mode of
substrate binding is unknown. How these enzymes achieve their specificities towards a
wide range of peptide and which interactions participate in the catalytic process in a
specific way are also questions still waiting for a satisfactory answer. Since there are no
structural data available of DPP III complexes, we started a project aiming at the 3D
structure determination – supported by thermodynamic characterization – of human DPP
III complexed with natural substrates and inhibitor peptides. These data should provide a
starting point for further drug design efforts. For isothermal titration calorimetry, a synthetic
hDPP III gene optimized for E.coli expression was utilized, while the hDPP III gene was
expressed as a secreted protein using the Baculovirus Expression Vector System in insect
cells for the crystallization experiments. In both expression systems, the proteins possess
an N-terminal His-tag. Purification was achieved through affinity and size exclusion
chromatography. In addition, active site variants were produced to prevent substrate
cleavage during crystallization. The samples were characterized by dynamic light
scattering assays and submitted to initial crystallization trials.
[1] D. Agić, M. Hranjec, N. Jajčanin, K. Starčević, G. Karminski-Zamola, M. Abramić,
Bioorg. Chem. 2007, 35, 153 – 169.
[2] P. K. Baral, N. Jajčanin-Jozić, S. Deller, P. Macheroux, M. Abramić, K. Gruber, J. Biol.
Chem. 2008, 283, 22316 – 22324.
67
Poster
Detection of vascular markers of patients with
gynecological malignancies
Miroslava Bilecová – Rabajdová1, Peter Urban1, Jana Mašlanková1,
Alexander Ostró2, Mária Mareková1
1
Department of chemistry, biochemistry, medical biochemistry and LABMED, Faculty of
medicine P. J. Šafárik University in Košice, Slovakia, [email protected]
2
2nd Department of Gynaecology and Obstetrics, Faculty of medicine P. J. Šafárik
University in Košice, Slovakia, [email protected]
Ovarian cancer is the most deadly disease from the cancers of female
reproductive organs. Survival time is about five years and the probability of relapse
varies between 87.8 % in the first stage to 18 % in the fourth stage. The first
symptoms are manifested only at an advanced stage with limited prospects for
treatment and a significant mortality. Our aim was to detect changes in gene
expression for tumour vascular markers like DR 6 and GPM6B, which should be
specific for ovarian cancer. Next goal was to determine specificity and sensitivity of
these markers in patients with different types of gynaecological cancers.
Subsequently we correlated the results from women with a confirmed diagnosis of
ovarian cancer against healthy women. After isolation of total RNA from peripheral
blood leukocytes (60 patients and 60 health women), we made reverse
transcription of mRNA into cDNA. Changes in gene expression of glycoprotein
M6B (GPM6B ) and death receptor 6 (DR6) were detected using PCR followed by
usual gel electrophoresis. As a housekeeping gene we used glyceraldehydes – 3 –
phosphate dehydrogenase (GAPDH). Numerical quantification was evaluated
using DataSyngene program. The expression of both markers was correlated with
the total biochemical background of patients with confirmed ovarian cancer and
also with levels of CA125. Compared to healthy women, we found increased levels
of mRNA for gene DR6, which corresponded with rising levels of CA125 and the
growth and spread of tumour. On the contrary, we also found increased levels of
GPM6B impaired compared to healthy women. We found that DR6 is good
predictive marker with different gene expression especially for ovarian cancer. It is
inevitable to spread count of patients. Monitoring the levels of vascular markers will
contribute to verification of results obtained with other markers (CA125). It is still
necessary to find a proper marker which allows searching for asymptomatic or
early stage patients (screening) with ovarian cancer or for the determination of
affectivity of cytostatic treatment.
Acknowledgments: This work was supported by grant project VEGA 1/0402/10
68
Poster
TPPP/p25: a new unstructured protein with GTPase
activity
Andrea Bodor1, András Perczel1, Á. Zotter2, J. Ovádi 2
1
Eötvös University, Institute of Chemistry, Laboratory of Structural Chemistry and Biology,
Budapest, Hungary, [email protected]
2
Hungarian Academy of Sciences, Institute of Enzymology, Karolina út 29, Budapest,
Hungary
Tubulin Polymerization Promoting Protein/p25 (TPPP, 25kDa) is an intrinsically
unstructured protein for which the supreme target is the microtubule system. In
normal human brain TPPP/p25 is expressed predominantly in oligodendrocyte; in
pathological inclusions TPPP/p25 co-accumulates with α-synuclein in both glial and
neuronal cells leading to synucleinopathies. Multinuclear NMR investigations
reveal the existence of a low signal dispersion part with intense peaks, in
accordance with an unstructured region; and an ordered core, which appears as
very low intensity peaks. SCS data obtained from the assignment of the 3D
measurements (HNCA, (H)CC(CO)NH, TOCSY-HSQC, NOESY-HSQC) for the
intense signals prove that the C-terminal and N-terminal parts are disordered.
Studying the biological relevance GTP binding was monitored by HSQC
measurements. Special binding positions can be the Rossmann fold sequence and
Switch II region, both situated at the C-terminal part. However, GTP binding occurs
in the P-loop, situated in the region. The specific hydolyitic activity of P25 was
followed by 31P NMR measurements. Real time kinetic analysis showed that GTP
hydrolysis leads to the formation of GMP and free inorganic phosphate, whilst GDP
concentration is maintained at steady-state condition. GDP alone doesn’t have
hydrolytic activity. This specific GTPase activity of P25 is comparable with that of
the non-activated small G protein, suggesting its involvement in multiple
physiological processes in addition to the regulation of GTP-mediated microtubule
assembly.
Acknowledgments: This work was supported by the Hungarian National Research
Foundation, and the János Bolyai Research Fellowship. 3D measurements at 950 MHz
and 750 MHz instruments were performed at the Oxford NMR Facility, in the EASTNMR
Research Infrastructure framework.
69
Poster
The influence of fluoride and iodide ions on the stability
of Trp-cage miniprotein: a computational study
Ferenc Bogár1*, Zoltán Násztor1, Balázs Leitgeb2, Botond Penke1,3
1
Supramolecular and Nanostructured Materials Research Group of HAS, University of
Szeged, Szeged, Hungary, [email protected].
2
Institute of Biophysics, Biological Reserch Center of HAS, Szeged, Hungary
3
Department of Medical Chemistry, University of Szeged, Szeged, Hungary
Since the famous observation of Hofmeister it is well know that solubility of proteins
can be influenced by adding salts to the solution. Ions can be ranked according to
extent of this influence (i.e. Hofmeister series). These series start with the
kosmotropic, structure stabilizing and end with the chaotropic structure
destabilizing ions. Despite the wide-spread use of Hofmeister effect, its
interpretation has remained a matter of debate. Together with experimental
investigations molecular modeling can help to understand the atomic level
processes behind this effect.
In a recent study, Dzubiella[1] pointed out that the effect of Hofmeister active salts
on the stability of simple model peptides (like charged Ala-based helices) can be
simulated using non-polarizable force fields. In this study we investigated the effect
of kosmotropic F − and chaotropic I − ions on the stability of a more protein-like
model peptide, the Trp-cage miniprotein. It is a 20-residue-long polypeptide and
shows several important characteristic features of proteins. It has a hydrophobic
core, a stabilizing salt bridge and a well-defined, stable secondary and tertiary
structure. In our investigations the Amber ff03 force field and the TIP3P water
model together with the ion parametrization of Joung et al.[2] were used in three
80ns long molecular dynamics simulations at 300K. In the first simulation the
model peptide was dissolved in water, while in the second and in the third cases
NaF and NaI were added to the solution in 1M final concentration. The atomic
fluctuations of the backbone as well as the side chains were used for the
characterization of stability. It is pointed out that, the chaotropic I − destabilized,
while the kosmotropic F − stabilized the structure of Trp-cage miniprotein. The
distribution of the ions around the hydrophobic and hydrophilic residues was also
investigated, as well as the strength of intramolecular and water-peptide H-bonds.
[1] J. Dzubiella, J. Phys. Chem. B 2009, 113, 16689–16694.
[2] I. S. Joung, T. E. Cheatham III, J. Phys. Chem. B 2008, 112, 9020–9041.
70
Poster
A possible interplay between two conserved Cys residues
of auxin-amidohydrolase BrILL2 from Brassica rapa L.
Ana Brcko1, Maja Brajlović1, Saša Kazazić2, Nina Jajčanin Jozić3,
Branka Salopek-Sondi1*
Ruđer Bošković Institute, Division of Molecular Biology, Zagreb, Croatia, [email protected],
[email protected]*.
1
Ruđer Bošković Institute, Division of Physical Chemistry, Zagreb, Croatia,
[email protected].
2
Ruđer Bošković Institute, Division of Organic Chemistry and Biochemistry, Zagreb,
Croatia, [email protected].
3
Auxin-amidohydrolases are a group of amidohydrolases from the peptidase M20D
family that modulate auxin levels in plants by releasing active plant hormones from
their conjugated storage forms. Based on sequence homology, auxinamidohydrolase BrILL2 from Chinese cabbage (Brassica rapa L.) contains two
highly conserved cysteine residues (Cys139 and Cys320). The BrILL2 enzyme
heterologously expressed in E. coli and purified via immobilised nickel affinity
chromatography preferentially cleaves alaninil-indole-3-propionic acid (IPAala) as a
substrate in an enzymatic assay in vitro. We have also determined that the wild
type BrILL2 enzyme is active only in the presence of Mn++, as a cofactor, and a
reducing agent such as dithiothreitol [1]. In the presence of various reducing agents
(DTT, β -mercaptoethanol, reduced glutathione, ascorbic acid and Cys) BrILL2, wt
and mutant Cys320Ser retain similar enzymatic activities, whereas they lose
activity upon alkylation with J-acetamide or without reducing agents. Site-directed
mutagenesis of Cys139 to Ser results in complete inactivation of the enzyme. We
confirmed by circular dichroism that, despite mutations, proteins preserve
secondary structure. Since enzymes are prone to aggregation in vitro, we have
applied gradient SDS-PAGE and Western blot analysis using anti-His antibodies
and compared the potential of the wt BrILL2 and the mutants Cys139Ser,
Cys320Ser and Cys139,320Ser for polymerization under reducing conditions vs.
oxidizing conditions. Furthermore, possible interaction between above mentioned
cysteine residues through intermolecular disulfide bonds formation was analyzed
by MALDI-TOF MS. We propose possible enzyme activation in vivo by dissociation
of polymers in the presence of natural reducing agents.
[1] B. Savić, S. Tomić, V. Magnus, K. Gruden, K. Barle, R. Grenković, J. Ludwig-Muller, B.
Salopek-Sondi, Plant Cell Physiol. 2009, 50, 1587–1599.
71
Poster
Diketone Cleaving Dioxygenase 1 – Computational Study
Sanja Tomić1, Grit Straganz2, Daniela Buongiorno2, Michael Ramek2,
Hrvoje Brkić3
1
Ruđer Bošković Institute, Zagreb, Croatia
2
Graz University of Technology, Graz, Austria
3
Medicinal Faculty, Osijek, Croatia, [email protected].
Diketone cleaving dioxygenase (Dke1) is a non heme Fe(II) enzyme (NHEE) with
an atypically ligated metal binding site. Dke1 catalyzes the oxidative C-C bond
scission in beta-dicarbonyl compounds by molecular oxygen.[1] The enzyme
employs the general mechanism of O2 activation in the NHEE surrounding. Upon
substrate ligation to the iron cofactor, one site of the six-coordinated metal center
vacates followed by O2 reduction - the rate determining step of the catalytic cycle.[2]
Mutational and spectroscopic analyses have shown that the H–bonding network in
the active site plays a crucial role in the rate of O2 reduction.[3] In order to elucidate
the structural basis of this effect, the active site was subjected to further mutational
analysis. The resulting variants were characterized regarding their O2 reduction
rates, and the molecular dynamic studies on Dke1 and its mutants were performed.
Standard force-field parameters in molecular mechanic programs typically fail to
account for the geometric rigidity of the NHEE metal binding sites that is found
experimentally.[4] As no applicable force field parameters for non-heme Fe(II) sites
were available, we deduced them by quantum mechanical calculations, performed
with the program GAMESS,[5] and incorporated them into the AMBER ff03 force
field used for the protein parametrisation. Molecular Dynamics (MD) simulations
(10 ns) and analysis of the results were performed within AMBER 10
(http://ambermd.org) suite. The simulations were performed for the acetylacetonate
ligated Dke1 and selected variants (F59A, F115A, F119A and Y70A) and the water
entrance into the enzyme active site was monitored. Thus we could identify the
primary small channel for water trafficking in the structure of Dke1 and
demonstrate that it is distinct from the larger, hydrophobic substrate entrance
channel. Furthermore, the structural impact of mutations in the active site on the
active site’s accessibility for water was investigated.
[1] M. Costas, M. P. Mehn, M. P. Jensen, L. Que, Chem. Rev. 2004, 104, 939–86.
[2] G. D. Straganz, B. Nidetzky, J. Am. Chem. Soc. 2005, 127, 12306–14.
[3] G. D Straganz, R. Adrienne, S. Egger, B. Nidetzky, Biochemistry 2010, 4, 996–1004.
[4] T. C. Pochapsky, S. S. Pochapsky, C. Hoefler, J. Liang, J. Biomol. NMR 2006, 34,
117–27.
[5] M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S. Gordon, J. H. Jensen,
S. Koseki, N. Matsunaga, K. A. Nguyen, S. J. Su, T. L. Windus, M. Dupuis, J. A.
Montgomery, J. Comput. Chem. 1993, 1347–63.
72
Poster
Molecular complexes of phloroglucinol derivatives with
pyridines
Dorota Stępień1, Michał K. Cyrański1
1
Faculty of Chemistry, University of Warsaw, Pasteura 1, Warsaw, Poland,
[email protected].
The rapid expansion in supramolecular chemistry over the past 25 years has
resulted in an enormous diversity of chemical systems, both rationally designed
and/or accidentally stumbled upon. [1] Phloroglucinol is a benzenetriol that has many
applications in synthesis of pharmaceuticals or explosive materials. It is also used
as a coupling agent in printing. The molecule may exist in two forms (1,3,5trihydroxybenzene and 1,3,5-cyclohexanetrione), which are in equilibrium. In this
communication we report novel crystal structures of molecular complexes of
phloroglucinol derivatives (1-3) with heteroaromatic nitrogen bases like 2,2’bipyridine, 4,4’-bipyridine or 1,2-bis(4-pyridyl)ethane. The latter molecules are well
known as a spacer molecules which are commonly used as ligands for the
preparation of the supramolecular assemblies of desired architectures and
properties.[2]
R
OH
HO
where,
1) R = CHO
2) R = COOCH3
3) R = NO2
OH
The structures of molecular complexes were investigated by X-ray diffraction on a
single crystal. The parent phloroglucinol derivatives existed always in benzenetriol
form and their structures were stabilized by intramolecular hydrogen bonding (see
below).[3] It has been found that the complexes were stabilized by a net of O-H...N
and O-H...O strong hydrogen bonds and van der Waals interactions.
[1] J. W. Steed, J. L. Atwood, Supramolecular Chemistry, John Wiley and Sons, Chichester, UK,
2009.
[2] K. K. Arora, M. S. Talwelkar, V. R. Pedireddi New J. Chem. 2009, 33, 57–63.
[3] W. Janowski, A. Kadzewski, M. Gdaniec Pol. J. Chem. 2007, 81, 1095-1108.
73
Poster
Post-SCF ab initio quantum chemistry characteristics of
8-oxogunine intermolecular interactions in B-DNA
Piotr Cysewski
Department of Physical Chemistry, Collegium Medicum, Nicolaus Copernicus University in
Toruń, Kurpińskiego 5, 85–950 Bydgoszcz, Poland, [email protected]
The intermolecular interactions between canonical and modified nucleobases are
significantly affected by local B-DNA chain conformations. Using particular
geometry even if it corresponds to mean value does not provide essential diversity
of structure-to-energy relationships. Meaningful dinucleotide steps d(XpY) in BDNA conformations require adequate sampling over configurational hyperspace,
especially for non-standard nucleobases. An approach for proper inclusion of
structural heterogeneities for energetic characteristics of any dinucleotide step was
successfully applied in previous investigations[1–6]. It was used for description of
energetic heterogeneities in canonical and oxidized central guanine triad of B-DNA
telomeric fragments[1,2], polymorphism-related heterogeneities of guanine stacking
in B- and A-DNA forms[3], characteristics of inter- and intra-strand stacking
interactions in d(CpG) and d(GpC) steps found in B-DNA, A-DNA and Z-DNA
crystals [4], quantification of all possible intra-strand stacking interactions between
nucleobases[5] and assessment of many-body contributions to d(GpG) and d(CpC)
dinucleotide steps[6]. The essential feature of applied approach is the
characteristics of statistically significant number of conformations leading to
distributions of intermolecular interaction energies (IIE) along with standard
deviations and other statistical parameters. All possible dinucleotide steps
comprising 8-oxoguanine were characterized using proposed approach.
Schematic
representation
of
intermolecular interactions in model
d(XpY) dinucleotide steps, where
X,Y denotes one of four nucleobases
or 8-oxoguanine and X’,Y’ stands
for corresponding complementary
base.
[1]
[2]
[3]
[4]
[5]
Cysewski P., Czeleń P., J. Mol. Mod. 2007, 13, 739–750.
Cysewski P, Czeleń P, J. Mol. Mod. 2009, 15, 607–613.
Cysewski P., J. Mol. Struct. THEOCHEM 2008, 865, 36–43.
Cysewski P., J. Mol. Mod. 2009, 15, 597–606.
Cysewski P., New J. Chem. 2009, 33, 1909–1917.
74
Poster
HNH motif as active centre of chimeric metallonucleases
for gene therapy
Anikó Czene1, Béla Gyurcsik1, Ida Noémi Jakab-Simon1,
Kyosuke Nagata2, Hans Erik Mølager Christensen3
1
University of Szeged, Department of Inorganic and Anaytical Chemistry, Szeged,
Hungary,
e-mail:[email protected],
[email protected],
[email protected]
2
University of Tsukuba, Dept. Infect. Biol., Grad. Sch. Comp. Human Sci. and Inst. Basic
Med. Sci., Tsukuba, Japan, [email protected]
3
Technical University of Denmark, Department of Chemistry, Lyngby, Denmark, e-mail:
[email protected]
Duchenne Muscular Dystrophy (DMD) is an X-linked lethal disease causing muscle
wasting in males. Therapeutic possibilities are limited, the current strategies will
end up with life-long treatment in the best case. [1] The goal of our work is to
design and prepare a new type of DNA sequence specific, functional and non-toxic
zinc-finger - HNH (HNH: His-Asn-His rich motif) metallonuclease, targeted towards
the mutation point in genomic DNA of a DMD patient. The HNH motif, a nuclease
domain with non-specific nuclease activity will be linked to a DNA-binding protein
with known or designed DNA sequence-specificity (ZF). The first step of this work
is to get deeper insight into the properties of the HNH nuclease domain. It is
abundant within the active centre of different nuclease enzymes, known to display
a ββα structure with a central metal ion binding site. As an example the bacterial
Colicin E7 (ColE7) functions in the presence of zinc(II). We expressed the 42
amino acid long C-terminal HNH motif of ColE7, and investigated its metal binding
ability and nuclease activity. Surprisingly, this protein did not exert cytotoxicity, and
the in vitro nuclease assay was also negative. In the knowledge of this we
searched for the minimal functional part of the nuclease domain. Several mutants
were designed, revealing the essential amino acid residues. The mutation of the Nterminal arginine to glycine allowed for only a minor residual nuclease activity,
pointing to the essential role of this amino acid in the control of ColE7 activity. The
methods for expression and purification of the GST fusion proteins have been
optimized. The stuctural and functional investigation of the mutants incubated with
metal ions and/or DNA were performed by fluorescence, CD and NMR spectroscopy, ESI-MS, crystallography and gel electrophoretic methods.
[1] A. Aartsma-Rus, G.-J.B. Van Ommen, RNA 2007, 13, 1
75
Poster
Alzheimer Risk Factors Age and Gender Induce Aβ
Aggregation by Raising Extraneuronal Zinc
Zsolt L. Datki 1*, Blaine R. Roberts 2, Akos Hunya 3, Adam Gunn 2, Eva
Kondorosi1, Paul A. Adlard 2, Viktor Szegedi 1, Gabor Juhasz 3, Dora Simon 3,
Livia Fulop 3, Istvan Foldi 3, Zsolt Bozso 3, Katalin Soos 3, Gabor Kozma 4,
Akos Kukovecz 4, Colin L. Masters 2, Zoltan Konya 4, Ashley I. Bush 2,5
1
Bay Zoltan Foundation for Applied Research, Institute for Plant Genomics, Human
Biotechnology and Bioenergy (BAYGEN), H–6726 Szeged, Hungary; [email protected]
2
Oxidation Biology Laboratory, Mental Health Research Institute, Parkville, Victoria 3052,
Australia
3
Department of Medical Chemistry, University of Szeged, H–6726 Szeged, Hungary
4
University of Szeged, Department of Applied and Environmental Chemistry
5
Department of Pathology, The University of Melbourne, Parkville 3010, Victoria, Australia
Evidence indicates that Aβ aggregation by Zn2+ released from glutamatergic
neurons induces amyloid neuropathology in Alzheimer's disease, a disorder where
the pathogenic mechanisms of its risk factors remain elusive. Using acute
hippocampal slices, we found that extracellular Zn2+-release induced by high K+
was significantly greater with older (65-week vs 10-week old) and female rats. This
elevation was driven by slower reuptake of extracellular Zn2+, and could be
recapitulated by mitochondrial intoxication. Zn2+:Aβ aggregates were toxic to the
slices, but Aβ alone was not. Accordingly, Aβ became toxic to slices treated with
high K+, and older and female slices were more vulnerable. These data indicate
that energy failure disturbing extracellular Zn2+ homeostasis could be the
mechanism in common for age and gender as major risks for Aβ deposition in
Alzheimer's disease.
76
Poster
Synthesis, antiinflammatory and anticancer activity of
binuclear and trinuclear copper (II) complexes
Zografia Boulsourani1, Seyedehraziyeh Hosseinian1, Evagellia Vassiliou1,
George Geromichalos3, Katia Repana3, Efthalia Yiannaki5, Catherine
Raptopoulou4, Dimitra Chatzipaulou-Litina2, Catherine Dendrinou-Samara1
1
Dept. General and Inorganic Chemistry, Aristotle University of Thessaloniki, Thessaloniki,
54124, GREECE [email protected]; 2Dept. of Pharmaceutical Chemistry, School of
Pharmacy, Aristotle University of Thessaloniki,; 3Cell Culture, Molecular Modeling and
Drug Design Lab., Symeonidion Research Center, Theagenion Cancer Hospital; Lab of
Hematology, Dept. Of Cytometry, Theagenion Cancer Hospital, Thessalonik; 4NCSR
"Demokritos", Institute of Materials Science, 15310 Aghia Paraskevi Attikis, GREECE
Curative properties of Cu-NSAIDs have led to the development of numerous Cu(II)
complexes of NSAIDs with enhanced anti-inflammatory activity and reduced
gastrointestinal toxicity. Their ability to influence copper metabolism has been a matter of
debate. Relatively, little is known about how they ultimately regulate the inflammatory
process and/or immune system, their pharmacokinetic and biodistribution profile, their
stability in biological media and pharmaceutical formulations. Moreover the relative
potency/efficacy of the Cu(II) monomeric versus Cu(II) dimeric or polymeric complexes is
limited and this is a subject of interest.
We have undertaken a study on the coordination chemistry and biological activities
of thiophen-2-yl saturated carboxylic acids [1], an emerging class of antioxidantsantiinflammatories, with Cu(II) in an attempt to model various properties in terms of
structure activity relationships in the presence/absence of additional ligands such as
tripodals of different nature and N-donors. A series of dinuclear and trinuclear copper
complexes have been synthesized and their pharmacochemical activity have been
examined. We present the synthesis, characterization and crystal structures of the
dinuclear
[Cu2(L)4(MeOH)2],
[Cu2(L)2(H2tea)2],
[Cu2(H2tea)2Cl2],
[Cu2(OH)(L)(bpy)2(NO3)(H2O)](NO3), [Cu2(H2tea)(H3tea)(L)(O3PPh)] as well as the
trinuclear linear [Cu3(L)4(H2tea)2] and [Cu3(L)2(H2tea)2(NO3)2] where L are the anions of
Thiophene 2- carboxylic acid or 2-(Thiophen-2-yl)-acetic acid; the tripodals H3tea=
triethanolamine, O3PPh= the anion of phenylphoshonate and N-donors bpy = bipyridine
and phen=phenanthroline.
In vitro studies of free ligands and their respective copper complexes, include: a)
soybean lipoxygenase inhibition, b) interaction with 1,1-diphenyl-2-picryl-hydrazyl stable
free radical, c) the ΗΟ˙ mediated oxidation of DMSO, d) inhibition of lipid peroxidation and
e) scavenging of superoxide anion radicals. Human colon, ovarian, breast and lung
cancer cell lines were selected for preliminary anti-cancer screening. For the cell cycle and
apoptosis experiments, were selected the more active compounds at IC50 concentrations.
Annexin V-FITC/ 7AAD double staining assay was adopted to quantify the apoptotic cells
formed upon treatment with the compounds.
[1] a) D. Panagoulis, E. Pontiki, E. Skeva, C. Raptopoulou, S. Girousi, D. Hadjipavlou-Litina, C.
Dendrinou-Samara, J. Inorg. Biochem. 101, 2007, 623–634 b) V. Tangoulis, D. Panagoulis, C. P.
Raptopoulou, C. Dendrinou-Samara, Dalton Trans., 2008, 1752 – 1760.
77
Poster
Protonation of some Supramolecular Compounds Based
on Pyridine Subunits
Ines Despotović1,*, Zvonimir B. Maksić2
1
2
Ruđer Bošković Institute, Zagreb, Croatia, [email protected]
Ruđer Bošković Institute, Zagreb, Croatia, [email protected],
Faculty of Science and Mathematics, Zagreb, Croatia
It is a common knowledge that neutral organic bases and superbases possess a
number of advantageous properties compared to their inorganic counterparts. They
exhibit higher solubility in most organic solvents, low sensitivity to moisture and
CO2, and very good stability at low temperatures. Therefore, they can be used in
mild chemical conditions [1]. Various structural and electronic motifs have been
used in tailoring strong and ultrastrong bases. Molecular suprastructures
containing pyridine building fragments are very good candidates for strong and
ultrastrong bases, bounding the proton inside the macrocycle in a bifurcated
manner [2].
In the present work we considered protonation of macrocyclic systems with three
(1 and 2) and four pyridine subunits (3). Gas phase molecular structures and
R1
R1
R1
R2
R2
N
N
N
N
N
N
R1
N
R2
R1
N
N
R2
R2
N
N
N
R2
N
N
R2
R2
R1
1
2
3
gas phase proton affinities as well as gas phase basicities were calculated by the
B3LYP/6–311+G(2df,p)//B3LYP/6–31G(d) method. It is found that supramolecules
1, 2 and 3 have high proton affinities of 290.3 kcal mol–1, 294.4 kcal mol–1 and
284.7 kcal mol–1, respectively, and corresponding superbasicities of 283.5 kcal
mol–1, 285,5 kcal mol–1 and 278.9 kcal mol–1, respectively. The reason behind are
strong bifurcated intramolecular bond in conjugate acids. Subsequent calculations
on the complexation of small cations and dications reveal some binding principle,
which might be useful for understanding cation affinities of biosystems.
[1] H. Oediger, F. Möller, K. Eiter, Synthesis 1972, 591– 598.
[2] I. Despotović, B. Kovačević, Z. B. Maksić, Org. Lett. 2007, 9, 1101– 1104.
78
Poster
Fast integrated optical switching by the protein
bacteriorhodopsin
László Fábián1, László Oroszi1, Elmar K. Wolff 2, Pál Ormos1,
András Dér1,*
1
Institute of Biophysics, Biological Research Center of the Hungarian Academy of
Sciences, P.O. Box 521, H–6701 Szeged, Hungary, [email protected]
2
Institute for Applied Biotechnology and System Analysis, University of Witten/Herdecke,
Herrhausenstrasse 44, 58455 Witten, Germany
One of the most important areas of application of integrated optics is
telecommunication, where devices built around active, nonlinear optical (NLO)
material can address data handling. Recently, we proposed a novel approach,
where the active role is performed by a protein (bacteriorhodopsin, bR) as an NLO
material that can be combined with appropriate IO-devices, like a slab optical
waveguide.
Our experiments have shown that under continuous illumination the refractive
index changes are consistent with a buildup of the population of the so-called M
intermediate of the bR photocycle. Such changes in the refractive index shift the
positions of the coupling peaks of the grating-coupled optical waveguide. The
refractive index change of the bR film during this process was determined to be
5*10-3 (at 632 nm) [1]. Consequently, the light intensity passing through an IOdevice (e.g. Mach-Zehnder interferometer) drops during the reaction of groundstate bR to the M intermediate, i.e. effective switching can be realized [2].
Here we present data supporting the possibility of switching based on a six orders
of magnitude faster (ps) photoreaction, involving the so-called K intermediate. The
results are expected to have important implications for ultrafast IO data processing
technology.
[1] P. Ormos, L. Fábián, L. Oroszi, J. J. Ramsden, A. Dér, Appl. Phys. Lett. 2002, 80,
4060–4062.
[2] A. Dér, S. Valkai, L. Fábián, P. Ormos, J. J. Ramsden, E. K. Wolff, Photochem.
Photobiol. 2007, 83, 393–396.
79
Poster
Mn2+/3+ and Fe2+/3+ Binding by Natural Siderophores and
Model Hydroxamic Acids
Etelka Farkas∗, Orsolya Szabó
Department of Inorganic and Analytical Chemistry, University of Debrecen, H–
4010, Debrecen, Hungary (e–mail: [email protected],
[email protected])
Microbial siderophores solubilize and transport iron(III) into the cells, where the
reduction of the metal centre is a crucial step in the mechanism of iron release.
Consequently, not only the complexes of iron(III), but also those of iron(II) are
interesting to study.
Investigation of the interaction of iron(II) with hydroxamate-based siderophores
or model hydroxamic acids was made in our lab to reach a better understanding of
the above mentioned biological process. Mono- and dihydroxamic acidsbind iron(II)
as usual, but unexpected oxidation of iron(II) occurs by desferrioxamine B (DFB) or
desferricoprogen (DFC) under strictly oxygen-free condition, and this reaction
might have importance in anaerobic microbial iron uptake. Unambiguously the
siderophores were found to be the oxidizing agent and one of their hydroxamic
acid groups is reduced to amide in the reaction. The oxidized metal ion is
complexed by the excess of the siderophore molecules while the reduced ligands
do not coordinate.[1]
Hydroxamate based siderophores also form strong complexes and exhibit high
reactivity with the 3+ oxidation form of the biologically important manganese,
suggesting a pathway by which manganese may disrupt iron-uptake.[2] In spite of
this interest, up to date, complexation of hydroxamic acids with manganese has not
received much attention and only very few results have been published so far.
In an our recent work, the complexation reactions of manganese(II)/(III) with
various hydroxamic acids, including DFB and its models and also with DFC, have
been investigated in solution and the obtained results have been compared to
those of iron(II)/(III).
Acknowledgements: The work was financially supported by OTKA-NKTH CK77586
[1] E. Farkas, É.A. Enyedi, L. Zékány, J. Gy, Inorg. Biochem. 2001, 83, 107–114.
[2] O. W. Duckworth, Ć. J. R. Bargar, Ć. G. Sposito, Biometals 2009, 22, 605–613.
80
Poster
Miniprotein structure: a chiroptical studies
Viktor Farkas1, Petra Rovó2, Gábor Tóth3, András Perczel 4*
1
Protein Modelling Group, Hungarian Academy of Sciences, Eötvös L. University,
2
Structural Chemistry and Biology Laboratory, Institute of Chemistry, Eötvös L. University
3
Department of Medical Chemistry, Faculty of General Medicine, University of Szeged,
Szeged, Hungary, [email protected]
4
Structural Chemistry and Biology Laboratory, Institute of Chemistry & Protein Modelling
Group, Hungarian Academy of Sciences, Eötvös L. University, Budapest, Hungary,
[email protected]
Type 2 diabetes is a disease characterised by high concentration of glucose in
blood due to insulin resistance. Glucagon-like peptide 1 (GLP–1) is an incretin
hormone involved in glucose homeostasis and stimulation of insulin release from
pancreatic β-cells through specific interaction with the GLP–1 receptor (GLP–1R).
Exendine-4 (Ex–4) is a naturally occurring GLP-1R agonist sharing 53% homology
with the human GLP–1, showing higher binding affinity to GLP–1R[1].
We have designed, synthesised and investigated the folding properties of several
Ex–4 derivatives by using chiroptical spectroscopy. Our model peptide, Tc6b
comes from our earlier studies which is structurally similar to the C-terminal
fragment of Ex–4[2]. The gradual N-terminal elongation of TC6b with the
corresponding
residues
of
Ex–4
(HGEGTFTSD–LSKQMEEEAV–
RLYIQWLKEGGPSSGRPPPS, C-term. extension highlighted) revealed a periodic
change in the helical content of this foldamer. We will present spectroscopic data
on the role of the length of the above mentioned extension in stabilizing the overall
molecular fold. In addition, shift of the molecular packing of the aromatic rings of
the key Trp and Tyr residues will be reported. For quantitative spectral analysis
was made possible by the use of the in house developed CCA+ deconvolutional
algorithm[3],[4].
[1] T. Perry, H.G. Nigel, Trends Pharmacol. Sci. 2003, 24, 377–383.
[2] P. Hudáky, P. Stráner, V. Farkas, Gy. Váradi, G. Tóth, A. Perczel Biochemistry 2008,
47, 1007–1016.
[3] I. Jákli, A. Perczel J. Pep. Sci. 2009, 15, 738–752.
[4] http://www.chem.elte.hu/departments/protnmr/cca/
81
Poster
Problems of controlling the aggregation of the synthetic
beta amyloid peptide for use in biological experiments
Lívia Fülöp1*, Dóra Simon1, Zsolt Bozsó1, Tamás Janáky1,
Gábor Kozma2, Ákos Kukovecz2, Botond Penke1
1
Department of Medical Chemisty, University of Szeged, Dóm tér 8. Szeged, Hungary,
[email protected]
2
Department of Applied and Environmental Chemistry, University of Szeged, Rerrich B. tér
1. Szeged, Hungary
In the last decade of the research of Alzheimer’s Disease (AD), a pronounced
paradigmal shift could be observed in terms of structure and toxicity relations of the
beta amyloid (Aβ) peptides involved in AD. The chase for the real pathogenic form
of Aβ has resulted in the discovery of numerous specific aggregates of the peptide
differing from mature fibrils, which are the constituents of the extracellular plaques.
These non-fibrillar structures are termed as Aβ oligomers.
Physico-chemical investigations, as well as in vitro and in vivo biological
applications require oligomer preparations of a relatively big volume, reproducible
aggregation grade and accurately determined concentration. Standardized
experimental set-ups utilized for the testing of Aβ apply complex buffers, which
fulfill the requirements of the biological tolerability (physiological ionic strength,
osmotic pressure, pH, etc.); the effect of the medium on the oligomerization
process has to be tested as well.
A new depsipeptide derivative, the so-called iso-amyloid peptide was synthesized
and utilized successfully for oligomer preparations in our laboratory1. This peptide
derivative owns advantageous structure properties in terms of solubility and
capability for aggregation compared to the unmodified sequence.
In the present work we aim to give a comprehensive study of the problems might
arise upon utilization of synthetic Aβ for preparation of oligomers; we compare the
aggregation properties of the unmodified Aβ 1–42 and the iso-Aβ 1–42 peptides,
test the effect of the buffer composition on the stability of the oligomers and
demonstrate the concentration dependence of the aggregation process.
[1] Z. Bozsó, B. Penke, D. Simon, I. Laczkó, G. Juhász, V. Szegedi, Á. Kasza, K. Soós, A.
Hetényi, E. Wéber, H. Tóháti, M. Csete, M. Zarándi, L. Fülöp, Peptides 2010, 31, 248–
256.
82
Poster
Structural and functional analyses of chlorite dismutases
from two nitrite-oxidizing bacteria
Paul. G. Furtmüller1, Julius Kostan2, Björn Sjöblom2, Georg Mlynek2,
Stephanie Füreder3, Michael Wagner3, Holger Daims3,
Christian Obinger1, Kristina Djinović-Carugo2
1
Department of Chemistry, Division of Biochemistry, BOKU – University of Natural
Resources and Applied Life Sciences, Vienna, Austria Email: [email protected]
2
Department for Structural and Computational Biology, Max F. Perutz Laboratories,
University of Vienna, Vienna, Austria
3
Department of Microbial Ecology, Vienna Ecology Centre, University of Vienna,
Althanstrasse 14, A–1090 Vienna, Austria
Chlorite dismutase (Cld) is a unique heme enzyme which transforms chlorite to
chloride and molecular oxygen (reaction: ClO2ˉ → Clˉ + O 2). Since bacteria with
Cld play significant roles in wastewater treatment and in the bioremediation of
industrially contaminated sites, it is of high interest to understand the molecular
mechanism of chlorite detoxification. Here we investigate highly active Clds from
Ca. Nitrospira defluivi and Nitrobacter winogradskyi, both key nitrifier in biological
wastewater treatment, by using a comprehensive structural, biochemical and
bioinformatics approach. We determined the crystal structures of both Clds and
showed that functional Cld of Nitrospira is a homopentamer whereas Cld of
Nitrobacter is a dimer [1–2]. Both enzymes possess a fold found in other Clds and
Cld-like enzymes. To investigate the Cld function in more detail, site-directed
mutagenesis of the catalytically important residue Arg173 was performed and two
enzyme mutants were structurally and biochemically characterized. We found that
Arg173 plays a key role in controlling ligand and substrate access to the active site
of the enzymes and in stabilizing higher oxidation states of intermediates that are
formed during the catalytic cycle. Additionally, we show that this residue is
conserved in all known active forms of Cld and propose it as a marker for Cld
activity in yet uncharacterized Cld-like proteins.
[1] J. Kostan, B. Sjöblom, F. Maixner, G. Mlynek, P.G. Furtmüller, C. Obinger, M. Wagner,
H. Daims, K. Djinović-Carugo, J. Struct. Biol. 2010, In press.
[2] G. Mlynek, B. Sjöblom, J. Kostan, S. Führeder, F. Maixner, P.G. Furtmüller, C. Obinger,
M. Wagner, H. Daims, K. Djinović-Carugo, Applied Environ. Microbiol. 2010, submitted
83
Poster
The binding of cadmium(II) to reduced form of
glutathione: Vibrational study
Martina Glušič1, Polona Ropret 2, Jože Grdadolnik 3*
1
National Institute of Chemistry, Ljubljana, Slovenia, [email protected].
2
Institute for the Protection of Cultural Heritage of Slovenia, Restoration Center, Ljubljana,
Slovenia, [email protected].
3
National Institute of Chemistry, Ljubljana, Slovenia.
EN FIST Centre of Excellence, Ljubljana, Slovenia, [email protected].
The binding of metal ions by proteins and peptides is of fundamental interest due
to the importance of metal ions in biological systems. Metals may be part of the
active sites of enzymes and stabilise the structure of proteins. However, at higher
concentration become toxic. Toxicity may result from the binding of metals to
sulfhydryl groups in proteins, leading to an inhibition of activity or disruption of
structure. Due to their high affinity to sulphur, the interaction of heavy metals with
sulfhydryl containing amino acids and proteins plays a major role in their
environmental and biochemical behaviour. Glutathione (GSH, γ-glutamyl-cysteinylglycine) is the most abundant low-molecular-weight thiol-containing compound in
the cells and plays important role in the complexation and elimination of toxic
heavy metals from organisms, shared with more complex peptides and proteins
such as metallothioneins and phytochelatins. The toxic effects of cadmium in the
form Cd2+ are well documented. Given the ubiquity of GSH in biology and the
affinity of sulfhydryl group for Cd2+, it seems likely that the complexation of Cd2+ by
GSH is important in the toxicology of Cd2+. We show that the privileged interaction
sites for the formation of complex GSH with Cd2+ is sulfhydryl group. We identify
the formation of mixed ligand complexes (nitrilotriacetic acid, penicillamine,
mercaptosuccinic acid and N-acetylcysteine) involving Cd2+ and GSH through the
determination of the sites of complexation by vibrational (Raman and FTIR)
spectroscopy.
84
Poster
Molecular modeling of phosphonium cyanine dyes in
complex with DNA
Marina Grabar, Sanja Tomić, Lidija Tumir, Ivo Piantanida,
Ivo Crnolatac
Institute Ruđer Bošković, Bijenička c. 54, 10 000 Zagreb, Croatia, [email protected]
Huge number of medicinal, biochemical and biological implications of DNA/RNA
targeting small molecules resulted in enormous scientific interest in them. Most
often these small molecules rely on one dominant non-covalent binding mode for
their interaction with double stranded (ds) DNA. The combination of different DNA
binding modes of one molecule as well as additional sterical and structural features
controlling three-dimensional recognition drew some attention with the aim of
developing new more selective drugs or biochemical markers as well as research
tools on a molecular level.
The molecular modelling results are compared with spectroscopic measurements
for new cyanine dyes in complexes with DNA. This is an example how molecular
modelling, particularly the long time molecular dynamics simulation (MD) can be
used to rationalize the experimental results on molecular interactions. The
molecular modelling was performed with AMBER101 suite. AMBER ff99SB2 and
GAFF1 force field were used to parameterize molecules. During 23.6 ns of MD
simulations at both room and elevated temperature the cyanine compounds
reoriented, but have not left the DNA minor groove in which they were initially
positioned (Figure 1). According to the calculated binding free energies the both
orientations are similarly possible. Moreover, at different dye/DNA ratio molecular
modelling results proved possibility of dye dimmer formation within DNA minor
groove. Thus, the obtained computational results rationalized the experimentally
determined affinity of cyanine dyes towards DNA.
Figure 1: The monomer in the DNA minor groove.
[1] http://amber.scripps.edu/
[2] V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, C. Simmerling, Proteins 2006,
65, 712–725.
85
Poster
Regulation of fungal chitinases containing LysM motifs
and adjacent LysM-proteins in Trichoderma atroviride
Sabine G. Gruber1, Gustav Vaaje-Kolstad2, Fabiola Matarese3,
Rubén López-Mondéjar4, Christian P. Kubicek1, Verena Seidl-Seiboth1*
1
Research Area Gene Technology and Applied Biochemistry, Institute of Chemical
Engineering, Vienna University of Technology, Getreidemarkt 9/166-5, 1060 Vienna,
Austria. [email protected], [email protected] *.
2
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life
Sciences, P.O. Box 5003, NO–1432 Ås, Norway.
3
Department of Tree Science, Entomology and Plant Pathology "G. Scaramuzzi", Plant
Pathology Section, Faculty of Agriculture, University of Pisa, Via del Borghetto 80, I–
56124 Pisa, Italy.
4
Department of Soil Water Conservation and Organic Waste Management, Centro de
Edafología y Biología Aplicada del Segura (CEBAS–CSIC), P.O. Box 164, 30100
Espinardo, Murcia.
Fungi have a plethora of chitinases, which can be phylogenetically divided into
three subgroups (A, B and C). Subgroup C (sgC) chitinases are especially
interesting due to their multiple carbohydrate binding modules, but they have not
been investigated in detail yet. In this study we analyzed sgC chitinases in the
mycoparasites Trichoderma atroviride and T. virens. Genomic analysis revealed
that the genes neighbouring sgC chitinases often encode proteins that are solely
composed of multiple LysM modules, which suggests that they support or enhance
the functions of sgC chitinases. We show that these LysM-proteins evolved by
birth/death evolution and that the respective genes are mainly coregulated with
their neighbouring sgC chitinase genes. The expression of sgC chitinase genes in
T. atroviride was induced during mycoparasitism of the fungal prey Botrytis
cinerea, but not Rhizoctonia solani and correspondingly only by fungal cell walls of
the former. Interestingly, only few sgC chitinase genes were inducible by chitin,
suggesting that non-chitinous cell wall components act as inducers. In contrast, the
transcriptional profile of the most abundantly expressed sgC chitinase gene tac6
indicated a role of the protein in hyphal network formation. This shows that sgC
chitinases have diverse functions and are not only involved in the mycoparasitic
attack. However, sequence analysis and 3D modelling revealed that TAC6 and
also its orthologue in T. virens have potentially detrimental deletions in the
substrate binding site and are thus probably not catalytically active enzymes. This
study provides first insights into fungal sgC chitinases and their associated LysM
proteins.
86
Poster
Selective inhibitors of human chymotrypsin C
developed by phage display
Dávid Héja1, Katalin Zboray1, Dávid Szakács1, András Szabó2,
Miklós Sahin-Tóth2, Gábor Pál1*
1
Department of Biochemistry, Eötvös University, 1117 Pázmány Péter sétány 1/c,
Budapest, Hungary, [email protected], [email protected], [email protected],
[email protected]
2
Department of Molecular and Cell Biology, Boston University, Boston, MA, USA,
[email protected], [email protected]
The digestive enzyme chymotrypsin C (CTRC) proteolytically regulates activation
of the most abundant pancreatic protease proenzyme, human cationic trypsinogen
and also regulates degradation of the activated form. Mutations in CTRC are risk
factors for chronic pancreatitis. The aim of the present work was to develop
selective peptide-inhibitors against CTRC. A chymotrypsin inhibitor, SGPI-2
(Schistocerca gregaria Protease Inhibitor-2) belonging to the Pacifastin family,
served as the parent molecule for an inhibitor library displayed on M13 phage.
Amino acid residues of the protease binding loop of the inhibitor are numbered as
P4-P3-P2-P1-P1'-P2'-P3'-P4'. The P1 amino-acid of the inhibitor interacts with the
primary specificity pocket of CTRC and the protease can cleave the peptide bond
between P1 and P1’. Positions P4, P2, P1, P1', P2' and P4' of the protease binding
loop were randomized and phages binding to CTRC were selected. A
characteristic sequence pattern with a clear consensus sequence was deduced
after DNA-sequencing 25 phage clones. Based on the selected sequence pattern
SGPI-2 variants were designed, recombinantly expressed, purified and tested
against the following human, chymotrypsin-like enzymes: human chymotrypsin B1,
B2, and C; elastase 2A, 3A and 3B; and chymotrypsin-like enzyme 1. The best
CTRC inhibitor exhibited an inhibitory binding constant (KI) of 20 pM and a
selectivity of 200-200,000-fold over the other 6 different pancreatic proteases. A
Leu at the P1 position and acidic residues (Asp or Glu) at the P4' position were
found to be important for high affinity binding, whereas an Asp residue at the P2'
position increased selectivity. Novel inhibitors against CTRC will be useful reagents
to study the role of CTRC in cellular and animal models of pancreatitis.
87
Poster
Structural investigation of palindromes of temporin
antimicrobial peptides by molecular dynamics methods
Balázs Leitgeb1*, Liza Hudoba1, Gábor Janzsó1, Gábor Rákhely1,2
1
Sciences, Szeged, Hungary, [email protected].
2
Department of Biotechnology, University of Szeged, Szeged, Hungary.
For several antimicrobial peptides, palindrome segments can be observed in their
sequences, which play an important role not only in the structure determination but
also in the biological effect. Few members of the temporin peptide family (i.e.
temporin C, temporin 1DYa, temporin 1Pra and temporin 1TSb), which are
synthesized in the skin of a wide range of North American and Eurasian frogs of
the genus Rana, belong to the antimicrobial peptides containing palindrome
sequences. In the case of these palindromes, a comprehensive structural
investigation was performed applying three different molecular dynamics (MD)
methods. First of all a detailed conformational analysis was carried out by means
of the simulated annealing protocol. For the conformers obtained from these
calculations, the occurring secondary structural elements were examined, and
different types of turn conformations, as well as helical structures of various lengths
were determined. In order to identify the low energy conformations, simulated
annealing coupled replica exchange molecular dynamics simulations were also
performed. Additionally, using the molecular dynamics calculations, the alteration
of structural properties as a function of time were studied as well. These MD
trajectories were analyzed to investigate the evolving characteristic structures, and
to determine their intramolecular H–bonding patterns. On the basis of the MD
simulations it could be concluded that all studied palindromes of temporin peptides
were mainly characterized by helical conformations in addition to the β-turn
structures.
This research was supported by the Hungarian Scientific Research Fund (OTKA PD
78554), and by the Gedeon Richter Plc. L. H. is a grantee of the Gedeon Richter Ph.D.
Scholarship.
88
Poster
Characteristic structural features of palindrome
sequences of indolicidin and tritrpticin
Liza Hudoba1, Gábor Janzsó1, Gábor Rákhely1,2, Balázs Leitgeb1*
1
2
Several antimicrobial peptides contain palindrome sequences with different
lengths, which play a relevant role not only in their structure determination but also
in their biological effect. Indolicidin purified from the cytoplasmic granules of bovine
neutrophils, and tritrpticin isolated from porcine neutrophils can serve as good
representatives of this kind of antimicrobial peptides. In the case of their
palindrome sequences, a comprehensive conformational analysis was performed
using the simulated annealing method. Since the palindromes of indolicidin and
tritrpticin contain two Pro amino acids and the cis-trans isomerism occurs for the
Xaa-Pro peptide bonds, four distinct isomers of these palindrome sequences were
modeled, respectively. The presence of various secondary structural elements was
investigated in the conformers derived from the calculations, and different types of
turn structures and helices were identified. In addition, a variety of intramolecular
interactions (i.e. H-bonds, aromatic-aromatic and proline-aromatic interactions)
were determined, which played an important role in the stabilization of the
secondary structures and the different conformational states of the palindrome
sequences. Concerning the palindrome of indolicidin, our results correspond to the
previous observations and data obtained from a detailed conformation analysis
performed on its whole sequence. Our findings with regard to the tritrpticins
palindrome correlate closely with the earlier conclusions stating that turn
conformations can be considered as the characteristic structural feature of this
antimicrobial peptide.
78554), and by the Gedeon Richter Plc. L. H. is a grantee of the Gedeon Richter Ph.D.
Scholarship.
89
Poster
Pair correlations in beta structures: from structural
databases to the real life
Imre Jákli1, Dóra Menyhárd Karancsiné2, András Perczel3
1
Protein Modeling Group HAS-ELTE, Institute of Chemistry, Eötvös Loránd University,
2
Protein Modeling Group HAS-ELTE, Institute of Chemistry, Eötvös Loránd University,
3
University, Budapest, Hungary, [email protected]
Beta sheets are the one of the most important secondary structure elements of
the natural proteins. Although the three dimensional structure of the protein is
determined by the amino acid sequence, 3D structure prediction methods are still
not precise enough. As the size of the system increases the estimation accuracy
decreases. Since stability and structure are closely related to each other, the
accurate knowledge of the stabilization forces is indispensable. The aggregation
potential of beta sheets makes their structure investigation important as these
sheets are hypothesized to be the seeds of aggregates (or plaques), associated
with conformational illnesses (e.g. Alzheimer and Creutzfeldt–Jakob disease).
The smallest building block of beta sheets was chosen as model system: two
amino acid pairs facing each other in the adjacent polypeptide chains. The
abundance of specific amino acid pairs can reveal the stabilization forces in beta
sheets. In order to perform a statistical analysis, a database of non homologous
proteins should be established. To complete this task a possible approach is to use
one of the non homologous protein sets already available (e.g. PDBSelect 1 or
“WHATIF culled data set”2). To make the statistical analysis user friendly and the
regular database update straightforward, a portable framework was developed in
Java. Quantum chemical calculations revealed that the side chain orientation also
influence the interaction energy of amino acid pairs. The side chain investigations
allowed only high quality and high resolution X–ray structure included in the
database. According to our findings the pair correlations are dependent on the
protein set, the resolution and the R factor of the X–ray structures involved.
[1] PDBselect 1992–2009 and PDBfilter-select; S. Griep, U. Hobohm, Nucleic Acids Res.
2010, 38, Database issue 318–319
[2] WHAT IF Culled datasets: http://swift.cmbi.kun.nl/whatif/select/.
90
Poster
Mechanism of hydrogen peroxide oxidation in
catalase-peroxidases
Christa Jakopitsch1, Jutta Vlasits2, Christian Obinger*3
1
Christa Jakopitsch, BOKU – University of Natural Resources and Applied Life Sciences,
Vienna, Austria, [email protected].
2
Jutta Vlasits, BOKU – University of Natural Resources and Applied Life Sciences,
3
Christian Obinger, BOKU – University of Natural Resources and Applied Life Sciences,
Monofunctional catalases and bifunctional catalase-peroxidases (KatGs) have
neither sequence nor structural homologies but both enzymes catalyze the
dismutation of hydrogen peroxide to dioxygen (2 H2O 2 → 2 H2O + O2). In typical
catalases the catalatic mechanism is well characterized with conventional
compound I [oxoiron(IV) porphyrin π-cation radical intermediate] being responsible
for hydrogen peroxide oxidation. The reaction pathway in KatGs is not as clearly
defined.
KatGs are unique among peroxidases in possessing a covalent adduct between a
tryptophan, a tyrosine and a methionine in the vicinity of the heme containing
active center. Mutation of any of these residues impairs the catalase activity
without affecting the peroxidase activity [1]. Recently it has been shown that in
KatGs, a radical is formed on this covalent adduct that persists only during the
turnover with hydrogen peroxide [2].
Here we demonstrate that the reaction of this redox intermediate with hydrogen
peroxide constitutes the second phase of the dismutation in which H2O2 acts as
two-electron reductant and dioxygen is released. KatGs use this radical site to
enhance the turnover and avoid release of superoxide [3].
[1] C. Jakopitsch, M. Auer, A. Ivancich, F. Rüker, P.G. Furtmüller, C. Obinger J. Biol.
Chem. 2003, 278, 20185–20191.
[2] J. Suarez, K. Ranguelova, A. A. Jarzecki, J. Manzerova, V. Krymoy, X. Zhao, S. Yu, L.
Metlitsky, G.J. Gerfen, R. S. Magliozzo J. Biol. Chem. 2009, 284, 7017–7029.
[3] J. Vlasits, P.G. Furtmüller, C. Jakopitsch, M. Zamocky, C. Obinger. BBA–Proteins
Proteomics. 2010, 1804, 799–805.
91
Poster
The multi-faceted melanin-concentrating hormone
Veronika Jancsik*, Emese É. Várkonyi
Szent István University Faculty of Veterinary Science, Department of Anatomy and
Histology, Budapest, Hungary, [email protected]
Melanin-concentrating hormone (MCH) is a cyclic polypeptide expressed in a wide
variety of animal species ranging from teleost fishes to primates. Its amino acid
sequence and the position of the functionally important disulphide bridge are both
highly conservative.
MCH acts either as a hormone, reaching the bloodstream by the hypothalamohypophyseal system, or as a neurotransmitter. Regulation of skin color, functioning
in numerous non-mammalian chordates but apparently non-existing in mammals is
the most prominent hormonal effect of MCH. In mammals MCH is produced by
neurons in the lateral hypothalamus. It acts primarily as neurotransmitter or
neuromodulator, playing a role in a variety of physiological functions involving the
control of food intake and energy homeostasis. This latter feature constitutes the
therapeutic potential of the MCH peptide system.
In our previous work we demonstrated [1] that in the mouse MCH neurons express
alpha-dystrobrevin, absent from other neurons in the adult brain. Alphadystrobrevin (a-DB) has been formerly identified as a component of the plasma
membrane associated dystrophin glycoprotein complex in several cell types. Our
recent immunohistochemical investigation shows that in MCH neurons a-DB is
present intracellularly, in close colocalization with MCH. Colocalization of the two
proteins occurs in perikarya as well as in the characteristic varicose processes of
this neuron type. Kinesin, the microtubule associated motor protein also
colocalizes with a-DB and MCH. On the basis of the above observations we
suggest that intracellular delivery of MCH is subject of a still unknown peculiar
regulation, in which a-DB plays a role.
[1] D. Hazai, C.F. Lien, F Hajós, K, Halasy, D.C. Górecki, V..Jancsik, Brain Res. 2008
1201, 52–59.
This work has been supported by the Hungarian Scientific Research Fund (OTKA, K
81419) for V. Jancsik.
92
Poster
Folding processes of alanine-based peptides containing
basic amino acids: helix and H–bond formation
Gábor Janzsó1, Ferenc Bogár2, Liza Hudoba1, Botond Penke2,3,
Gábor Rákhely1,4, Balázs Leitgeb1*
1
2
Supramolecular and Nanostructured Materials Research Group of the Hungarian
Academy of Sciences, University of Szeged, Szeged, Hungary.
3
Department of Medical Chemistry, University of Szeged, Szeged, Hungary.
4
Ala-based peptides are important model systems for molecules showing a high
propensity to form helical structures. In this study, Lys- and Arg-containing Alabased peptides (i.e. Ace-(AAAAX)nA-NH 2 and Ace-(AXAAA) nA-NH 2, where A is
Ala, X denotes Lys or Arg, and n=1–4) were modeled using molecular dynamics
methods. The helicity evolution of these oligopeptides were examined with regard
to the whole conformation, as well as on a per-residue basis. Our results indicated
that the longer sequences possessed larger propensities to form -helical structure
for the Lys-containing molecules (i.e. AK peptides), as compared to those for the
Arg-containing molecules (i.e. AR peptides). The investigation of per-residue
helicities pointed out that continuous helical segments could be observed in the
case of AK peptides, except for the longest ones, where two helical parts were
detected. The AR peptides showed different features regarding the helicities,
namely, segments of higher and lower helicities alternated along the sequences.
The regions with lower helicities are situated around the Arg residues. Beside the
helix formation, the evolution of various intramolecular H–bonds (i.e. local and nonlocal interplays) were studied. The←i+3
i
and i←i+4 H –bonds were considered as
local interactions, while every other H–bonds, including the ones formed between
the backbone CO and sidechain NH groups, were considered as non-local
interactions. The appearance of←i+4
i
H –bonds correlated well with the overall
helicities of certain sequences, indicating their helix-stabilizing role. Nevertheless,
our results revealed that the non-local H–bonds affected the evolution of helical
conformations, as well as the folding processes, pointing out their destabilizing
role.
78554).
93
Poster
Folding processes of alanine-based peptides containing
basic amino acids: folding time, pathways and stability
Gábor Janzsó1, Ferenc Bogár2, Liza Hudoba1, Botond Penke2,3,
Gábor Rákhely1,4, Balázs Leitgeb1*
1
2
Academy of Sciences, University of Szeged, Szeged, Hungary.
3
4
Ala-based peptides are important model systems for molecules showing a high
propensity to form helical structures. In this study, Lys- and Arg-containing Alabased peptides (i.e. Ace-(AAAAX) nA-NH 2 and Ace-(AXAAA) nA-NH 2, where A is
Ala, X denotes Lys or Arg, and n=1–4) were modeled using molecular dynamics
methods. To describe the folding processes of each peptide, characteristic folding
times were calculated, which proved to be proportional to the length of
oligopeptides. Based on these results, it could be concluded that for the molecules
possessing Arg residues (i.e. AR peptides), it took generally more time to reach
their maximum regarding the average helicities, as compared to the Lys-containing
molecules (i.e. AK peptides). In order to demonstrate the folding processes of
oligopeptides, four individual trajectories were selected, for which a principal
coordinate analysis was performed. These plots indicated that the AK peptides
adopted a fully helical conformation relatively fast, while the trajectories of AR
peptides were found to be punctuated with many transition states, throughout the
whole simulation time. The degree of observed helicities depends not only on the
rate of helix formation, but also on the stability of evolved helices. To examine the
structural stability of helical conformations, the atomic fluctuations of backbone
heavy atoms were calculated.
78554).
94
Poster
Replica exchange molecular dynamics simulations of
Aβ1-42 and its isopeptide
Gábor Janzsó1, Balázs Leitgeb1, Gábor Rákhely1,2, Botond Penke3,4,
Ferenc Bogár3*
1
Sciences, Szeged, Hungary.
2
3
Academy of Sciences, University of Szeged, Szeged, Hungary, [email protected].
4
Aβ1-42 is the main component of amyloid plaques found frequently in the central
nervous system of Alzheimer's disease patients. After it is cleavage from amyloid
precursor protein, the conformation of this peptide goes through major changes
during its aggregation, from monomers through oligomers to fibrils. The
experimental investigation of the aggregation process of Aβ1-42 is a difficult task,
mainly because of the ill-defined initial aggregation state of this oligopeptide. A
possible solution for this problem is the application of the hardly aggregating
A
25
26
isopeptide, containing an ester bond formed between the Gly and Ser residues,
which transforms to the normal Aβ1-42 structure at physiological conditions. Thus,
using this isopeptide, the aggregation process can be initiated by pH switching.
According to the recent experimental studies, characteristic differences were
detected between the aggregation propensities of Aβ1-42 and its isopeptide form. In
this study, we investigated the structural features of Aβ 1-42 and Aβ isopeptide
monomers at pH = 2 and pH = 7.4 by means of replica exchange molecular
dynamics simulations. Since the Aβ1-42 in its monomer form has no well-defined
structure, the conformer populations derived from the calculations were analyzed.
The occurring secondary structural elements were identified along the whole
sequences, with the exception of the isopeptide unit including Gly25 and Ser 26
residues, where any conventional secondary structures could not be defined. To
characterize the connections and distances between the amino acid residues,
contact maps were produced. For evaluating the stability of evolved structures, the
atomic fluctuations of the Cα atoms were calculated. In conclusion, we identified
those structural differences which may explain the origins of dissimilarity of the
aggregation properties of two Aβ forms.
95
Poster
Novel possibilities of FTIR spectroscopy in bioscience:
From interactions at nanobioconjugates to bacterial cells
Alexander A. Kamnev*, Lev A. Dykman, Anna V. Tugarova
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy
of Sciences, Saratov, Russia; [email protected]
Modern biochemistry and microbiology tend to use a wide range of instrumental
techniques, among which Fourier transform infrared (FTIR) spectroscopy is an
important versatile tool[1–3]. Using the plasmon-resonance (PR) based effect of
surface-enhanced infrared absorption (SEIRA) of biomacromolecules adsorbed on
gold nanoparticles (AuNP), we earlier developed a novel spectroimmunochemical
approach[4]. It has further showed promise in controlling the quality of AuNP
functionalised by proteinaceous macromolecules (used as biomarkers) and in the
SEIRA-based detection of biospecific interactions of various kinds (e.g., antigen–
antibody; lectin–polysaccharide), when one type of the interacting biomolecules
(either recognising or target molecules) is conjugated with AuNP.
Another highly promising direction is the application of the diffuse reflectance (i.e.,
DRIFT) mode of FTIR spectroscopy for studying fine structural modifications of
intracellular microbial biopolymers induced by environmental factors[1–3]. It was
found that, besides the accumulation of carbon and energy storage compounds,
polyhydroxyalkanoates (PHA), represented in some soil bacteria by homopolymer,
poly-3-hydroxybutyrate (PHB), various stress conditions induce fine changes in the
secondary structure of cellular proteins[1]. Moreover, DRIFT spectroscopy appears
to be sensitive to the degree of crystallinity of intracellular PHB (accumulated or
consumed at various stages of bacterial growth under stresses), which is related to
the rate of its enzymatic hydrolysis (i.e., its degree of digestibility inside the cell).
Supported by NATO (Grant ESP.NR.NRCLG 982857) and INTERTECH (Moscow).
[1] A. A. Kamnev, J. N. Sadovnikova, P. A. Tarantilis, M. G. Polissiou, L. P. Antonyuk,
Microb. Ecol. 2008, 56, 615–624.
[2] A. A. Kamnev, Spectroscopy 2008, 22, 83–95.
[3] A. A. Kamnev, A. V. Tugarova, L. P. Antonyuk, P. A. Tarantilis, L. A. Kulikov, Yu. D.
Perfiliev, M. G. Polissiou, P. H. E. Gardiner, Anal. Chim. Acta 2006, 573–574, 445–452.
[4] A. A. Kamnev, L. A. Dykman, P. A. Tarantilis, M. G. Polissiou, Biosci. Rep. 2002, 22,
541–547.
96
Poster
Spectroscopic approaches for studying the effects of
abiotic factors on microbial molecular signalling
Alexander A. Kamnev1, Roman L. Dykman1, Krisztina Kovács2,
Ernő Kuzmann2, Attila Vértes 2
1
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian
Academy of Sciences, Saratov, Russia; [email protected]
2
Eötvös Loránd University, H–1117 Budapest, Hungary; [email protected]
It has been well documented that microbial consortia use a “chemical language” for
communication between its members, which involves a number of small diffusible
molecules of specific structure excreted by microbial cells into the growth medium
(see, e.g.[1] and references therein). It is clear that all possible physicochemical
(abiotic) effects of environmental factors on such extracellular signalling molecules
also represent direct interferences in the microbial signalling processes. For
instance, the chemical reactivity of particular metal species (or their possible
catalytic effects) could result in complexation or redox degradation of biogenic
organics involved in microbe–host interactions or intercellular microbial
communication via the remote exchange of molecular signals[1–3]. As a continuation
of our related studies, in this work it has been shown that alkylresorcinols (ARs),
microbial autoregulators with adaptogenic functions, can be gradually oxidised in
the presence of iron(III) in weakly acidic media (simulating acidic soil conditions).
The course of Fe(III) reduction and iron complexation were quantitatively monitored
using 57Fe Mössbauer spectroscopy both in the solutions (rapidly frozen prior to
measurements) and in the dried solids, while the concomitant AR oxidation was
studied using UV spectrophotometry.[3] The rate of the redox process was found to
strongly depend on the length and/or position of the alkyl substituent in the AR
aromatic ring (AR oxidation was much more rapid with 4-n-hexyl than with 5methyl), testifying to the role of the AR molecular structure in its reactivity.
Supported under the Agreement on Scientific Cooperation between the Russian and
Hungarian Academies of Sciences for 2008–2010 (Project No. 45).
[1] A. A. Kamnev, K. Kovács, E. Kuzmann, A. Vértes, J. Mol. Struct. 2009, 924–926, 131–
137.
[2] K. Kovács, V.K. Sharma, A.A. Kamnev, E. Kuzmann, Z. Homonnay, A. Vértes, Struct.
Chem. 2008, 19, 109–114.
[3] A. A. Kamnev, K. Kovács, R.L. Dykman, E. Kuzmann, A. Vértes, Bull. Russ. Acad. Sci.
Phys. 2010, 74, 394–398.
97
Poster
ICV injected Aβ-peptide induces dysfunctions in
hippocampus and in spatial memory: model for
Alzheimer's disease
Ágnes Kasza1*, Viktor Szegedi2, Gábor Juhász3, Zsuzsanna Frank4,
Zsuzsa Penke5, Botond Penke6
1
Department of Medical Chemistry, University of Szeged, Hungary, [email protected]
2
Bay Zoltán Foundation for Applied Research – BAYGEN Institute, Szeged, Hungary
3
Department of Medical Chemistry, University of Szeged, Hungary
4
Department of Medical Chemistry, University of Szeged, Hungary
5
Neurobiology of Learning, Memory and Communication, University Paris Sud, France
6
Department of Medical Chemistry, University of Szeged and Bay Zoltán Foundation for
Applied Research – BAYGEN Institute, Szeged, Hungary, [email protected]
Alzheimer’s
disease
(AD)
is
a
progressive
neurodegenerative
disorder,
characterized pathologically by β-amyloid (Aβ) plaques and neurofibrillary tangles
especially in the hippocampal (HC) area. The abnormal accumulation of these
proteins induce many degenerational processes, such as astrogliosis, neuronal
loss, impaired long-term potential (LTP), and eventuate cognitive deficit, typically in
spatial memory.
The purpose of the present study was to establish a reliable AD rat model with
intracerebroventricular (ICV) Aβ1–42 oligomer injection bilaterally into the lateral
ventricles. The control group was injected with hydrocarbonate buffered saline
(HCBS), and there were six Aβ1–42-treated groups. The effects on the spatial
memory dysfunction were tested by using the Morris water maze (MWM)
behavioral task, and further characterized by multi-electrode array (MEA)
measurements for LTP recordings, which was supplemented with histological
procedures in the HC region.
The results demonstrated that animals obtaining the Aβ1–42 oligomer samples
exhibited significant differences compared to the control group both in vivo and ex
vivo. The histological studies provided supporting data for our behavioral and
electrophysiological results. These findings together are in accord with our
hypothesis that ICV injected Aβ1–42 oligomers impair the spatial memory.
98
Poster
Biological effect of halogenated anthracenes on
biochemical processes
Galina A. Kudryasheva1*, Elena V. Nemtseva2
1
Department of Biophysics, Siberian Federal University, 79 Svobodny Prospect,
Krasnoyarsk, 660041, Russia, [email protected].
2
Lab. of Photobiology, Institute of Biophysics SB RAS, 50/50 Akademgorodok,
Krasnoyarsk, 660036, Russia, [email protected].
Influence of halogenated compounds on enzymatic reactions is of great interest
because iodine and bromine atoms are important components in living organisms,
and halogenated compounds can exert a toxic impact on human organism and
environment. The hypothesis was proposed that the biological effect of
homologous halogenated compounds is a function of substituent weight[1] (like
external heavy atom effect in photophysics[2]). The aim of this work was to study
the patterns in changes of biochemical reaction characteristics after addition of
halogenated homologues.
Bioluminescent system of coupled enzymatic reactions catalyzed by bacterial
luciferase and NAD(P)H:FMN-oxidoreductase was applied as model biochemical
process. The reaction rate in this system is proportional to the intensity of
emission. To effect bioluminescent reaction the following homologues we used:
anthracene (A), 9-chloroanthracene (9-ClA), 9-bromoanthracene (9-BrA), 9iodoanthracene (9-IA) from Sigma. The interaction with proteins was studied using
bovine serum albumin (BSA) and amylase.
The kinetic and spectral characteristics of bioluminescence for coupled enzyme
system NAD(P)H:FMN-oxidoreductase-bacterial luciferase were registered in the
presence of the halogenated anthracens of different concentration. Dependence of
quenching effect on the weight of haloid substitutes was revealed, thus
demonstrating the external effect of heavy atom in the bioluminescent reaction.
Interactions of the anthracenes with BSA and amylase were studied. Emission and
excitation fluorescence spectra, fluorescence anisotropy in the presence and in the
absence of the proteins were registered. Dissociation constants (Kd) of antraceneprotein complexes were estimated using fluorescence anisotropy technique.
Anthracens-amylase binding were explained in terms of electrostatic interaction: Kd
for A and 9-IA are higher than those for 9-ClA and 9-BrA. Binding with BSA was
assumed to be promoted by hydrophobic interaction.
[1] T.N. Kirillova, N.S. Kudryasheva, Anal. Bioanal. Chem. 2007, 387, 2009–2016.
[2] M. Kasha, J. Chem. Phys. 1952, 20, 71–74.
99
Poster
Antiproliferative activity, uptake and intracellular
distribution of urea-phenanthridinium conjugates
Saška Marczi1, Marijana Radić Stojković2, Ivo Piantanida2,*,
Ivan Mihaljević1,3, Ljubica Glavaš-Obrovac1,3,*
1
Clinical Institute of Nuclear Medicine and Radiation Protection, University Hospital Centre
Osijek, Osijek, Croatia, [email protected], [email protected],
[email protected]
Laboratory for Study of Interactions of Biomacromolecules, Ruđer Bošković Institute,
Zagreb, Croatia, [email protected], [email protected]
2
3
School of Medicine, J.J. Strossmayer University of Osijek, Osijek, Croatia
* corresponding authors
We investigated possible changes in biological activity as a result of introduction of
permanent positive charge on a series of previously studied bis-urea
phenanthridine derivatives[1,2].
Growth inhibition effects of neutral and positively charged bis-urea
phenanthridinium derivatives were analysed in vitro on human pancreatic
(MIAPaCa-2), larynx (HEp-2), cervical (HeLa) and colon carcinoma cells (Caco-2,
HT-29) and human normal fibroblasts (BJ) using MTT assay. Uptake and
intracellular distribution of methylated derivative 8 (1-(5,6-dimethylphenanthridin-8yl)-3-[6-(3-(5,6-dimethylphenanthrydin-8-yl)ureido)hexyl]urea hydrogensulfate) was
tested.
Results indicated enhanced anticancer potency of methylated in respect to nonmethylated analogues. One positively charged compound, derivative 8,
demonstrated selectivity between tumour and normal cells. The same compound in
10–3 M concentration entered and dyed HeLa cells in yellow fluorescence after 10
min of incubation.
The results show how applying structural modifications to conventional molecules
can achieve an enhanced antitumor activity and improvement of compound’s
selectivity[3].
[1] M. Radić Stojković, I. Piantanida, Tetrahedron 2008, 64, 7807–7814.
[2] P. Čudić, M. Žinić, V. Tomišić, V. Simeon, J.-P. Vigneron, J.-M. Lehn, J. Chem. Soc.
Chem. Commun. 1995, 1073–1075.
[3] M. Radić Stojković, S. Marczi, Lj Glavaš-Obrovac, I. Piantanida, Eur. J. Med. Chem.
2010, 45, 3281–3292.
100
Poster
Molecular modeling of crystallization processes of trans
and cis bis(L-valinato)copper(II) from aqueous solution
Marijana Marković, Jasmina Sabolović*
Institute for Medical Research and Occupational Health, Ksaverska cesta 2, P.O.
Box 291, HR–10001 Zagreb, Croatia, [email protected], [email protected]
Heating of cis aquabis(L-valinato)copper(II) at 90°C and subsequent
recrystallization of the heated compound from aqueous solutions yielded
anhydrous trans bis(L-valinato)copper(II). To resolve if the heating affected the
structural changes that steered the crystallization process, conformational analyses
were performed for cis- and transconformers in vacuo and in crystal by molecular
mechanics, and in aqueous solution by molecular dynamics simulations using the
force field FFWa-SPCE[1]. The conformational analyses yielded that transconformers were the most stable in vacuo, but cis-conformers could form more
favorable intermolecular interactions than trans-ones, and both cis- and transconformers were predicted to exist simultaneously in aqueous solution. The unit
cell packing predictions suggest that cis-isomer required water molecules to form
more energetically stable crystal lattice packings than trans-isomer, in accordance
with their X–ray crystal structures[2]. Fifty-nanosecond molecular dynamics
simulations completed at 298 K and 370 K for the system containing 84 complexes
and 84 water molecules suggested thoroughgoing cis- to trans- transformation at
higher temperature. Prevalence of trans-conformers in water may explain the
crystallization of trans-isomer. The association of the complexes (crystallization
nucleus) in aqueous solution is predicted to proceed firstly from dimers up to
pentamers by weak Cu-to-Ocarboxylato bonds. These aggregations bind together via
water molecule’s layer until they acquire appropriate positions for noncovalent
bonding alike in real crystal structures.
[1] J. Sabolović, V. Gomzi, J. Chem. Theory Comput. 2009, 5, 1940–1954.
[2] M. Marković, N. Judaš, J. Sabolović, Cryst Growth Des. 2010, submitted
101
Poster
Steady-state kinetic analysis of E. coli purine nucleoside
phosphorylase active site mutants
Goran Mikleušević1, Marta Narczyk2, Lucyna Magnowska2,
Beata Wielgus-Kutrowska2, Agnieszka Bzowska2, Marija Luić*1
1
2
Department of Biophysics, Institute of Experimental Physics, University of Warsaw,
Warsaw, Poland, [email protected]
In order to get close insight into very complex mechanism of Escherichia coli
purine nucleoside phosphorylase (PNP) kinetic measurements of a wild type
enzyme and its Arg24Ala mutant towards phosphate as substrate were performed
and discussed. The key role of Arg24 during phosphorolysis was studied at
different pH and at constant nucleoside (7-methylguanosine and adenosine)
concentration. The comparative analysis of the catalytic sites of wild type enzyme
and Arg24Ala mutant based on kinetic data will be presented. E. coli PNP, in
contrast to human homologue, has a broad substrate specificity[1] which makes this
enzyme a good candidate in gene therapy against solid tumours [2]. Therefore,
understanding of its catalytic mechanism is of utmost importance.
[1] A. Bzowska, E. Kulikowska, D. Shugar, Pharmacol Ther. 2000, 88, 349–425.
[2] Y. Zhang, W. B. Parker, E. J. Sorscher, S. E. Ealick, Curr Top Med Chem. 2005,5,
1259–74.
102
Poster
Infrared spectrum of the acetic acid dimer in the
O–H stretching region
Nađa Došlić*,1, Milena Petković2, Jurica Novak3
1
2
Faculty of Physical Chemistry, Belgrade, Serbia, [email protected]
3
DNA base pairs are characterized with multiple hydrogen bonds. Thus, carboxylic
acid dimers serve as prototypes for Watson-Crick base pairs. The acetic acid dimer
comprises a double hydrogen bond, and at the same time small side groups that
take into account the effect of lateral substituents. Consequently, the infrared
spectrum of this system is remarkably complex in the O–H stretching region. The
reason for this phenomenon is twofold. First, in addition to the most stable
structure, the two conformers constructed by single and double methyl rotation
(first and second order saddle point, respectively) are present in the sample due to
the low barrier for this type of motion. Secondly, anharmonic couplings are
noticeably strong, resulting in pronounced mode interactions. Particularly intriguing
is the breakdown of the infrared-Raman exclusion rule – although the most and the
least stable conformers possess a center of symmetry, in all three cases the
strongest coupling by far is the one between the antisymmetric (IR active) and the
symmetric (Raman active) O–H stretching motion.
The MultiConfigurational Time Dependent Hartree procedure
handling the resulting 8-dimensional models [2].
[1]
was used for
[1] M. H. Beck, A. Jäckle, G. Worth, H.-D. Meyer, Phys. Rep. 2000, 324, 1–105.
[2] M. Petković, J. Novak, N. Došlić, Chem. Phys. Lett. 2009, 474, 248–252.
103
Poster
Pyridine and quinoline as privileged moieties in drug
design: potential AChE reactivators, antifungal and HIV
integrase inhibitors
Katarzyna M. Majerz-Maniecka1, Robert Musioł2, Agnieszka Skórska-Stania1,
Josef Jampilek3, Barbara J. Oleksyn1, Jarosław Polański2
1
Faculty of Chemistry, Jagiellonian University, Krakow, Poland, [email protected]
Institute of Chemistry, University of Silesia, Katowice, Poland
3
Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno,
Czech Republic
2
The pyridine and quinoline moieties play an important role in many natural
compounds, especially alkaloids. The activity of these molecular fragments is
mainly due to their aromatic character and the presence of the nitrogen atom with
the lone electron pair, which may be protonated and form hydrogen bonds e.g. with
anionic acceptors or with water molecules. Because of their properties both these
moieties may be considered as privileged and they are introduced to synthetic
compounds in order to equip them with special ability to interact with selected
macromolecules.
In this contribution the results of our crystallographic and docking studies on three
groups of new compounds containing these moieties and synthesized as potential
antifungal agents [1], AChE reactivators [2], and HIV–1 integrase inhibitors [3,4]
will be described. The most important conclusions concern the intermolecular
interactions, which may decide about the biological activity of the investigated
compounds. These interactions are mainly hydrogen bonds and π – π stacking.
The title molecular fragments show similar behaviour towards their environment
both in the crystals containing only the small molecules and in the crystals of the
proteins in complexes with the potential drugs.
[1] R. Musiol, J. Jampilek, V. Buchta, H. Niedbala, B. Podeszwa, A. Palka, K. MajerzManiecka, B. Oleksyn, J. Polanski, Bioorgan. Med. Chem. 2006, 14, 3592–3598.
[2] A. Skórska-Stania, M. Śliwa, K. Musilek, K. Kuca, J. Jampilek, R. Musiol, B. J. Oleksyn,
J. Dohnal, Struct. Chem. 2010, 21, 495–501.
[3] K. Majerz-Maniecka, R. Musiol, W. Nitek, B. J. Oleksyn, J. F. Mouscadet, M. Le Bret,
Bioorg. Med. Chem. Lett. 2006, 16, 1005–1009.
[4] K. Majerz-Maniecka, R. Musiol, A. Skorska-Stania, D. Tabak, P. Mazur, K. Serafin,
B. J. Oleksyn, J. Polanski “Investigation of three quinoline-based scaffolds. Intermolecular
interactions in crystals and molecular docking” (in preparation)
104
Poster
Structural and electronic effects on the reactions of
metallochlorophylls formation
Łukasz Orzeł1, Dorota Rutkowska-Żbik2, Leszek Fiedor3,
Grażyna Stochel1
1
Faculty of Chemistry, Jagiellonian University, Kraków, Poland, [email protected].
2
Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,
Niezapominajek 8, 30–239 Kraków, Poland, [email protected].
3
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków,
Poland, [email protected].
Incorporation of metal ions into tetrapyrrolic systems results in the formation of
complexes of crucial biological importance and/or potentially applicable in various
branches of medicine and technology. Despite many years’ standing studies, some
mechanistic aspects of these reactions remain still unclear. Particular uncertainty
concerns chlorophylls that reveal distinct electronic and structural features. Recent
studies indicated that solvent[1] and counter ion[2] determine kinetics of these
reactions. Moreover, in case of redox-active metal ions they control reaction
course, leading to either metallochlorophyll formation or macrocycle degradation.
To clarify the structure–reactivity relationships in metalation reactions of
porphyrinoids the interactions of a series of eight chlorophyll-based ligands was
prepared in which both the peripheral groups and the degree of saturation of the
macrocycle were systematically varied. The reactions of these ligands with reactive
Zn2+ and inert Pt2+ ions were investigated in methanol and acetonitrile using
absorption spectroscopy. The kinetic data clearly show that the solvent has a
crucial role in the activation of the incoming metal center. The electronic factors
revealing in the size of the delocalized π–electron system influence both rigidity
and nucleophilicity of the macrocycle. The role of peripheral substituents is of less
importance, except for strongly electron-withdrawing groups, that reduce the
chelating ability of tetrapyrrole. The results of kinetic studies were correlated with
the static descriptors of chlorophylls and porphyrins reactivity obtained from
quantum chemical calculations (DFT).
[1] Ł. Orzeł, R. van Eldik, L. Fiedor, G. Stochel, Eur. J. Inorg. Chem. 2009, 2393–2406.
[2] Ł. Orzeł, L. Fiedor, M. Wolak, A. Kania, R. van Eldik, G. Stochel, Chem. Eur. J. 2008,
14, 9419 – 9430.
105
Poster
Relationship of the heme iron stereochemistry and
Mössbauer hyperfine parameters in different
oxyhemoglobins
Michael I. Oshtrakh1*, Aron L. Berkovsky2, Amit Kumar3,
Suman Kundu3, Alexander V. Vinogradov4, Tatiana S. Konstantinova4,
Vladimir A. Semionkin1,5
1
Faculty of Physical Techniques and Devices for Quality Control, Ural Federal University,
Ekaterinburg, 620002, Russian Federation, e–mail: [email protected]
2
Hematological Scientific Center of the Russian Academy of Sciences, Moscow, 125167,
Russian Federation, e–mail: [email protected]
3
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road,
New Delhi – 110021, India, e–mail: [email protected]
4
Faculty of Internal Diseases Propedeutics, Ural State Medical Academy, Repin str., 3,
5
Faculty of Experimental Physics, Ural Federal University, Ekaterinburg, 620002, Russian
Federation, e–mail: [email protected]
Normal oxyhemoglobins from human, rabbit and pig with different primary
structures and oxygen affinities as well as oxyhemoglobin from patients with
chronic myeloleukemia and multiple myeloma were studied using Mössbauer
spectroscopy with a high velocity resolution (see[1]). Mössbauer spectra were fitted
using two models: 1 – using one quadrupole doublet (without accounting for the
heme Fe electronic structure variations in α– and β–subunits of oxyhemoglobins)
and 2 – using two quadrupole doublets with equal areas (accounting for the heme
Fe electronic structure variations in α– and β–subunits of oxyhemoglobins). In both
models small variations of Mössbauer hyperfine parameters (quadrupole splitting
and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins.
These results were related to different heme Fe stereochemistry obtained from X–
ray crystal structures of the three hemoglobins published in the Protein Data Bank.
Furthermore, small variations of Mössbauer hyperfine parameters for patients’
oxyhemoglobins were related to possible variations in the heme Fe
stereochemistry.
This work was supported in part by the Russian Foundation for Basic Research (grant #
09-02-00055-a).
[1] V. A. Semionkin, M. I. Oshtrakh, O. B. Milder, E. G. Novikov, Bull. Rus. Acad. Sci.:
Phys. 2010, 74, 416–420.
106
Poster
Comparative analysis of human liver ferritin, chicken liver
and spleen, and pharmaceutically important ferritin
models using Mössbauer spectroscopy
Michael I. Oshtrakh1*, Arina V. Alenkina1,2, Nikolai V. Sadovnikov3,
Vladimir A. Semionkin1,2
1
Faculty of Physical Techniques and Devices for Quality Control, Ural Federal University,
2
Faculty of Experimental Physics, Ural Federal University, Ekaterinburg, 620002, Russian
Federation, e–mail: [email protected]
3
Faculty of Physiology and Biotechnology, Ural State Agricultural Academy, K. Liebknecht
str., 42, Ekaterinburg, 620075, Russian Federation
Ferritin is an iron storage protein consisted of ferric hydrous oxide core surrounded
with a protein shell. This iron core may vary in various tissues, animals and in
normal and pathological cases. Some ferritin models are used for treatment of iron
deficiency anemia. In this work a comparative study of human liver ferritin, normal
and leukemia chicken liver and spleen as well as commercial pharmaceutical
products Imferon, Maltofer® and Ferrum Lek which modeling ferritin was
performed using Mössbauer spectroscopy with a high velocity resolution (technical
details see in[1]). Mössbauer spectra were fitted using two models: 1 – using one
quadrupole doublet (homogeneous iron core) and 2 – using two or more
quadrupole doublets (heterogeneous iron core). Small variations of Mössbauer
hyperfine parameters (quadrupole splitting and isomer shift) were found within the
model 1 for normal human ferritin, normal and leukemia chicken liver and spleen
as well as for Imferon, Maltofer® and Ferrum Lek. Variation of iron content in
chicken tissues was also observed. In the case of model 2 Mössbauer spectra of
various samples were better fitted with different number of quadrupole doublets.
These results demonstrated small variations in the iron cores of human liver
ferritin, normal and leukemia chicken liver and spleen as well as in the iron cores of
ferritin models.
This work was supported in part by the Russian Foundation for Basic Research (grant #
09-02-00055-a).
[1] V. A. Semionkin, M. I. Oshtrakh, O. B. Milder, E. G. Novikov, Bull. Rus. Acad. Sci.:
Phys. 2010, 74, 416–420.
107
Poster
Theoretical modeling of Pt-histamine complex hydrolysis
and interactions with guanine and adenine
Wojciech P. Oziminski1*, Piotr Garnuszek2, Aleksander P. Mazurek 3
1
National Medicines Institute, Warsaw, Poland, [email protected]
2
3
Platinum complexes are the most important metal-based antitumor agents. After
the spectacular Rosenberg’s discovery of cisplatin there have been synthesized
several thousands of platinum complexes and some of them turned out to possess
interesting properties like higher activity, lower toxicity or better solubility than the
original cisplatin. In our Instutute we have been studying platinum(II)–histamine
complexes for several years. We performed the synthesis[1], in vitro tests, which
showed promising antitumor activity of these complexes[2–3], and also molecular
modeling studies[4].
The hydrolysis of platinum agent is very important step in drug path across the
living organism as the hydrolysed cation aqua-complexes are the active species
taking part in alkylation of DNA chains and causing cytotoxic effect to tumor cells.
In the current project we model both thermodynamic and kinetic aspects of the
hydrolysis of Pt-histamine complex using MPW1PW91/6–311G** (Stuttgart SDD
basis set for platinum) theoretical model established in our previous
investigations[4]. Results show that the energetic cost of hydrolysis of Pt-histamine
complex is lower than cisplatin (about 6.4 kcal/mol vs 7.3 kcal/mol) and the
activation energy is also lower (22.6 kcal/mol vs 25.3 kcal/mol). Similar
relationships characterise the alkylation process of N7 nitrogen atom of DNA base
guanine, which is well known as the primary target for alkylation agents. The
energetic cost is lower (5.6 kcal/mol vs 8.5 kcal/mol) than for cisplatin and also the
activation barrier is lower (19.0 kcal/mol vs 21.5 kcal/mol). Similar study for
adenine base is being carried out.
[1] P. Garnuszek, J. K. Maurin, J. Witowska-Jarosz, B. Ptasiewicz-Bak, Inorg. Chim. Acta
2002, 338C, 119–126.
[2] P. Garnuszek, Nucl. Med. Rev. Cent. East Eur. 2004, 7, 113–116.
[3] P. Garnuszek, U. Karczmarczyk, M. Maurin, Nucl. Med. Biol. 2008, 35, 605–613.
[4] W. P. Ozimiński, P. Garnuszek, E. Bednarek, J. Cz. Dobrowolski, Inorg Chim Acta
2007, 360, 1902–1914.
108
Poster
Neutral or positively charged new tetramer structures: a
computational study of xanthine and uric acid derivatives
Gábor Paragi1*, Lajos Kovács2, Zoltán Kupihár2,
Célia Fonseca Guerra3, F. Matthias Bickelhaupt 3
1
Academy of Sciences, Szeged, Hungary, [email protected]
2
Department of Medicinal Chemistry at the University of Szeged, Szeged, Hungary
3
Department of Theoretical Chemistry at the Free University of Amsterdam, Amsterdam,
Netherland
New tetramer structures based on 9-methylxanthine (Xa) (Figure 1a), 9-methyluric
acid (Ua) (Figure 1b), and 9-methylxanthine protonated at N7 (XaH+) were
examined by high level density functional calculations.
O
HN 1
O
N
6
HN 1
7
5
8
2
4
3
O
5
7
4
9
H
8
2
9
3
N
N
H
H
N
6
O
N
N
H
CH3
1a
O
CH3
1b
Tetrads and dimers were constructed and optimized at BLYP/TZ2P level of theory.
The optimized structures were decomposed in several ways and the interaction
energy of the fragments was determined. The calculated binding energies show
that systems with positive charges [(XaH+-Xa) 2, (XaH+-Ua)2,] contain low barrier
hydrogen bonds which according to the dimer investigations provide an extra
strong interaction. Cation and anion binding capacity of the new systems were also
investigated and stable structures were found without the necessity of stacking
geometry. The dimer fragmentation of tetrads shows large asymmetry without extra
ion in the positively charged complexes but ions (anion around the LBHB region
and cation in the center of the tetrad) can diminish this asymmetry.
109
Poster
Calculation of the Asparagine pKa Values in Water Using
COSMO, COSMO–RS and Modified Cluster–Continuum
Models
Nena Peran1*, Zvonimir B. Maksić2
Quantum Organic Chemistry Group, Ruđer Bošković Insitute, Zagreb, Croatia,
[email protected].
1
Quantum Organic Chemistry Group, Ruđer Bošković Insitute, Zagreb, Croatia,
[email protected],
2
Faculty of Science, Department of Physical Chemistry, University of Zagreb, Zagreb,
Croatia.
Asparagine is one of the 20 most common natural amino acids on Earth due to its
irreplaceable role as important building block of proteins. Since its side chain’s
functional group is carboxamide, it can form hydrogen bonds which predominantely
determine its function in the structure of protein.
In water solution, asparagine assumes a zwitterionic form possessing the NH3+ and
COO– groups which are stabilized by solvation with water molecules [1]. The
standard thermodynamic cycle for calculating pK a values includes energies of the
equilibrium gas phase geometries, which are corrected by solvation Gibbs free
energy. Since a few water molecules are needed to stabilize zwitterionic form of
asparagines in gas phase, its clusters with one and two water molecules were
investigated. They revealed the important part of the hydrogen bonding pattern of
asparagine zwitterion in water, which is essential part of the modified cluster–
continuum approach in calculating pKa values.
The geometries of Asn in solution and corresponding energies were estimated by
using COSMO and COSMO–RS solvation models. Calculated pK a values show
good accordance with experiment.
[1] J. H. Jensen, M. S. Gordon, J. Am. Chem. Soc. 1995, 117, 8159–8170.
110
Poster
Car–Parrinello simulation of hydrogen bond dynamics in
sodium hydrogen bissulfate
Gordana Pirc, Jernej Stare, Janez Mavri
National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia,
[email protected]
The vibrational dynamics and structure of hydrogen-bonded crystalline ferroelectric
material, sodium hydrogen bissulfate (Na3H(SO4)2), was studied [1]. Molecular dynamics
simulation was preformed with the Car–Parrinello method, followed by an a posteriori
quantization of the OH stretching motion. In order to calculate the OH stretching envelope,
we adopted an approach, based on snapshots structures taken from the CPMD trajectory
[2]. We pointwise calculated the proton potentials, and solved the 1D vibrational
Schrödinger equation for each of the snapshot potentials. The calculated spectrum was in
very good agreement with the experiment. [3] The effects of deuteration were also
considered.
Sodium hydrogen bissulfate unit cell (atom colors: red is oxygen, yellow is sulfur, blue is sodium,
and white is hydrogen) and corresponding OH stretching envelope (red curve) with distribution of
anharmonic OH stretching transitions (blue vertical lines) obtained from proton potentials extracted
from snapshot structures of the CPMD simulation.
The probability density obtained from the averaged wavefunction allowed for calculation of
potential of mean force along the proton transfer coordinate. We demonstrated that for the
present system the free energy profile for both proton and deuteron transfer processes are
barrierless.
All the calculated time-averaged geometric parameters were in reasonable agreement with
the experimental neutron diffraction data [4].
111
Poster
It is worth to stress that the methodology for the quantization of proton motion and
calculation of potential of mean force for the proton transfer process is applicable to a
variety system, including enzymes in the framework of QM/MM methodology [5, 6].
[1] G. Pirc, J. Stare, J. Mavri, J. Chem. Phys. 132, 224506 (2010).
[2] J. Stare, J. Panek, J. Eckert, J. Grdadolnik, J. Mavri, D. Hadži, J. Phys. Chem. A 112, 1576
(2008).
[3] F. Fillaux, A. Lautié, J. Tomkinson, G. J. Kearley, Chem. Phys. 154,135 (1991).
[4] W. Joswig, H. Fuess, G. Ferraris, Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst.
Chem. 38, 2798 (1982).
[5] M. H. M. Olsson, J. Mavri, A. Warshel, Philos. Trans. R. Soc. London Ser. B 361, 1417
(2006).
[6] S.C.L. Kamerlin, J. Mavri, A. Warshel, FEBS Lett. 584, 2759 (2010).
112
Poster
Assessing the Role of Loops on Cation Movement within
DNA G-quadruplexes
Primož Šket1,2, Janez Plavec1,2,3
1
Slovenian NMR center, National Institute of Chemistry, Hajdrihova 19, SI–1000 Ljubljana,
Slovenia; [email protected].
2
EN->FIST Centre of Excellence, Dunajska 156, SI–1000 Ljubljana, Slovenia.
3
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva cesta
5, SI–1000 Ljubljana, Slovenia.
DNA is mostly known for its function in storage and duplication of genetic
information as a double helical structure. Guanine-rich DNA sequences can fold
into four-stranded G-quadruplex structures composed of G-quartets, planar arrays
of four guanines held together by eight Hoogsteen hydrogen bonds. G-rich
sequences are overrepresented in the promoter regions of a number of genes,
including oncogenes, ribosomal DNAs as well as in telomeric DNA regions and
immunoglobulin heavy chain switch regions of higher vertebrates. G-quadruplexes
can be used as suitable building blocks for functional materials and biotechnology
due to their structural variability, high temperature stability and the feasibility of
controlling their dynamic behaviour. However, in contrast to double-helical DNA the
rules for self-assembly of G-quadruplexes are yet to be understood.[1–2]
The present study expands the notion that a d(TG4T) forms solely a tetramolecular
G-quadruplex with parallel orientation of the four strands and four G-quartets with
all residues in anti orientation. NMR experiments reveal the equilibrium of two
monomeric forms in the presence of K+, Na+ and 15NH4+ ions. A major form
consists of four G-quartets, whereas a minor form exhibits an additional T-quartet
at the 5’-end. Three 15NH4+ ion binding sites are identified between four G-quartets
within the major monomeric form, while an additional 15NH4+ ion binding site is
established between G- and T-quartets at the 5’-end of the minor form. 15NH4+ ions
are shown to move faster between the interior of tetramolecular structures and bulk
solution in comparison to monomolecular and bimolecular G-quadruplexes.[3]
[1] M. Webba da Silva, M. Trajkovski, Y. Sannohe, N. Ma’ani Hessari, H. Sugyiama, J.
Plavec, Angew. Chem. Int. Ed. 2009, 48, 916.
[2] M. Trajkovski, P. Šket, J. Plavec, Org. Biomol. Chem. 2009, 7, 4677.
[3] P. Šket, J. Plavec, J. Amer. Chem. Soc. 2010, accepted.
113
Poster
Mining databases for the analysis of catechol and
azanaftalene polypharmacology
Agata Kurczyk, Pawel Mazur, Barbara Janik, Andrzej Bak,
Tomasz Magdziarz, Jaroslaw Polanski*
Institute of Chemistry, University of Silesia, Katowice, Poland, [email protected]
In recent years chemoinoformatics has seen an explosion of molecular information
resources available, e.g. more than 50 million compounds were synthesized and
catalogued. Moreover, in silico molecular simulations, an increasingly important
component of current medicinal chemistry, further contributes to this. A number of
molecular databases are publicly available and can be used in drug design, e.g.
PubChem and ZINC databases contain ca. 37 and 8 mln compounds, respectively.
Here we report an application of a novel and unique molecular and structural
database managing system, MoStBioDat1, available as a public domain package2.
for the analysis of large ligand libraries. MoStBioDat is not only the dual purpose
storage/extraction database platform maintaining the high-standards of data
integrity and reliability, but consistent environment providing software-based
solutions for the massive in silico protocols parallely analyzing small molecule
ligand and protein data.
Thus, we analyzed intramolecular hydrogen bonded motifs in catechols searching
within the combined data of available databases. This revealed topological
incoherence among accessible structural and molecular data, suggesting that one
should exercise special caution while analyzing data resulting from molecular
database mining in HTS applications involving hydrogen-bonding effects.
The concept of privileged structures is an idea in medicinal chemistry that certain
structural features produce biological effects more often than others. By screening
databases we can estimate the population of such (sub)structural motifs. However,
we cannot be sure if overpopulation of a certain structural feature, in fact, does
result from its real polypharmacological advantages in biological systems or a
chemist synthetic preferences. Thus, in another MoStBioDat application we
compared the results of synthetic vs. drug databases screening for the analysis of
the privileged azanaftalene motifs.
[1] A. Bąk, J. Polański, A. Kurczyk, Molecules, 2009, 14, 3436; A. Bąk, J. Polański,
T. Stockner and A. Kurczyk, Comb. Chem. & HTS., 2010, 13, 366;
www.chemoinformatyka.us.edu.pl.
[2] A. Bak, MoStBioDat; www.chemoinformatyka.us.edu.pl.
114
Poster
Cloning and molecular analysis of katG genes from the soil fungi
Chaetomium globosum and Chaetomium cochliodes
Mária Bučková1, Jana Godočíková1, Marcel Zámocký1,2,
Christian Obinger2, Bystrík Polek1
1
Institute of Molecular Biology, Slovak Academy of Sciences, SK–84551 Bratislava,
Slovakia Email: [email protected]
2
BOKU, Muthgasse 18, A–1190 Vienna, Austria
Catalase-peroxidases are bifunctional heme peroxidases involved in efficient dismutation
of hydrogen peroxide and are found in majority of microorganisms obtained from
environmental samples, particularly from contaminated soils. Previously, we have shown
the occurrence of multiple KatG isoenzymes in the Comamonadaceae bacteria isolated
from waste waters and contaminated soil [1]. In further investigations we have focused on
the occurrence of katG genes in ascomycetous soil fungi from the order Chaetomiaceae.
Two complete genes were cloned and sequenced from Chaetomium globosum and
Chaetomium cochliodes. Detailed sequence analysis of these intronless genes coding for
intracellular catalase-peroxidases confirmed the previously formulated hypothesis on
horizontal gene transfer of ancestral katG gene(s) between Bacteroidetes and
Ascomycetes [2]. Our recent qRT-PCR results reveal inducibility of Chaetomia katG
expression mainly with peroxyacetic acid and paraquat. Furthermore, we have analysed
the subcellular location of KatG and found it mainly in the peroxisomal fraction in
accordance with the presence of peroxisomal targeting signal (PTS1) in both obtained
Chaetomia katG sequences on their C-termini. The importance of observed subcellular
location is underlined with the fact that the fungus Chaetomium cochliodes is a producer of
the rare antibiotics chaetomin highly active against Gram-positive bacteria. It was shown
recently that final steps of biosynthesis of related antibiotics are located in peroxisomes [3].
We are currently analyzing the genomic region preceding katG gene in the genomic DNA
of both mentioned sac fungi to get hints on the presence and location of promoter regions.
This analysis will explain the inducible regulation of catalase-peroxidase expression in
response to various oxidative stress stimuli.
[1] J. Godočíková, M. Zámocký, M. Bučková, C. Obinger, B. Polek, Arch. Microbiol. 2010,
192, 175–184.
[2] M. Zámocký, P.G. Furtmüller, C. Obinger, Bioch. Soc. Transact. 2009, 37, 772–777.
[3] M. J. Koetsier, A. K. Gombert, S. Fekken, R. A. Bovenberg, M. A. van den Berg, J. A.
Kiel, P. A. Jekel, D. B. Janssen, J. T. Pronk, I. J. van der Klei, J. M. Daran, Fungal
Genet. Biol. 2010, 47, 33–42.
Acknowledgments This work was supported by Grants APVV-0444-07 and VEGA
2/0084/08
115
Poster
Interaction of the LC8 dynein light chain with myosin Va
and EML3: structural studies
László Radnai1, Péter Rapali1, Alíz Tichy-Rács1, Csaba Hetényi2,
Veronika Harmat3, Weixiao Wahlgren4, Gergely Katona4,
László Nyitray 1*
1
Dept. of Biochemistry, 2Dept. of Genetics, and 3Inst. of Chemistry, Eötvös Loránd
University, Budapest, Hungary, 4Dept. of Chemistry, University of Gothenburg, Sweden
[email protected]
LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic protein with
dozens of binding partners. Initially it was described as a subunit of the dynein and
myosin Va (myoVa) motor proteins, and supposed to play a cargo adaptor role.
Recent results suggest that DYNLL is a hub protein regulating other proteins by
complex formation. Eight residues linear peptides fit into the two parallel binding
grooves formed at the opposite edges of the subunit interface of the homodimeric
protein. The binding motifs have diverse sequences and affinities to DYNLL (Kd
from 100 nM to 50 μM). The most conserved residue is a Gln, which is substituted
by Met in myoVa. The binding partners are usually dimeric in vivo; therefore, they
bind to DYNLL as bivalent ligands with high avidity. Here we present the 3D
structures of two DYNLL-partner complexes solved by X-ray crystallography. The
structure of the binding motif of myoVa complexed to DYNLL (1.8 Å resolution)
shows that this non-canonical peptide binds into the same groove as other
partners. This fact indicates that DYNLL holds the two heavy chains of myoVa in
close proximity, thereby stabilizing its coiled-coil tail region and possibly regulating
its transport functions; however, it seems unlikely that DYNLL could be a cargo
adaptor. Reduced affinity of myoVa compared to the canonical motifs can be
attributed to the loss of one hydrogen bond of the conserved Gln. A novel DYNLL
binding partner, EML3 (echinoderm microtubule associated protein like) was
identified by an in vitro evolution method. The EML3 peptide has the highest know
affinity to DYNLL. We present crystal structures of the DYNLL-EML3 peptide
complex (1.3 Å resolution) and an artificially dimerized (by a Leu-zipper) EML3
motif complexed to DYNLL (2.9 Å resolution). The EML3 motif binds to DYNLL by
forming the usual antiparallel β-strand. Based on these structures and molecular
dynamics simulations we conclude that an optimal hydrogen bond network
between the motif and the binding groove as well as interaction between a Val at
the N-terminal end of the motif and a His sidechain of DYNLL are responsible for
the increased stability of this complex. (Supported by OTKA K61784, NK81950)
116
Poster
In vitro evolution of dynein light chain (DYNLL) binding
peptides via phage display
Peter Rapali, Laszlo Nyitray, Gabor Pal*
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary
[email protected]
The highly conserved homodimer dynein light chain (DYNLL) is a eukaryotic hub
protein. It is a micromolar binder of linear epitopes having the loose consensus
sequence, [DS]KX[TVI]Q[TV][DE]. The binding motif is frequently located in
intrinsically disordered regions in the vicinity of coiled coil structures. DYNLL might
be a dimerization engine regulating its partners involved in cancer development,
transcription regulation and apoptosis. Gene knockout or knockdown of DYNLL
causes cell death through apoptosis suggesting that it should be a potential drug
target protein. In order to explore weather the binding motif of DYNLL is
thermodynamically optimal we applied an in vitro evolution approach, phage
display. A naive peptide library was displayed on M13 phage in a bivalent manner
using a Leu-zipper. Seven positions were totally randomized, while the naturally
conserved glutamine was fixed (XXXXXQXX). The in vitro selected consensus
sequence, VSRGTQTE is similar to the natural one, but is extended by an
additional binding determinant, a Val, which increases the affinity tenfold.
Dimerization through the Leu-zipper further increases the affinity into the sub
nanomolar range. Interestingly, we identified a human protein, EML3 that contains
the phage-selected consensus sequence located in a disordered region directly Cterminal to a coiled coil structure. The results show that natural evolution of the
DYNLL binding motif was driven towards a biologically rather than
thermodynamically optimal affinity. Nevertheless, the highest affinity binding motif
evolved by phage display does exist at least in one human protein. The phageselected peptide presented here could be used as an in vivo molecular trap and/or
as a competitive inhibitor for therapeutic purposes.
117
Poster
A bacterial model for identification of putative
transporters
Katalin Revesz, Tamas Meszaros, Miklos Csala
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Budapest,
Transport processes greatly influence the activity of enzymes localized in the
lumen of the endoplasmic reticulum (ER). The majority of these transport
processes have been found to be protein-mediated. However, attempts to identify
the transporter proteins have only been successful in a few cases.
Conjugation with glucuronate, an important step of biotransformation, is catalyzed
by the UDP-glucuronyl-transferases in the lumen of the ER. The conjugated
products are polar and charged compounds, which are known to leave the ER
lumen via yet-unidentified glucuronide transporters.
Our aim was to develop a bacterial model system, which is suitable for
identification of membrane proteins having glucuronide transporter activity.
However, the bacterial model described in this study may also be used as a
general tool to examine transporter proteins.
Several human transporters have been identified through bacterial homology. Our
work is based on the known sequence of the bacterial glucuronide permease
encoded by the gusB gene of E. coli.
We found a putative protein in the human EST database, which has 38 % amino
acid sequence similarity to GusB. In silico analysis predicted the human protein to
be a transmembrane protein having a conserved domain typical to carbohydrate
transporters and an ER retention signal.
We used E. coli strains bearing a null mutation in the chromosomal gusB gene and
plasmid constructs for expression of wild type or loss-of-function point mutant gusB
used as a positive and negative controls, respectively. An E. coli strain expressing
the putative human glucuronide transporter has been also constructed.
The transporter activity is assessed in these bacteria using a variety of artificial
glucuronides. Once the expressed protein has glucuronide transporter activity, the
substrates enter the cells and are hydrolyzed by the cytoplasmic glucuronidase
enzyme. Deconjugated aglycones can be easily detected by photometry,
fluorimetry or HPLC. After validation of the positive and negative control cells, the
transporter activity of the human protein will be investigated.
118
Poster
Synthesis and DNA/RNA binding studies of cationic
peptides
Giovanni N. Roviello*, Domenica Musumeci, Cristian D’Alessandro,
Enrico M. Bucci, Carlo Pedone
Istituto di Biostrutture e Bioimmagini – CNR, Naples, Italy, [email protected]
Several polyamino acids are known to act as nucleic acid compacting devices and
in some cases show also a significant antimicrobial activity[1]. In this work we
describe the synthesis, purification and characterization of a novel positivelycharged peptide based on modified amino acids suitable for the Fmoc solid phase
synthesis. Furthermore some structural and interaction characteristics of this
polycationic peptide were investigated in order to explore its possible application in
biotechnology.
[1] M. Takehara, M. Saimura, H. Inaba, H. Hirohara, FEMS Microbiol. Lett. 2008, 286,
110–117.
119
Poster
Structure and dynamic studies of exendin-4: A new model
for ligand-receptor interaction
Petra Rovó1, Viktor Farkas1, Pál Stráner1, Beáta Huszka1,
András Perczel1,2*
1
Eötvös University, Institute of Chemistry, Structural Chemistry and Biology Laboratory,
2
Eötvös University, Institute of Chemistry, Protein Modelling Group, Budapest, Hungary,
[email protected]
Exendin-4 (Ex-4) a 39-amino-acid peptide secreted by the lizard Heloderma
suspectum displays similar receptor-binding properties as the glucagon-likepeptide-1 (GLP-1) hormone[1]. Both Ex-4 and GLP-1 bind the GLP-1 receptor and
potentiate insulin secretion by pancreatic β-cells in a glucose-dependent manner.
Since Ex-4 posses a longer in vivo half-life than GLP-1 due to its resistance for the
protease dipeptidyl peptidase IV (DPP IV) Ex-4 and its analogues have received
much attention as potential tool for the treatment of Type 2 diabetes.
We have used NMR and ECD spectroscopy to investigate the structural and
dynamical properties of Ex-4 in free and in a receptor bound mimicking state.
Identification of important segments with characteristic dynamical motion was
accomplished by the substitution of amino acids at the sites that seems to
influence the overall structure and dynamics of Ex-4. In order to further understand
the nature of receptor-peptide interaction, we designed a variety of truncated
analogues to quantify the contribution of each dynamical segment in the structure
stabilization that highly determine the receptor-binding affinity.
Our data show that the free form of Ex-4 exhibits three distinct dynamic regions:
the unstructured N-terminal region (residue 1-9), the mostly structured helical
region (residue 10-28) and the less regular and more fluxional C-terminal region
(residue 29-39). In 30 vol-% trifluoroethanol the dynamics of the N-terminal
segment is unchanged while both the helix and the C-terminal region gain more
rigidity forming the helix-stabilizing C-cap structure known as Trp-cage. With the
mutational stabilization of the Trp-cage segment of Ex-4 the overall fold stabilizes
and acquires similar dynamical properties as Ex-4 has in the flouroalcohol medium.
Our findings suggest a more delicate mode of receptor-binding of Ex-4.
[1] R J. Eng, W. A. Kleinman, L. Singh, G. Singh, J. P. Raufman, J. Biol. Chem. 1992, 267, 7402.
120
Poster
A comparison of drug-binding ability of bovine and
human serum albumin using fluorescence spectroscopy
Joanna Równicka-Zubik1, Anna Sułkowska1, Iwona Zubik-Skupień1,
Agnieszka Szkudlarek1, Małgorzata Maciążek-Jurczyk1,
Barbara Bojko1, Wiesław W. Sułkowski 2
1
Department of Physical Pharmacy, Faculty of Pharmacy, Medical University of Silesia,
Jagiellońska 4, 41–200 Sosnowiec, Poland, [email protected],
2
Department of Environmental Chemistry and Technology, Institute of Chemistry,
University of Silesia, Szkolna 9, 40–006 Katowice, Poland, [email protected]
Determination of the binding parameters such as binding constants, quenching
constants, number of binding sites for the independent class of binding sites or
fractional accessible protein fluorescence for sulindac (SDK) in complex with
human (HSA) or bovine serum albumin (BSA) were studied using fluorescence
technique. Sulindac is a non-steroidal anti-inflammatory drug useful in the
treatment in acute or chronic inflammatory conditions. Serum albumin, the most
abundant of the plasma protein, plays a main function in transport of various
endogenous and exogenous compounds such as bilirubin, hormones, drugs etc.
We compare binding parameters for the system BSA-SDK and HSA-SDK excited
both 280 nm and 295 nm. Binding (KB) and quenching (K Q) constants for analyzed
complexes were calculated on the basis of Scatchard and Stern-Volmer method,
respectively. The more intensive interaction was observed in the complex BSASDK (KB= 95.24x103 [M–1] nm and the K Q=45.18x103 [M–1]). The results suggest
that not only Trp 214 occurring in both albumins BSA and HSA was engaged in
the interaction with sulindac but also tryptophan located in 135 position in BSA
molecule.
For all analyzed systems one class of binding site was found. For complex HSASDK a binding site of SDK was determine in subdomain IIA and for BSA-SDK – in
subdomain IIA and/or IB.
121
Poster
Determination of the binding site of antirheumatoid drugs in
serum albumin. Fluorescence and 1H NMR studies
Małgorzata Maciążek-Jurczyk1, Anna Sułkowska1,
Joanna Równicka-Zubik1, Agnieszka Szkudlarek1, Barbara Bojko1,
Wiesław W. Sułkowski2
1
Department of Physical Pharmacy, Medical University of Silesia, Sosnowiec, POLAND,
[email protected].
2
Department of Enviromental Chemistry and Technology, Institute of Chemistry, University
of Silesia, Katowice, POLAND, [email protected]
The monitoring of a drug concentration in blood serum is necessary in multidrug
therapy. The aim of the study was to determine binding sites, a strength and kind
of interaction between human (HSA) and bovine (BSA) serum albumin and
methotrexate (4-amino-10-methylfolic acid, MTX) and acetylsalicylic acid (2 –
acetoxybenzoic acid, aspirin, ASA) used in combination therapy of rheumatoid
arthritis (RA) and accompanying cardiovascular disorders. Analysis of binary and
ternary systems in high and low affinity binding sites was made with the use of
quenching fluorescence technique and proton nuclear magnetic resonance ( 1H
NMR), respectively. Fluorescence analysis showed that both MTX and ASA formed
complex in the high affinity binding site of SA in subdomain IIA. For each site the
drug – serum albumin binding parameters in the absence and presence of the
second drug were estimated. The analysis of the quenching and association
constants suggests that the competition between MTX and ASA occurs in a
primary binding site in subdomain IIA. The analysis of 1H NMR spectra of MTX and
ASA in the presence of serum albumin allowed for observation of π–π interactions
between aromatic rings of the drugs and the rings of amino acids located in
hydrophobic subdomain of the protein. On the basis of obtained data the
stoichiometric molecular ratio values characterizing the equilibrium of the complex
were also evaluated. The analysis of parameters obtained from 1H NMR spectra of
ternary system pointed to the influence of particular drug on interaction between
the second drug and serum albumin in low affinity binding site.
Methotrexate and aspirin can compete for the same or different binding sites. The
competition can lead to the increase of a free fraction of drugs and then it causes
the enhancement of their adverse effect.
122
Poster
Interaction of plasmalogens with singlet oxygen and
oxidizing free radicals
Agnieszka Broniec1, Anna Pawlak1, Marta Wrona-Król1, Andrzej Żądło1,
David H. Thompson2, Tadeusz J. Sarna 1*
1
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Krakow, Poland, [email protected]
2
Department of Chemistry,
[email protected]
Purdue
University,
West
Lafayette,
Indiana,
USA,
Plasmalogens are unique glycerophospholipids that contain in the sn-1 position
vinyl ether bond. Although it has been postulated that plasmalogens are
physiological antioxidants, the exact mechanism of their antioxidant action remains
mostly unknown.
In this study, we examined the effect of several
plasmenylcholines on oxidation of their unsaturated diacyl analogs and cholesterol,
induced by singlet oxygen and free radicals in models systems, by measuring the
accumulation of cholesterol hydroperoxides using HPLC-EC(Hg), and by
monitoring rates of oxygen consumption using ESR-oximetry and oxygen
electrode. Rates of the interaction of plasmenylcholines with singlet oxygen and
free radicals were determined by measuring lifetimes of these “reactive oxygen
species” using time-resolved singlet oxygen phosphorescence and pulse
radiolysis.
Our data show that plasmenylcholines quenched singlet oxygen with the rate
constants being one-two orders of magnitude higher than those observed for the
other lipids. The interaction was primarily chemical in nature. Only strongly
oxidizing radicals, such as bromide radical, exhibited much higher reactivity with
plasmenylcholine than with methyl esters of polyunsaturated fatty acids. No
difference in reactivity of plasmenylcholines and their diacyl analogs were
observed when relatively mild peroxyl radicals were tested. Plasmenylcholines,
present in liposomal membranes at concentration up to 30 mol%, either did not
inhibit radical-induced peroxidation of the membrane cholesterol or PUFA, or
actually accelerated it.
In conclusion, antioxidant action of plasmalogens in membranes to protect against
photosensitized oxidation is mostly due to the ability of their vinyl ether bond to
interact with singlet oxygen.
Supported in part
2040/B/P01/2007/33)
by
Ministry
of
Science
and
Higher
Education
(grant
123
Poster
Hydroxypyridinecarboxylic acids as possible chelating
agents in the therapy of neurodegenerative disorders
Éva Sija1*, Annalisa Dean2, Tamás Kiss1
1
Department of Inorganic and Analytical Chemistry, University of Szeged, P.O. Box 440,
H–6701 Szeged, Hungary, [email protected]
2
Department of Chemical Sciences, University of Padova, via Marzolo 1, I–35131 Padova,
Italy
Several neurodegenerative diseases are characterized by modified metal
ions homeostasis. The biochemical processes of these diseases are very
complicated, however, the metal ions have very important roles in the development
of illnesses. Restoring the balance of metal ion homoeostasis may result significant
improvement in the treatment. Regulating the concentrations of metal ions by
suitable chelators could be the key step in the process.
The Alzheimer’s disease (AD) is one of the main neurodegenerative
disorders. The hallmark of AD is the accumulation of amyloid-β peptide in the
brain. Amyloid-β peptide (Aβ) originates from the larger amyloid precursor protein
(APP)[1]. The peptide misfolding and oligomerisation of produced peptides are
induced by metal ions. The Aβ aggregates can be cytotoxic influence therefore
they initiate the axon degradation. The formed supramolecular structures can
generate reactive radicals and H2O2 through a metal dependent reactions[2].
In this present work we have performed chemical measurements to evaluate
the examined hydroxypyridinecarboxylic acids as new possible metal targeting
agents in the therapy against neurodegenerative diseases. Thermodynamic
studies are aimed to determine the number, the stoichiometry and the stability
constant of the metal (Fe3+, Al3+, Cu2+ , Zn2+)- chelator complexes in aqueous
solutions. The formations of metal-chelator complexes are characterized by pHpotentiometric titrations.
Having been the most promising drug candidates selected, as the next step,
information about their fate in the biological system is needed. As they intended as
oral drugs, after absorption, they are expected to be transported in blood stream.
Their binary interactions with the plasma proteins primarily with albumin might be
important. They have been determined by UV-Vis and fluorescence
measurements.
Ackowledgements. The work was supported by the Hungarian Research Fund (OTKA K77833)
[1] H. Vural, H. Demirin, Y. Kara, I. Eren, N. Delibas, J. Trace Elem. Med. Bio. 2010, 24, 169–173.
[2] M. P. Cuajungco, K. Y. Fagét, Brain Res. Rev. 2003, 41, 44–56.
124
Poster
Protein chip based interactome analysis of Aβ indicates
an inhibition of the cellular translation machinery
Dóra Simon1*, Lívia Fülöp1, Zsolt Bozsó1, Róbert Rajkó2, Zsolt László
Datki3, Tamás Janáky1, Botond Penke1,3, Dezső Virók3
1
Department of Medical Chemistry, University of Szeged, Szeged, Hungary,
[email protected]
2
Faculty of Engineering, University of Szeged, Szeged, Hungary
3
Institute for Plant Genomics, Human Biotechnology and Bioenergy (BAY–GEN), Szeged,
Oligomeric amyloid beta is currently in the focus of amyloid beta (Aβ) mediated
toxicity and the pathogenesis of Alzheimer's disease. Mapping the amyloid beta
interaction partners could help to discover novel pathways in AD pathogenesis. To
identify the amyloid beta interaction partners we applied a protein chip with 7000
unique recombinantly expressed human proteins. The protein chip results showed
that oligomeric amyloid beta binds to multiple proteins. This promiscuous binding
phenotype indicates that multiple protein interactions mediate the toxicity of the
oligomeric amyloid-beta at the plasma membrane and also in intracellular
compartments. Gene ontology functional analysis of the binding partners showed
that one of the most highly impacted cellular systems was the protein translation
machinery. The interaction of oligomeric amyloid beta with ribosomes was
confirmed by ELISA and the translation inhibitory activity was demonstrated in an
in vitro translation system.
125
Poster
Does nonplanarity of nucleic acid bases affect NMR
parameters?
Zuzana Sochorová Vokáčová1, Lukas Trantírek2, Vladimir Sychrovský3
1
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, Prague, Czech Republic, [email protected].
2
Department of Chemistry, Utrecht University, Utrecht, The Netherlands.
The glycosidic torsion angle χ is used as a measure of nucleobase orientation with
respect to sugar ribose in nucleosides and it is one of the major determinants of
nucleic acid structure. The deformations from planar nucleobase arrangement in
purine and pyrimidine nucleosides due to variation of the glycosidic bond were
investigated with the computational methods and the calculated trends were
correlated with the high resolved X–ray data[1].
The Karplus equation describes the correlation between value of 3J–coupling and
corresponding dihedral angle in nuclear magnetic resonance spectroscopy.[2] Our
study showed that angular argument of Karplus equations for the 3J(C8/6–H1’) and
J(C4/2–H1’) couplings assigned to the χ torsion, must reflect deformation from
planar spatial arrangement of atoms around nitrogen N9/1 involving in the χ
torsion. We showed that also deformation from ideal tetrahedric arrangement of
atoms around carbon C1’ must be reflected. (Conception of ideal tetrahedric and
planar spatial arrangement consist in using of constant phase shift in Karplus
equation.[3]).
3
Our study showed that out-of-plane deformation (pyramidalization) depends
significantly on the χ torsion, namely the orientation of the pyramidalization (up and
down). These deformation affect mostly the phase factor in the Karplus equation
for structural interpretation of the 3J(C8/6–H1’) coupling. The 3J(C4/2–H1’) coupling
is effected slightly and only by deformation at the C1’ arrangement.
[1] V. Sychrovský, S. Foldýnová-Trantírková, N. Špačková, K. Robeyns, L. Van Meervelt,
W. Blankenfeldt, Z. Vokáčová, J. Šponer, L. Trantírek, Nucleic Acids Res. 2009, 37, 7321–
7331.
[2] M. Karplus, J. Am. Chem. Soc. 1963, 85, 2870
[3] S. S. Wijmenga, B. N. M. van Buuren, Prog. NMR Spectrosc. 1998, 32, 287.
126
Poster
Influence of the Primary Structure of Enzymes on the
Formation of CaCO3 Polymorphs: A Comparison of Plant
and Bacterial Ureases
Ivan Sondi1*, Branka Salopek-Sondi 2
1
Center for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb,
Croatia, [email protected]
2
Department for Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia,
[email protected]
Precipitation processes of carbonate particles of different size, shape, and surface
properties have been intensively investigated due to their importance in geo- and
biosciences, and in numerous industrial applications. This study describes and
discusses a conceptually new method for the precipitation of calcium carbonate
polymorphs, effectuated through catalytic decomposition of urea by urease
enzymes in solutions of calcium salts [1–2]. Specifically the influence of primary
structures of plant (Canavalia ensifomis) and bacterial (Bacillus pasteurii) ureases
on the nucleation and growth of carbonate precipitates was investigated. Despite
the same catalytic function in decomposition of urea, these ureases exert different
influence on the crystal phase formation and on development of unusual
morphologies of calcium carbonate polymorphs. These differences may be
explained by dissimilarity in amino acid sequences of two examined ureases and
their different role in nucleation and physico–chemical interactions with the surface
of the growing crystals [3–4]. Finally, this study has demonstrated an example how
different organisms in nature have ability to produce proteins with the same
function, and how with the slight manipulation in their primary structure they take
control over the crystal phase formation and growth processes of biogenic calcium
carbonate precipitates.
[1] I. Sondi, E. Matijević, J. Colloid. Interface Sci. 2001, 238, 208–214.
[2] I. Sondi, E. Matijević, Chem. Mater. 2003, 15, 1322–1326.
[3] I. Sondi, B. Salopek-Sondi, Langmuir. 2004, 21, 8876–8882.
[4] I. Sondi, S. D. Škapin, B. Salopek-Sondi, Cryst. Growth. Des. 2008, 8, 435–441.
127
Poster
Stability engineering of the Fc fragment of human IgG1 by
targeted mutagenesis
Gerhard Stadlmayr1,*, Gordana Wozniak-Knopp2,*,
Christoph Hasenhindl1, Florian Rüker2, Christian Obinger1
1
Department of Chemistry, BOKU University of Natural Resources and Applied Life
Sciences Vienna, Division of Biochemistry, Christian Doppler Laboratory for Antibody
Engineering, Muthgasse 18, 1190 Vienna, Austria, e–mail: [email protected]
2
Department of Biotechnology, BOKU University of Natural Resources and Applied Life
Sciences Vienna, Christian Doppler Laboratory for Antibody Engineering, Muthgasse 18,
1190 Vienna, Austria
These authors contributed equally to this work
Monoclonal antibodies (mAbs) are the most successful biologically produced
therapeutics today. FcabTM, the antigen binding IgG1-Fc fragment turned out as an
alternative small-size antibody format [1]. In silico-guided mutagenesis of the Fc
fragment based on the FOLD X algorithms [2] was applied with the aim to generate
a more robust scaffold for further loop engineering applications. Single mutations
and combinations of mutations were introduced by site-directed mutagenesis and
expressed in Pichia pastoris, purified and investigated for biophysical properties.
Biophysical analysis included differential scanning calorimetry (DSC), circular
dichroism spectrometry (CD) and the verification of the presence of wild-type like
effector functions like binding to Protein A, CD16a, and FcRn.
Fcab is a trademark of f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H.
[1] G. Wozniak-Knopp, S. Bartl, A. Bauer, M. Mostageer, M. Woisetschlager, B. Antes, K.
Ettl, M. Kainer, G. Weberhofer, S. Wiederkum, G. Himmler, G. C. Mudde, F. Ruker,
Protein Eng Des Sel. 2010, 23, 289–97.
[2] R. Guerois, J. E. Nielsen, L. Serrano, J. Mol. Biol. 2002, 320, 369–87.
128
Poster
Enhancing solubility of proteins isolated from inclusion
bodies: Expression and NMR study of the GLP-1 receptor
Pál Stráner1,2, András Perczel1,2
1
University, H–1117 Budapest, Hungary, [email protected]
2
Protein Modeling Group MTA-ELTE, Institute of Chemistry, Eötvös Loránd University,
P.O. Box 32, H–1538 Budapest, Hungary
The glucagon-like-peptide-1 receptor (GLP–1R) belongs to Family B1 of the seven
transmembrane G protein-coupled receptors and its natural agonist ligand is the
paracrine hormone GLP–1. It is secreted by the duodenum in response to high
glucose levels, which subsequently triggers the release of insulin from pancreatic
cells. The ability of GLP–1 to induce insulin secretion in dependence on high
glucose level renders this component potentially useful in the treatment of noninsulin dependent diabetes mellitus. The extracellular domain of the GLP–1
receptor (nGLP–1R) can specifically bind this peptide[1].
When expressed in Escherichia coli, nGLP–1R accumulates in insoluble inclusion
bodies. The protein refolded in vitro, gives correctly folded state, but it is a
roundabout and inefficient step. Therefore we choose thioredoxin as a fusion
partner of the nGLP–1R. Thioredoxin is a small monomeric protein which facilitates
the soluble and biologically active expression of a number of mammalian growth
factors and cytokines[2]. Furthermore, the expression of the nGLP–1R ligands was
also achieved by an ubiquitin fusion system. Here we demonstrate that the
thioredoxin fusion system increases the yield of soluble protein after refolding. We
are also presenting early biophysical (VCD, NMR) results on nGLP–1R and its
interaction with ligands.
.
[1] C. R. Underwood, P. Garibay, L. B. Knudsen, S. Hastrup, G. H. Peters, R. Rudolph, S.
Reedt-Runge, J. Biol. Chem. 2010, 285, 723–730.
[2] D. Sachdev, J. M. Chirgwin, Protein. Expr. Purif., 1998, 12, 122–132.
129
Poster
Phage Display of Single Chain Ecotin on the Surface of
M13
Dávid Szakács, Dávid Héja, Gábor Pál*
Department of Biochemistry, Eötvös University, 1117 Pázmány Péter sétány 1/c,
Budapest, Hungary
*Corresponding author: [email protected].
Ecotin is a periplasmic, homodimer, serine-protease inhibitor protein from E. coli.
Ecotin inhibits several very different serine-proteases (e.g. trypsin, chymotrypsin,
elastase, urokinase and fXa) with KI values of 10–9 – 10–13M. Such potent inhibition
of proteases having such different specificities is a unique feature of ecotin among
the family of canonical, substrate-like serine-protease inhibitors. The ecotin
monomer has two different protease binding sites: a primary and a secondary site.
One ecotin dimer binds two protease molecules simultaneously. In the complex,
both proteases contact both ecotin monomers. Each protease binds to the primary
site of one monomer and to the secondary site of the other monomer. This
interaction network appears to be responsible for the “pan-specificity” of ecotin.
Combinatorial mutagenesis combined with directed evolution using phage display
would be an ideal approach to elucidate the roles of individual binding site residues
on ecotin. However, such an approach requires a system, which guaranties that
ecotin is displayed on phage in a native-like homodimer form. We created a
recombinant Single Chain (SC) ecotin variant where the C-terminus of one
monomer is connected to the N-terminus of the other monomer with a suitable
linker. The SC ecotin is functionally equivalent with the wild type inhibitor. We
tested the display efficiency of SC ecotin in all combinations of two types of
promoters and two types of coat proteins. We demonstrated that SC ecotin is
functionally displayed on phage. As we aimed to mutate the two halves of the SC
ecotin gene independently using synthetic mutagenesis oligos, we constructed an
SC ecotin gene, in which one half of the SC ecotin gene contained a near maximal
amount of silent mutations. We confirmed that in this way we can indeed mutate
the two halves of SC ecotin independently. Thus, we developed a display system,
with which the background of the unique “pan-specificity” phenomenon can be
tackled using directed evolution of the primary and secondary binding sites. In
addition, the system will allow for evolving ecotin variants with altered specificity
against various proteases. Most importantly, this way novel inhibitors can be
evolved against proteases, which have no known selective natural inhibitors.
130
Poster
Strength of individual hydrogen bonds in G-C and A-T
Crick-Watson base pairs
Halina Szatyłowicz1*, Nina Sadlej-Sosnowska2
1
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00–664
Warsaw, Poland, Email: [email protected]
National Medicines Institute, 30/34 Chełmska Street, 00–725 Warsaw, Poland, Email:
[email protected]
2
From the point of view of medicinal and supramolecular chemistry, the nucleic acid
base pairs seem to be the most significant systems with multiple H–bonds. Two
aspects of the H–bond “network” should be distinguished: (i) strength of the
individual interactions and (ii) cooperativity (anticooperativity) effects.
Strengthening of the H–bonds within guanine-quartet network relative to those in
the isolated dimer is an example of the cooperative effect.[1] Enhancement of the
strength of the intermolecular interactions was confirmed by evaluation of the
average energy per single hydrogen bond. Therefore, the question concerning
contributions of the individual H–bonds to the total energy of the system is of
paramount importance.
H
O
N
a
H
H
N
H
H
H
N
H
N
H
b
H
N
N
N
N
N
H
c
O
H
Guanine-Cytosine (G-C)
N
N
H
H
N
N
H
a
H
b
CH3
O
H
H N
c
N
O
H
Adenine-Thymine (A-T)
In order to disentangle the particular contributions, several different philosophies
have been applied[2] but the obtained results significantly differ. A new approach to
the estimation of the individual H–bonds’ energies in the A-T and G-C WatsonCrick base-pairs, based on the Natural Bond Orbital[3] analysis, is proposed.
[1] R. Otero, M. Schöck, L.M. Molina, I. Lœgsgaard, I. Stensgaard, B. Hammer, F.
Besenbacher, Angew. Chem. Int. Ed. 2005, 44, 2270–2275.
[2] A. Asensio, N. Kobko, J.J Dannenberg, J. Phys. Chem. A 2003, 107, 6441–6443. J.
Grunenberg, J. Am. Chem. Soc. 2004, 126, 16310–16311. H. Dong, W. Hua, S. Li, J.
Phys. Chem. A 2007, 111, 2941–2945.
[3] F. Weinhold, C.R. Landis, Valency and Bonding. A Natural Bond Orbital Donor–
Acceptor Perspective, Cambridge University Press: Cambridge, 2005.
131
Poster
Functional models for catechol oxidase and
phenoxazinone synthase
Imola Cs. Szigyártó, László I. Simándi
Chemical Research Center, Hungarian Academy of Sciences, Institute of Nanochemistry
and Catalysis, Budapest, Hungary, [email protected]
Oxidase type metalloenzymes use dioxygen to perform vital catalytic oxidations in
living organisms. Catechol oxidase (E.C.1.10.3.1), this copper-containig dinuclear
enzyme catalyzes the oxidation of catechol derivatives to quinones. Catecholase
are also involved in the formation of melanin pigments and in the enzymatic
browning of fruits. Phenoxazinone synthase (also called o-aminophenol oxidase)
(EC 1.10.3.4), is a type 2 copper-containing oxidase isolated from Streptomyces
antibioticus. This enzyme is involved in the last stages of the biosynthesis of
Actinomycin D a naturally occurring antineoplastic agent, used clinically for the
treatment of certain types of cancer. Functional catecholase and o-aminophenol
oxidase models based on copper, iron, cobalt and zinc have been investigated to
shed light on the reaction mechanism involved and to help design „bioinspired”
catalyst systems for various reactions requiring activation of dioxygen.
We have extended our work using manganese(II) complexes as a functional model
system for oxidase enzymes. This dioximato dimer complex catalyzes the oxidation
of 3,5-di-tert-butylcatechol to the corresponding o-quinone, and the conversion of
2-aminophenol to 2-amino-3H-phenoxazinone-3-one[1–2]. Beside these activities we
are investigating the complex as a model of superoxide dismutase or catalase
enzymes.
[1] I. Cs. Szigyártó, L. I. Simándi, L. Párkányi, L. Korecz, G. Schlosser, Inorg. Chem. 2006,
45, 7480–7487.
[2] I. Cs. Szigyártó, T. M. Simándi, L. I. Simándi, L. Korecz, N. Nagy, J. Mol. Catal. A:
Chem. 2006, 251, 270–276.
Acknowledgement: This work was supported by the Hungarian Research Fund (OTKA
Grant K60241)
132
Poster
Binding of ibuprofen to bovine serum albumin
Anna Sułkowska1, Agnieszka Szkudlarek1, Joanna Równicka-Zubik1,
Iwona Zubik-Skupień1, Małgorzata Maciążek-Jurczyk1, Barbara Bojko1,
Wiesław W. Sułkowski2
1
Department of Physical Pharmacy, Faculty of Pharmacy, Medical University of Silesia,
Jagiellońska
4,
41–200
Sosnowiec,
Poland,
[email protected],
[email protected], [email protected], [email protected],
[email protected], [email protected]
2
Department of Environmental Chemistry and Technology, Institute of Chemistry,
University of Silesia, Szkolna 9, 40–006 Katowice, Poland, [email protected]
Bovine serum albumin (BSA) is a transporting protein which has the ability to bind
various exo- and endogenous ligands. BSA is a single-chain transporting protein of
581 amino acids with 20 tyrosyl residues, and two tryptophans located in position
135 and 214. It contains 17 disulfide bridges and free thiol group (Cys 34).
Ibuprofen ((RS)-2-(4-(methylpropyl)phenyl)propanoic acid; IBU) belongs to nonsteroidal anti-inflammatory drugs (NSAIDs). IBU is commonly used for relieving
muscular and skeletal pain. The binding of Ibuprofen with bovine serum albumin
was investigated by fluorescence spectroscopy. Two wavelength (λex = 280 nm
and λex = 295 nm) were used to have the possibility to observe tryptophanyl and
tyrosyl groups. On the basis of obtained data the interaction between BSA and IBU
was proved. Binding (KB) and quenching (K Q) constants for analyzed complexes
were calculated on the basis of Scatchard and Stern-Volmer method. Binding
constants for complex IBU-BSA equal to KB = 60.84x103 M–1 (for 280 nm) and the
KB = 85.59x103 M–1 (for 295 nm). Quenching constants for the same complex were
KQ = 10.67x103 M–1 and KQ = 1.90x103 M–1. IBU quenches the BSA fluorescence
by 27,68 and 21,98 for excitation λex = 280 nm and λex = 295 nm, respectively for
the molar ratio IBU:BSA 50:1. Ibuprofen binds to BSA in one class of binding sites
and a number molecule of IBU bound to one molecule of BSA equals 1.07 and
1.03 for λex = 280 nm and λex = 295 nm, respectively .
133
Poster
Synthesis and crystal structures of novel
pentacyclo[5.4.0.02,6.03,10.05,9]undecane hydrazones
Jelena Veljković, Marina Šekutor, Kata Mlinarić-Majerski*,
Krešimir Molčanov, Biserka Kojić-Prodić
Ruđer Bošković Institute, Bijenička cesta 54, PO Box 180, 10 002 Zagreb, Croatia,
[email protected]
It has been known for some time now that the toxicity of hydrazines is a limiting
factor in their use in medicine as they can cause severe liver degenerations. [1] As
polycyclic cage moieties have been extensively used to modify the potency of
biologically active compounds,[2] we plan to investigate a potential biological activity
of this type of compounds as we expect less toxic side effects in vivo due to their
pronounced lipophilic character.
NH2NPh2
aps MeO H
or EtOH
O
O
NPh2
NHNPh2
N NPh2
O
re fl ux, 5 h
+
NPh2
+
N
N
NHNPh2
+
N
N
N
NPh2
NPh2
NPh2
1
2
3a
3b
3c
Starting from diketone 1[3], pentacyclo[5.4.0.02,6.03,10.05,9]undecane-8,11-dione
bis(diphenylhydrazone) 3 was synthesized and the obtained mixture of isomers
was successfully separated and fully characterized. We also obtained the crystal
structures of isomers 3b and 3c, thus confirming our assumptions regarding the
configuration and stability of the isomers and thereby enabling the biological
studies with the pure isomers of a known structure.
[1] M. Bodansky, J. Biol. Chem. 1924, 58, 799–811.
[2] W. J. Geldenhuys, S. F. Malan, J. R. Bloomquist, A. P. Marchand, C. J. Van der
Schyf, Med. Res. Rev. 2005, 25, 21–48.
[3] A. P. Marchand, R. W. Allen, J. Org. Chem. 1974, 39, 1596.
134
Poster
Determination of the binding affinities of DNA with
chalcone derivates and their influence on mitochondria
Miroslava Štefanišinová1*, Mária Kožurková2, Vladimíra Tomečková1,
Mária Mareková1
1
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of
Medicine, PJ Šafárik University, Tr. SNP 1, 040 66 Košice, Slovak Republic,
[email protected].
2
Department of Biochemistry, Institute of Chemistry, Faculty of Science, PJ Šafárik
University, Moyzesova 11, 040 66 Košice, Slovak Republic.
Substituted chalcone (1) and its cyclic chalcone analogues: indanone ( 2), tetralone
(3), benzosuberone (4) with dimethylamino substituent in 2, 4 position are
interesting as drugs with antitumor activities [2]. The binding of small molecules to
DNA has been of great interest due to the understanding of the drug-DNA
interaction and designing of new efficient drugs targeted to DNA [1]. The purpose
of this study was to investigate the interaction of control, ischemic mitochondria
and DNA with novel ligands (1–4) using by spectroscopy methods. Interaction of
chalcone derivatives (1–4) with the outer mitochondrial membrane was
investigated by fluorescence polarization. Ligand - DNA binding affinities and
binding constants (K) of the DNA-drug complexes were determined by UV-Vis and
fluorescence and CD spectroscopy. CD experiment was performed to define the
orientation of the compounds with respect to the DNA helix and showed the
stabilization of the right-handed B-form of DNA by intercalation. The compounds
have no circular dichroism spectrum when are free in the solution but they induced
CD spectrum when they are in the complex with DNA. Generally, these
compounds bound to DNA, they show significant decrease of fluorescence and
bathochromic shift of excitation and emission maxima compared to the spectral
characteristics of the free form of ligands in solution phase. Binding constant (K)
was determined by spectrofluorimetric titrations. The strong hypochromism,
extensive broadening, red-shifting and increased stability of DNA double helix were
observed when these derivatives where bound to DNA.
VEGA 1/0402/10, 1/0053/08
[1] R. Langer, Science 2001, 293, 58.
[2] J. R. Dimmock, N. M. Kandepu, A. J. Nazarali, T. P. Kowalchuk, N. Motaganahalli, J.
W. Quail, P. A. Mykytiuk, G. F. Audette, L. Prasad, P. Perjési, T. M. Allen, C. L. Santos,
J. Szydlowski, E. De Clercq, J. Balzirini, J. Med.Chem. 1999, 42, 1358 – 1366.
135
Poster
Topological variability of G-quadruplexes in oncogenic
promoters region
KatarinaTluckova*, Lubos Bauer, Viktor Viglasky
Department of Biochemistry, Institute of Chemistry, Faculty of Natural Sciences, P. J.
Safarik University, Kosice, Slovakia, [email protected].
G-quadruplexes are highly stable alternative DNA structures formed by tetrads of
guanines that interact via Hoogsteen hydrogen bonds and are stabilized by
monovalent cations [1]. In the human genome, the number of distinct sites with a
potential to form G-quadruplex is estimated at more than 360 000[2]. G-quadruplexforming sequences have been identified in eukaryotic telomeres, as well as in nontelomeric genomic regions, such as gene promoters, recombination sites, and DNA
tandem repeats [3]. The main aim of interest is G-quadruplex structures that form in
gene promoter regions, which have emerged as potential targets for anticancer
drug development. This has led to suggestions that formation or resolution of
specific quadruplex structures may contribute to the regulation of gene expression,
and prompted the design of therapeutics targeted to these structures [4].
Here we present recent studies on G-quadruplex structures that form in the
promoter regions of some important protooncogenes (c-Myc, VEGF, Hif-1, Ret,
Kras, Bcl-2, c-Kit and PDGF). Our results were obtained by polyacrylamide gel
electrophoresis, temperature gradient gel electrophoresis and circular dichroism
spectroscopy at different salt conditions, different pH and presence of substances
imitating the cellular crowding condition (polyethylene glycol PEG 200).
The identification of G-quadruplex structures in promoter regions provides us with
new insights into the fundamental aspects of G-quadruplex topology and DNA
sequence structure relationships.
[1] S. Neidle, S. Balasubramanian, Quadruplex Nucleic Acids, RSC Publishing,
Cambridge, UK, 2006.
[2] J. L. Huppert, S. Balasubramanian, Nucleic Acids Res. 2005, 33, 2908–2916.
[3] A. K. Todd, M. Johnston, S. Neidle, Nucleic Acids Res. 2005, 33, 2901–2907
[4] L.H. Hurley, D.D. Von Hoff, A. Siddiqui-Jain, D. Yang, Semin. Oncol. 2006, 33, 498–
512.
136
Poster
The interactions of bis-phenanthridinium–nucleobase
conjugates with double stranded DNA
Lidija–Marija Tumir1*, Ivo Piantanida1, Marina Grabar2, Sanja Tomić2
1
Laboratory for Study of Interactions of Biomacromolecules, Division of Organic Chemistry
and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia, [email protected]
2
Laboratory for Chemical and Biological Crystallography, Division of Physical Chemistry,
Interactions of various intercalator–nucleobase conjugates with targeted
polynucleotides were the objective of intensive research in the last two decades,
whereby some of conjugates exhibited selective binding at complementary abasic
sites in double stranded DNA / RNA[1]. Study of previously prepared
bisphenanthridinium-nucleobase conjugates by spectroscopic methods and MD
stimulations revealed strong intramolecular stacking of phenanthridinium units, as
well as at least partial nucleobase insertion between them[2]. Recently, bisphenanthridinium–adenine derivative 3 showed not only high affinity towards
nucleotides (which is characteristic for analogous bis-intercalands[3]) but also
selectivity toward complementary nucleotide (UMP).[2]
N
N
NH
N
H2N
NH
N
NH
N
N
N
N
N
N
N
HN
N
O
HN
HN
N
HN
N
NH
O
NH2
N
N
2
N
NH2
N
1
NH
N
N
3
N
4
Figure 1. Phenanthridinium–nucleobase conjugates 2–4 and reference 1.
Here presented spectroscopic study (fluorimetric and CD titrations, thermal melting
experiments) revealed that interactions of 1–4 with various ds-DNA strongly
depend on type and number of nucleobases attached to phenanthridinium units, as
well as on specific differences in secondary structure of studied polynucleotides.
[1] F. Thomas, J. Michon, J. Lhomme, Biochemistry 1999, 38, 1930–1937. and ref. cited
therein.
[2] L.–M. Tumir, M. Grabar, I. Piantanida, S. Tomić, Tetrahedron 2010, 66, 2501–2513.
[3] I. Piantanida, B. S. Palm, P. Čudić, M. Žinić, H.-J. Schneider, Tetrahedron Lett. 2001,
42, 6779–6783.
137
Poster
Ischemic-reperfusion injury of the small intestine and
changes in gene expression
Peter Urban1, Miroslava Bilecová - Rabajdová1, Jana Mašlanková1,
Jarmila Veselá2, Mária Mareková1
1
Department of chemistry, biochemistry, medical biochemistry and LABMED, Faculty of
medicine P. J. Šafárik University in Košice, [email protected]
2
Department of histology and embryology, Faculty of medicine P. J. Šafárik University in
Košice, Slovakia, [email protected]
IR stress significantly affects the endoplasmic reticulum (ER), which
dysfunction induce responses through activation of kinases which stimulate antiapoptic mechanism like activation of grp78 (BIP), or pro-apoptic mechanism by
activation of gadd153 (CHOP). Currently, many authors also highlight the important
role of serotonin, whose levels in blood and urine reflects changes in
gastrointestinal tract function (Lesurtel et al., 2008). It is expected that this
hormone plays an important role in regeneration of intestinal epithelial cells. Our
goal was to analyze the effects of IR injury of the small intestine epithelium of rats
after 1 hour ischemia and subsequent reperfusion times in periods 1h, 24 h and for
30 days. We studied expression of pro (gadd153) and anti (grp78) apoptic genes.
We have also been monitoring the levels of serotonin in urine which were
subsequently correlated with the degree of ER damage. After isolation of RNA and
reverse transcription into cDNA, we measured changes in gene expression by
PCR. For the determination of the levels of serotonin in urine samples collected
from rats after 1h ischemia and subsequent reperfusion in different time periods
was used High performance liquid chromatography (HPLC) with the UV – Vis
detector at wavelengths 220 and 280 nm. After one hour ischemia and 1h
reperfusion was detected significantly increased level of mRNA for gadd153 gene.
In contrast, mRNA levels of grp78 gene showed an increased level against to
controls at 24h after ischemia. In serotonin levels detected by HPLC after 1h and
24h reperfusion there were no significant changes. After 30 days of reperfusion,
the level of serotonin in the urine increased by 14%, what correlates with
proapoptic signalization of gadd153. So far there is no effective treatment
procedure of the ischemic small intestine and it is therefore necessary to study
changes in the damaged tissue on the molecular level and try to define possible
pathways which could lead to tissue protection.
Acknowledgments: This work was supported by grant project APVV -0252-07.
[1] M. Lesurtel, C. Soll, R. Grof, P.A. Clavien, Cell Mol. Life Sci. 2008, 65, 940–952.
138
Poster
Transition metal complexes of non-proteinogenic histidine
analogue amino acids and their tripeptide derivatives
Katalin Várnagy1, Dóra Kiss1, Zsuzsanna Kovács1, Katalin Ősz1,
Daniele Sanna2, Eugenio Garribba3, Giovanni Micerac,3
1
Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér
1, H–4032 Debrecen, Hungary ([email protected])
2
Istituto C.N.R. di Chimica Biomolecolare, Sezione di Sassari, Trav. La Crucca 3, I–07040
Sassari, Italy.
3
Department of Chemistry, University of Sassari, Via Vienna 2, I–07100 Sassari, Italy.
The growing interest in the synthesis and studies of non-proteinogenic α-amino
acids is due to their biological and toxicological properties. The substitution of the
naturally occurring amino acids in peptides with their structural analogues can
produce dramatic changes in the biological activity of peptides.
The imidazole group of histidine is, however, essential for the metal binding activity
of metalloenzymes. Its substitution with structural analogues in the enzyme could
significantly change the metal binding ability, too. To know the effect of these
amino acids the complexes of several biologically important metal ions (Cu(II),
Ni(II), Zn(II), VO(IV)) and different histidine analogue compounds were studied by
means of potentiometry and UV–Vis, CD, 1H NMR and EPR spectroscopies.
Our data reveal that the coordination ability of histidine analogue amino acids and
their tripeptide derivatives is similar to that of simple amino acids and tripeptides.
The interaction of aromatic rings, however, can be established in all the metal
complexes formed in pyridine containing ligands systems. Depending on the type
of the aromatic ring and the metal ion, the effect of other aromatic ring could be
observed in certain metal complexes, by means of UV–Vis, 1H NMR or EPR
spectroscopic methods.[1] As a conclusion we can state that
• the stability of the metal complexes follows the expected order:
Cu(II) > Ni(II) > Zn(II) > VO(IV)
• the presence of heteroaromatic rings in amino acid analogues and their
tripeptide derivatives can influence the amino acid or tripeptide like coordination
mode, and their effects decrease in order
pyridyl > triazolyl ~ thiazolyl > thienyl rings.
Acknowledgements: This work was supported by the Hungarian Scientific Research Fund (K
72956)
[1] K. Várnagy, E. Garribba, D. Sanna, I. Sóvágó, G. Micera, Polyhedron, 2005, 24, 799.
139
Poster
Dipeptidyl peptidase III from human symbiont Bacteroides
thetaiotaomicron: isolation and characterization
Bojana Vukelić1, Branka Salopek-Sondi 2, Igor Sabljić1,
Jasminka Špoljarić1, Dejan Agić3, Marija Abramić1
Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb,
Croatia, [email protected], [email protected], [email protected]
1
2
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia, [email protected]
3
Department of Chemistry, Faculty of Agriculture, The Josip Juraj Strossmayer University,
Osijek, Croatia, [email protected]
The flow of data on primary structures of proteolytic enzymes enabled a new
system of their classification based on evolutionary families. Peptidase family M49
(dipeptidyl peptidase III family) has been recently recognized among
metallopeptidases, based on the unique structural motif, hexapeptide HEXXGH
which harbors the predicted active site residues.
The dipeptidyl peptidase III (DPP III), first discovered in the pituitary gland, was
considered to be exclusively eukaryotic enzyme involved in intracellular peptide
catabolism, with an implied role in defense against oxidative stress and painmodulatory system. However, the new data of complete microbial genome
sequences revealed in 2003 the first prokaryotic orthologs of M49 family, including
putative DPP III from human gut symbiont Bacteroides thetaiotaomicron.
Bacterial orthologs allowed us to define 5 evolutionary conserved sequences in
proteins of DPP III family using bioinformatics. In order to investigate the properties
of bacterial M49 peptidases, which show low homology with well-characterized
mammalian enzymes, we overexpressed heterologously the full length cDNA
encoding DPP III from bacterium B. thetaiotaomicron (675 amino acids) and
purified it by a three-step procedure. Isolated bacterial DPP III was shown to be a
monomeric acidic protein (Mr~72 000, pI 5.0–5.2) which preferred characteristic
DPP III synthetic substrate Arg-Arg-2-naphthylamide. Comparison with the human
counterpart revealed lower specific activity, pH optimum and stability of the
bacterial DPP III, which seemed to be much more prone to oxidation. Both DPPs III
were very sensitive to sulfhydryl reagent p-hydroxy-mercuribenzoate.
This is the first report on the experimental characterization of the metallopeptidase
M49 from bacteria. Further studies are needed to clarify the (nutritional) benefit
which DPP III type of enzyme brings to our gut flora.
140
Poster
Modeling studies of potato NTPDase1: an insight into the
catalytic mechanism
Andrzej Wojtczak1, Anna Kozakiewicz2
1
Institute of Chemistry, N. Copernicus Univ., Torun, Poland, [email protected].
2
Institute of Chemistry, N. Copernicus Univ., Torun, Poland, [email protected].
NTPDases are the enzymes hydrolyzing nucleotide triphosphates, differing in both
the substrate specificity and products of the reaction. NTPDase1 (apyrase)
hydrolyzes ATP or ADP to AMP and Pi ions. Neither the molecular structure nor
the mechanism for these enzymes is known. The modeling studies of NTPDase1
from S. tuberosum have been performed based on weak sequence similarity to the
exopolyphosphatase 1T6C [1]. Also the similar model of human NTPDase3 was
obtained based with the same template [2]. The sequence analysis revealed that
the plant NTPDase1 belongs to the actin superfamily. The enzyme is built up with
4 subdomains [3]. The major difference between the template 1T6C and apyrase is
subdomain 4, which comprises of only few residues in exopolyphosphatase, while
in apyrase 72 residues are arranged in the alpha-helical domain. The role of that
domain in apyrase is unclear. In other enzymes of the superfamily (actin, Hsp70) it
is involved in a formation of protein-protein complexes. The active site of apyrase
is located in the central cleft of the molecule, and is formed by 5 ACRs (Apyrase
Conserved Regions). The potential ATP binding site was identified based on the
structure alignment with actin-ATP and exopolyphosphatase-ATP complexes. The
CHARMM forcefield energy minimization was performed to give the reasonable
position of ATP. The analysis allowed to identify the residues involved in the ATP
hydrolysis.
The Pgamma cleavage involves Wat-Thr127-Glu170 and such
mechanism is similar to that of actin. The ADPase activity of apyrase involves
another pair Thr55-Glu78, and that is specific for apyrase. The obtained model
gives a basis for understanding the substrate specificity for different NTPDases. It
is also consistent with the biochemical data on proteins belonging to the actin
superfamily (structure, mechanism) and other NTPDases (point mutations).
[1] O. Kristensen, M. Laurberg, A. Liljas, J.S. Kastrup, M. Gajhede, Biochemistry 2004, 43,
8893–8900.
[2] V.V. Ivanenkov, J. Meller, T.L. Kirley, Biochemistry 2005, 44, 8998–9012.
[3] A. Kozakiewicz, P. Neumann, M. Banach, M. Komoszynski, A. Wojtczak, Acta Biochim.
Polonica 2008, 55, 141–150.
141
142
LIST OF PARTICIPANTS
143
List of participants
Abramić, Marija
Bijenička cesta 54, Zagreb, 10 000,
Croatia
[email protected]
Belogurova, Nadezda
Institute of biophysics SB RAS
Akademgorodok 50, Krasnoyarsk, 660036
Russia
[email protected]
Alexandrova, Maria
Siberian Federal University,
Baumana 24-2, Krasnoyarsk, 660028,
Russia
[email protected]
Bertoša, Branimir
Croatia
[email protected]
Andrushchenko, Valery
Institute of Organic Chemistry and
Biochemistry, Academy of Sciences
Flemingovo nam. 2., Prague, 16610,
Czech Republic
[email protected]
Bezerra, Gustavo A.
University of Graz
Humboldtstrasse 50/3, Graz, A-8010,
Austria
[email protected]
Árus, David
University of Szeged
Dom ter. 7., Szeged, 6720,
Hungary
[email protected]
Bilecova – Rabajdova, Miroslava
Medical Biochemistry and LABMED
Bernolakova 10, Košice, 04 001,
Slovakia
[email protected]
Auer, Markus
BOKU University of Natural Resources
and Applied Life Sciences
Muthgasse 18, Vienna, A-1190,
Austria
[email protected]
Bodor, Andrea
Eötvös University, Institute of Chemistry
Pázmány Péter s. 1/A, Budapest, 1117,
Hungary
[email protected]
Balogh, Zsofia
Semmelweis University
Üllői út 26., Budapest, 1085,
Hungary
[email protected]
Bodor, Erik
University of Florida
1445 16th St, Apt 1403, Miami Beach, FL 33139
USA
[email protected]
Ban, Nenad
Institute of Molecular Biology and Biophysics
Schafmattstr. 20, 8093 Zürich
Switzerland
[email protected]
Bodor, Nicholas S.
Center for Drug Discovery, University of Florida
P.O. Box 100497 ,Gainesville, FL 32610
USA,
[email protected].
Batta, Gyula
University of Debrecen
Egyetem ter 1, Debrecen, H-4010,
Hungary
[email protected]
Bogár, Ferenc
Dom ter 8, Szeged, 6720,
Hungary
[email protected]
Bauer, Lubos
Safarik University
Moyzesova 11, Kosice, 04001,
Slovakia
[email protected]
Bozsó, Zsolt
Dóm tér 8, Szeged, H-6720,
Hungary
[email protected]
144
Brcko, Ana
Croatia
[email protected]
Fabian, Laszlo
Biological Research Center of the Hungarian
Academy of Sciences
Temesvari krt. 62., Szeged, H-6726, Hungary
[email protected]
Brkić, Hrvoje
Medical faculty
J. Huttlera 4, Osijek, 31000,
Croatia
[email protected]
Farkas, Etelka
Egyetem ter 1, Debrecen, 4225,
Hungary
[email protected]
Bujnicki, Janusz M.
International Institute of Mol. and Cell Biology,
ul. Ks. Trojdena 4, 02-109 Warsaw,
Poland
[email protected]
Farkas, Viktor
Eotvos University, Institute of chemistry
Pazmany P.s. 1/a, Budapest, H-1117,
Hungary
[email protected]
Cyrański, Michał K.
Faculty of Chemistry, University of Warsaw,
Pasteura 1, Warsaw, 00-664
Poland
[email protected].
Frank, Zsuzsanna
Dóm tér 8, Szeged, 6720,
Hungary
[email protected]
Cysewski, Piotr
Collegium Medicum, N. Copernicus University
KurpiĹ„skiego 5, Bydgoszcz, 85-950,
Poland
[email protected]
Fülöp, Livia
Dóm tér 8, Szeged, H-6720,
Hungary
[email protected]
Czene, Aniko
Dom square 7, Szeged, 6720,
Hungary
[email protected]
Furtmüeller, Paul
BOKU-University of Natural Resources and
Applied Life Sciences
Muthgasse 18, Vienna, 1190,
Austria
[email protected]
Datki, Zsolt
Dóm tér 8, Szikra utca 2, Szeged, 6720,
Hungary
[email protected]
Geremia, Silvano
University of Trieste
Via L.Giorgieri 1, Trieste, 34127,
Italy
[email protected]
Dendrinou-Samara, Katerina
Aristotle University
Thessaloniki, 54124,
Greece
[email protected]
Glušič, Martina
National Institute of Chemistry
Hajdrihova 19, Ljubljana, 1001,
Slovenia
[email protected]
Despotović, Ines
Croatia
[email protected]
Grabar, Marina
Rudjer Boskovic Institute
Croatia
[email protected]
145
Gruber, Sabine
TU WIEN
Getreidemarkt 9/166, Vienna, 1060,
Austria
[email protected]
Janzso, Gabor
Academy of Sciences
Temesvari krt. 62, Szeged, H-6726,
Hungary
[email protected]
Gyurcsik, Bela
Hungary
[email protected]
Jaskolski, Mariusz
A. Mickiewicz University
Grunwaldzka 6, Poznan, 60-780,
Poland
[email protected]
Hasenhindl, Christoph
University of Natural Resources and Applied Life
Sciences
Alserbachstrasse 25/17, Vienna, 1090, Austria
[email protected]
Kamnev, Alexander A.
Institute of Biochemistry and Physiology of
Plants and Microorganisms, Russian Academy
of Sciences
13 Prospekt Entuziastov, Saratov, 410049,
Russia
[email protected]
Heja, David
Eötvös University
Kőzet utca 3, Budapest, H1037,
Hungary
[email protected]
Kasza, Ágnes
Dóm tér 8, Szeged, 6720,
Hungary
[email protected]
Hudoba, Liza
Academy of Sciences
Temesvari krt. 62, Szeged, H-6726,
Hungary
[email protected]
Krygowski, Tadeusz Marek
Warsaw University
Pasteura 1, Warsaw, 02093,
Poland
[email protected]
Jákli, Imre
Eotvos Lorand University, Institute of Chemistry
Pazmany Peter setany 1/A, Budapest, H-1117,
Hungary
[email protected]
Kudryasheva, Galina
Siberian Federal University
Akademgorodok 18-92, Krasnoyarsk, 660036,
Russia
[email protected]
Jakopitsch, Christa
BOKU – University of Natural Resources and
Muthgasse 18, Vienna, 1190,
Austria
[email protected]
Kudryasheva, Nadezhda
Inst.of Biophysics SB RAS
Akademgorodok 18-92, Krasnoyarsk, 660036,
Russia
[email protected]
Jancsik, Veronika
SZIE Faculty of Veterinary Science
Istvan utca 2, Budapest, H-1078,
Hungary
[email protected]
Lewinski, Krzysztof
Faculty of Chemistry, Jagiellonian University
ul. Ingardena 3, Kraków, 30-060,
Poland
[email protected]
146
Luck, Linda A.
State University of New York at Plattsburgh
319 Hudson Hall, SUNY, Plattsburgh, NY,
12901,
USA
[email protected]
Mohar, Barbara
Hajdrihova 19
SI-1000 Ljubljana,
Slovenia
[email protected]
Luić, Marija
Croatia
[email protected]
Naseer, Abeer
20 Beech Lane, Earley, Reading, RG65PT,
Berkshire
[email protected]
Macheroux, Peter
Graz University of Technology
Petersgasse 12, Graz, 8010,
Austria
[email protected]
Nasztor, Zoltan
Hungary
[email protected]
Maksić, Mirjana
Croatia
[email protected]
Novak, Jurica
Croatia
[email protected]
Maksić, Zvonimir
Croatia
[email protected]
Nyitray, Laszlo
Eötvös University
Pazmany P. s. 1/c, Budapest, 1117,
Hungary
[email protected]
Marczi, Saška
Universitiy Hospital Centre Osijek
Gornjodravska obala 90c, Osijek, 31000,
Croatia
[email protected]
Obinger, Christian
Department of Chemistry, BOKU
Muthgasse 18, Vienna, A-1190,
Austria
[email protected]
Marković, Marijana
Institute for Med. Res. and Occupational Health
Ksaverska c. 2, Zagreb 10001,
Croatia
[email protected]
Oleksyn, Barbara J.
Faculty of Chemistry, Jagiellonian University
ul. R. Ingardena 3, Krakow, 30-060,
Poland
[email protected]
Meszaros, Tamas
University
Tűzoltó u. 37-47., Budapest, 1094,
Hungary
[email protected]
Orzel, Lukasz
Jagiellonian University
Ingardena 3, Krakow, 30060,
Poland
[email protected]
Mikleušević, Goran
Croatia
[email protected]
Oshtrakh, Michael
Ural Federal University
Mira str., 19, Ekaterinburg, 620002,
Russia
[email protected]
147
Oziminski, Wojciech
National Medicines Institute
30/34 Chelmska street
00-725 Warsaw,
Poland
[email protected]
Pirc, Gordana
Slovenia
[email protected]
Pál, Gábor
Eötvös Loránd University
1/C Pázmány Péter street, Budapest, 1117,
Hungary
[email protected]
Plavec, Janez
Slovenia
[email protected]
Palecek, Emil
Institute of Biophysics, ASCR
Kralovopolska 135, 612 65 Brno
Czech Republic
[email protected]
Polanski, Jaroslaw
University of Silesia
Szkolna 9, Katowice, PL-40-006,
Poland
[email protected]
Paragi, Gabor
Hungarian Academy of Sciences, University of
Szeged
Hungary
[email protected]
Polek, Bystrik
Institute of Molecular Biology, Slovak Academy
of Sciences
Dubravska cesta 21, Bratislava, SK-85101,
Slovakia
[email protected]
Pavkov-Keller, Tea
Max-Planck Institute of Biophysics
Max-von-Laue-Strasse 3., Frankfurt, 60438,
Germany
[email protected]
Radnai, László
Pázmány Péter sétány 1/C, Budapest, 1117,
Hungary
[email protected]
Penke, Botond
Hungary
[email protected]
Ramek, Michael
Graz University of Technology
Rechbauerstrasse 12, Graz, A-8010,
Austria
[email protected]
Peran, Nena
Rudjer Boskovic Institute
Croatia
[email protected]
Rapali, Péter
Pázmány péter sétány 1/C, Budapest, 1117,
Hungary
[email protected]
Perczel, András
Pázmány Péter sétány 1/A, H-1117 Budapest,
Hungary
[email protected]
Revesz, Katalin
Semmelweis University
ĂśllĹ‘i Ăşt 26., Budapest, 1085,
Hungary
[email protected]
Picone, Delia
University of Naples Federico II
via Cintia, Naples, I-80126,
Italy
[email protected]
Roviello, Giovanni N.
IBB-CNR, Naples
Via Mezzocannone 16, Naples, 80134,
Italy
[email protected]
148
Rovo, Petra
Pazmany P. s. 1/a, Budapest, H-1117,
Hungary
[email protected]
Sondi, Ivan
Croatia
[email protected]
Rownicka-Zubik, Joanna
Medical University of Silesia
Jagiellonska 4, Sosnowiec, 41-200,
Poland
[email protected]
Stadlmayr, Gerhard
Boku University of Natural Resources and
Muthgasse 18, Wien, A-1190,
Austria
[email protected]
Sabolović, Jasmina
Institute for Med. Res. and Occupational Health
Ksaverska c. 2, P. O. Box 291, Zagreb, 10001,
Croatia
[email protected]
Stochel, Grażyna
Faculty of Chemistry Jagiellonian University
Ingardena 3, Krakow, 30-060,
Poland
[email protected]
Salopek-Sondi, Branka
Croatia
[email protected]
Stoka, Veronika
J. Stefan Institute
Jamova 39, Ljubljana, Sl-1000,
Slovenia
[email protected]
Sarna, Tadeusz
Jagiellonian University
Gronostajowa 7, Krakow, 30-387,
Poland
[email protected]
Straganz, Grit
TU Graz
Petersgasse 12, Graz, 8010,
Austria
[email protected]
Sija, Eva
Dóm tér 7., Szeged, H-6700,
Hungary
[email protected]
Stráner, Pál
Pázmány Péter sétány 1/A, Budapest, 1117,
Hungary
[email protected]
Simon, Dora
Dom ter 8., Olajos u. 1/H, Szeged, 6720,
Hungary
[email protected]
Szakacs, David
Eotvos Lorand University
Pázmány Péter sétány 1/c, Budapest, 1117,
Hungary
[email protected]
Smith, David
Croatia
[email protected]
Szalaine Agoston, Bianka
Eotvos Lorand University
Pazmany P. s. 1/A, Budapest, 1117,
Hungary
[email protected]
Sochorova Vokacova, Zuzana
Institute of Org. Chem. and Biochem., AS CR
Flemingovo nĂˇm. 2., Praha 6, 16610,
Czech Republic
[email protected]
Szatylowicz, Halina
Warsaw University of Technology
Noakowskiego 3, Warsaw, 00-664,
Poland
[email protected]
149
Szigyarto, Imola
Chemical Research Center
Pusztaszeri u. 59-67, Budapest, 1025,
Hungary
[email protected]
Tumir, Lidija-Marija
Croatia
[email protected]
Szkudlarek, Agnieszka
Medical University of Silesia
Jagiellonska 4, Sosnowiec, 41-200,
Poland
[email protected]
Urban, Peter
P. J. Šafárik University
Tr. SNP ÄŤ.1, Košice, 04 001,
Slovakia
[email protected]
Šekutor, Marina
Croatia
[email protected]
Várnagy, Katalin
Egyetem tér 1., Debrecen, H-4032,
Hungary
[email protected]
Špoljarić, Jasminka
Croatia
[email protected]
Vianello, Robert
Croatia
[email protected]
Štefanišinová, Miroslava
PJ Šafárik University
Trieda SNP 1, Košice, 04011,
Slovakia
[email protected]
Viglasky, Viktor
Safarik University
Moyzesova 11, Kosice, 04001
Slovakia
[email protected]
Tlučkova, Katarina
Safarik University
Moyzesova 11, Kosice, 04001,
Slovakia
[email protected]
Volarević, Siniša
University of Rijeka
Brace Branchetta 20, Rijeka, 51000,
Croatia
[email protected]
Tomić, Sanja
Croatia
[email protected]
Vukelić, Bojana
Croatia
[email protected]
Toth, Gabor
Hungary
[email protected]
Weygand-Đurašević, Ivana
University of Zagreb, Faculty of Science
Horvatovac 102a, Zagreb, 10000,
Croatia
[email protected]
Tramontano, Anna
Sapienza University
P.le Aldo Moro, 5, 00185 Rome,
Italy
[email protected]
Wojtczak, Andrzej
Institute of Chemistry, N. Copernicus University
Gagarina 7, Torun, 87-100,
Poland
[email protected]
150
Žagar, Ema
Slovenia
[email protected]
Zechner, Rudolf
Institute of Molecular Biosciences, Karl
Franzens University Graz
Heinrichstr. 31, A-8010 Graz
Austria
[email protected]
Zámocký, Marcel
BOKU University
Muthgasse 18, Wien, A-1190,
Austria
[email protected]
151
152
INDEX OF CONTRIBUTIONS
153
Index of contributions
Abramić, Marija
67, 140
Frank, Zsuzsanna
98
Alexandrova, Maria
60
Fülöp, Livia
76, 82, 125
Andrushchenko, Valery
57
Furtmüeller, Paul
38, 39, 62, 83
Árus, David
61
Geremia, Silvano
49
Auer, Markus
62
Glušič, Martina
84
Balogh, Zsofia
63
Grabar, Marina
85, 137
Ban, Nenad
29
Gruber, Sabine
86
Batta, Gyula
37
Gyurcsik, Bela
56, 75
Bauer, Lubos
55, 64, 136
Hasenhindl, Christoph
128
Belogurova, Nadezda
65
Heja, David
87, 130
Bertoša, Branimir
66
Hudoba, Liza
88, 89, 93, 94
Bezerra, Gustavo A.
67
Jákli, Imre
90
Bilecova-Rabajdova,
Miroslava
68, 138
Jakopitsch, Christa
39, 91
Bodor, Andrea
69
Jancsik, Veronika
92
Bodor, Nicholas S.
44
Janzso, Gabor
88, 89, 93, 94, 95
Bogár, Ferenc
70, 93, 94, 95
Jaskolski, Mariusz
27
Bozsó, Zsolt
125
Kamnev, Alexander A.
96, 97
Brcko, Ana
71
Kasza, Ágnes
98
Brkić, Hrvoje
72
Krygowski, Tadeusz M.
58
Bujnicki, Janusz M.
54
Kudryasheva, Galina
99
Cyrański, Michał K.
73
Kudryasheva, Nadezhda
42, 60, 65
Cysewski, Piotr
74
Lewinski, Krzysztof
26
Czene, Aniko
75
Luck, Linda A.
52
Datki, Zsolt L.
76, 125
Luić, Marija
40, 102
Dendrinou-Samara,
Katerina
77
Macheroux, Peter
36, 67
Despotović, Ines
78
Maksić, Zvonimir
78, 110
Fabian, Laszlo
79
Marczi, Saška
100
Farkas, Etelka
80
Marković, Marijana
101
Farkas, Viktor
51, 81, 120
Meszaros, Tamas
63, 118
154
Mikleušević, Goran
40, 102
Rownicka-Zubik, Joanna
121, 122, 133
Mohar, Barbara
43
Sabolović, Jasmina
101
Nasztor, Zoltan
70
Salopek-Sondi, Branka
71, 127, 140
Novak, Jurica
103
Sarna, Tadeusz
123
Nyitray, Laszlo
50, 116, 117
Sija, Eva
124
Obinger, Christian
38, 39, 62, 83, 91,
115, 128
Simon, Dora
76, 82, 125
Oleksyn, Barbara J.
104
Smith, David
53
Orzel, Lukasz
105
Sochorova Vokacova,
Zuzana
126
Oshtrakh, Michael
106,107
Sondi, Ivan
127
Oziminski, Wojciech
108
Stadlmayr, Gerhard
128
Pál, Gábor
47, 50, 87, 117,
130
Stochel, Grażyna
48, 105
Palecek, Emil
24
Stoka, Veronika
33
Paragi, Gabor
109
Straganz, Grit
72
Pavkov-Keller, Tea
34
Stráner, Pál
51, 120, 129
Penke, Botond
70, 82, 93, 94, 95,
98, 125
Szakacs, David
87, 130
Peran, Nena
110
Szalaine Agoston, Bianka
28
Perczel, András
28, 51, 69, 81, 90,
120, 129
Szatylowicz, Halina
58, 131
Picone, Delia
41
Szigyarto, Imola
132
Pirc, Gordana
111
Szkudlarek, Agnieszka
121, 122, 133
Plavec, Janez
113
Šekutor, Marina
134
Polanski, Jaroslaw
104, 114
Špoljarić, Jasminka
140
Polek, Bystrik
115
Štefanišinová, Miroslava
135
Radnai, László
50, 116
Tlučkova, Katarina
55, 64, 136
Ramek, Michael
72
Tomić, Sanja
66, 72, 85, 137
Rapali, Péter
50, 116, 117
Toth, Gabor
51, 81
Revesz, Katalin
118
Tramontano, Anna
35
Roviello, Giovanni N.
119
Tumir, Lidija-Marija
85, 137
Rovo, Petra
51, 81, 120
Urban, Peter
68, 138
155
Várnagy, Katalin
139
Wojtczak, Andrzej
141
Viglasky, Viktor
55, 64, 136
Zámocký, Marcel
39, 62, 115
Volarević, Siniša
31
Zechner, Rudolf
32
Vukelić, Bojana
140
Žagar, Ema
46
Weygand-Đurašević,
Ivana
30
156

Here - Index of

Transcription

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