SOCIEDAD ARGENTINA DE BIOFÍSICA XLIII Reunión Anual

Transcription

XLIII RA – SAB 2014
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SOCIEDAD ARGENTINA
DE
BIOFÍSICA
XLIII Reunión Anual
3 – 5 de Diciembre 2014
Sierra de la Ventana, Pcia. de Buenos Aires
ARGENTINA
_____________________________________________________
Sanchez Fornillo, Néstor Edgardo
SAB 2014: XLIII Reunión Anual de la Sociedad
Argentina de Biofísica / Néstor E. Sanchez Fornillo ;
Marcelo D. Costabel. 1 ed. – Bahía Blanca – Sociedad
Argentina de Biofísica 2014
ISBN 978-987-27591-3-1
1 Biología. Investigación. I. Sanchez Fornillo, Nestor E.
II. Costabel, Marcelo III. Título CDD 571.4
Fecha de Catalogación: 01/12/2014
Quedan prohibidos, dentro de los límites establecidos en la ley y
bajo apercibimiento legalmente previsto, la reproducción total o
parcial de esta obra por cualquier medio o procedimientos ya sea
electrónico o mecánico, el tratamiento informático, el alquiler o
cualquiera otra forma de cesión de la obra sin la autorización
previa y por escrito de los titulares del copyright.
Diagramación y Edición: Néstor Edgardo Sanchez Fornillo
Diseño de Tapa: Marcelo Costabel.
Asistencia Técnica Web: Juan Pablo Acierno - Mauricio Sica
COMISIONES Y COMITÉS
_____________________________________________________
COMISION DIRECTIVA DE LA
SOCIEDAD ARGENTINA DE BIOFÍSICA
AÑO 2014
PRESIDENTE
Gerardo Fidelio
Universidad Nacional de Córdoba
VICEPRESIDENTA
Gabriela Amodeo
Universidad de Buenos Aires
Presidente Saliente
Luis Gonzalez Flecha
Secretario
Mauricio Sica
Centro Atómico Bariloche
Tesorero
Lía Pietrasanta
Vocales Titulares
Karina Alleva
Rosana Chehín
Universidad Nacional de Tucumán
Vocales Suplentes
Rodolfo Rassia
Universidad Nacional de Rosario
Florencia Martini
Universidad de Buenos Aires.
COMISIONES Y COMITÉS
_____________________________________________________
COMITÉ CIENTÍFICO
Dra. Gabriela Amodeo
Dra. Silvia Antollini
Universidad Nacional del Sur
Dra. Cecilia Bouzat
Dra. Betina Córsico
Universidad Nacional de La Plata
Dr. Marcelo Costabel
Dra. Verónica Dodero
Dra. Lía Pietrasanta
Comité organizador (SAB-Bahía Blanca)
Dr. Marcelo Costabel
Dra. Silvia Antollini
Dra. Cecilia Bouzat
Dra. Verónica Dodero
Dr. Jeremías Corradi
Ing. Néstor Sánchez Fornillo
Bioq. Fernando Zamarreño
Colaboradores
Lic. Ma Julia Admundarain
Bioq. Giorgina Herrera
Bioq. Daniel A. Peñalva
Dra. Tania Veuthey
Lic. Juan F. Viso
ÍNDICE GENERAL
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PALABRAS COMITÉ ORGANIZADOR…………………………..
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AUSPICIANTES……………………………………………………………
8
PROGRAMA……………………………………………………………….
10
CONFERENCIAS Y SIMPOSIOS…………………………………….
22
POSTERS
Bioenergética, Transferencia Electrónica………………….
Bioenergetics, Electronic Transfer…………………………….
54
Enzimología……………………………………………………………….
Enzimology…………………………………………………………………
58
Lípidos y Biomembranas……………………………………………
Lipids and Biomembranes…………………………………...……
61
Nuevas Técnicas y Aplicaciones en Biofísica……………..
New Techniques and Applications in Biophysics……….
85
Proteínas y Ácidos Nucleicos……………………………………..
Proteins and Nucleic Acids…………………………………………
95
Señalización y Dinámica Intracelular…………………………
Signaling and Intercellular Dynamics………………………..
129
Teoría y Modelado de Sistemas Biológicos……………….
Theory and Modelling of Biological Systems…………….
136
Transportadores, Receptores y Canales…………….……..
Transporters, Receptos and Channels……………………….
157
INDICE DE AUTORES………………………………………………….
179
PALABRAS DEL COMITÉ ORGANIZADOR
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Pasó mucho desde aquel “Club de la Membrana”…
Hoy, en las reuniones SAB, no sólo se escucha de lípidos y
de cómo organizarlos en estructuras más complejas; las proteínas, y
otras macromoléculas pasaron a ser, también, parte fundamental de
las preguntas que intentamos responder. Más aún, además del
cambio en los sistemas que estudiamos, las técnicas y la metodología
han variado sustancialmente con el correr de los años; el espectro
experimental ha crecido en variantes y precisión y los modelos
computacionales han adquirido un protagonismo esencial en la
búsqueda de respuestas donde el experimento aún no puede llegar.
Inexorablemente nuestra SAB se ha transformado en un mundo
donde la biología, la bioquímica, la física, la química, la informática y
sus variadas combinaciones se aglutinan para ofrecernos
herramientas de discusión que nos permitan describir un universo
cuanto menos fascinante.
En función de esto, si quisiéramos resumir hoy nuestra
reunión SAB en una palabra, quizás esa palabra sea Consiliencia. Es
por esto que, disfrutando de un entorno maravilloso donde se mezclan
las sierras, los campos y los riachos de una Ventania pródiga, seamos
capaces de entretejer ideas y dejemos que esa consiliencia desarrolle
sus frutos y nos dé el marco adecuado para intentar dar forma a un
proyecto de ciencia diferente, donde nuestros jóvenes encuentren su
lugar y su oportunidad sin necesidad de mezquindades, y sepan que
el trabajo con inteligencia puede tener su premio.
Unas últimas palabras para todos los que comparten estos
días. Gracias a todos los que aportaron su trabajo, su tiempo, su
opinión, su consejo, su reflexión, y también su crítica en pos de una
organización no perfecta; pero seguramente empeñada en el
bienestar de todos. Y gracias a cada uno de los que hoy llegaron
hasta aquí: los conferencistas, simposistas y asistentes en general sin
los cuales, obviamente, todo esto no tendría sentido.
En fin… en un contexto económico difícil, fue arduo armar el
rompecabezas, pero llegamos a este punto con la esperanza puesta
en que todos los que hoy comparten la reunión vuelvan a su terruño
con una sonrisa y sientan el placer de que cada minuto en Sierra de la
Ventana valió la pena.
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AUSPICIANTES
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8
PROGRAMA
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PROGRAMA
9
PROGRAMA
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Miércoles 3 de diciembre
9:00 - 12.00 hs
REGISTRO
11:50 – 12:00 hs
APERTURA
12:00 - 13.00 hs
CONFERENCIA 1 – CONFERENCIA
DE APERTURA
“Insertion of a transmembrane
helix, and its uses in targeting
tumors”
Dr. Donald M. Engelman
Department of Molecular Biophysics and
Biochemistry, Yale University, Estados Unidos
13.00 - 14.30 hs
Almuerzo /Colocación de
posters
14.30 - 16.00 hs
SIMPOSIO 1: POSTERS
SELECCIONADOS
Coordinadora: Dra. Gabriela
Amodeo
16.00 - 17.00 hs
CONFERENCIA 2
“Dynamic Aspects of
Peptide/Protein-DNA Interaction”
Dr. Norbert Sewald
Department of Organic and Bioorganic
Chemistry University of Bielefeld, Alemania
17:00 - 17.30 hs
Café
17.30 - 19.30
SIMPOSIO 2: INTERACCIONES
MOLECULARES
Coordinadora Dra. Betina Córsico
“Regulation of Voltage gated
sodium channels by Calmodulin”
Dra. Sandra Gabelli
10
PROGRAMA
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Johns Hopkins University School of
Medicine, Baltimore, Estados Unidos
“Sterols in membranes: structural
features that rule the interaction
with phospholipids”
Dr. Jorge Wenz
Instituto de Investigaciones Bioquímicas de
Bahía Blanca, Universidad Nacional del Sur
Argentina
“Lipid and protein aggregates
activate receptors of the innate
system”
Dr. Jean Mari Ruysschaert
Free University of Brussels-Belgica
“Structural
Characterization
of
Heparin-induced GAPDH Protofibrils
Preventing α-synuclein Oligomeric
Species Toxicity”
Dra. Rosana Chehin
INSIBIO-CONICET-UNT, Tucumán, Argentina
19.30 – 20:30 hs
CONFERENCIA 3 – CONFERENCIA
GREGORIO WEBER
“Solvent accessibility profiling in
proteins”
Dr. José María Delfino
Instituto de Química y Fisicoquímica,
Universidad de Buenos Aires, Argentina
20.30 - 22.30 hs
SESIÓN DE POSTERS Y CENA
SNACK
11
PROGRAMA
_____________________________________________________
Jueves 4 de diciembre
8.30 - 9.30 hs
SIMPOSIO 3: JÓVENES
INVESTIGADORES
Coordinadora Dra. Silvia Antollini
“Synaptic gain-of-function effects
of mutant CaV2.1 channels in a
mouse model of familial
hemiplegic migraine are due to
increased basal [Ca2+]”
Dr. Mariano Di Guilmi
IFIBYNE, UBA- CONICET
“ABA-1A: a Nematode Polyprotein
Allergen (NPA) of Ascaris suum.
Structure and binding properties.”
Dra. Gisela Franchini
INIBIOLP-CONICET, Fac.de Ciencias
Médicas, Universidad de La Plata
“Membrane role in the rational
design of HIV fusion inhibitors”
Dr. Axel Hollmann
Instituto de Medicina Molecular, Faculdade
de Medicina, Universidade de Lisboa,
Portugal; Laboratory of Biointerfaces and
Biomimetic Systems- CITSE- CONICET,
Argentina
12
PROGRAMA
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“Effects of nitric oxide on heart
mitochondrial calcium handling”
Dra. Tatiana Zaobornyj
Institute of Biochemistry and Molecular
Medicine (IBIMOL, UBA-CONICET), School of
Pharmacy and Biochemistry, University of
Buenos Aires, Buenos Aires, Argentina
9.30 – 10:30 hs
CONFERENCIA 4 – PREMIO SAB
10.30 – 11:00 hs
Café
11.00 - 12.00 hs
CONFERENCIA 5
“Triatoma virus as viral model and
biotechnological tool: A simple
machine able to do complex tasks”
Dr. Diego Guerin
Unidad de Biofisica (CSIC-UPV/EHU),
Universidad del País Vasco (EHU), Leioa,
Vizcaya, España
12.00 - 13.00 hs
CONFERENCIA 6
“TRPC channels and Ca2+ entry into
cells”
Dr. Lutz Birnbaumer
Instituto de Investigaciones Biotecnológicas
(IIB-INTECH), Universidad Nacional de San
Martín, Argentina
13.00 - 14.30 hs
Almuerzo /Colocación de
posters
13
PROGRAMA
_____________________________________________________
14.30 - 16.30 hs
SIMPOSIO 4: MODELOS
MATEMÁTICOS APLICADOS A
SISTEMAS BIOLÓGICOS
Coordinadora Dra. Verónica
Dodero
“Mathematical models for
cytoskeletal transport and cellular
viscoelasticity”
Dr. Sebastián Bouzat
Grupo de Física Estadística e
Interdisciplinaria, Centro Atómico Bariloche
(CNEA). Argentina.
“Using Machine Learning for
Predictive Modeling on Systems
Biology and Molecular Informatics”
Dr. Ignacio Ponzoni
Instituto de Ciencias e Ingeniería de la
Computación, Universidad Nacional del
Sur, CONICET, Bahía Blanca, Argentina
“Water at the nanoscale: Towards
new principles and design
elements for biophysics”
Dr. Gustavo Appignanesi
INQUISUR-UNS-CONICET and
Departamento de Química, Universidad
Nacional del Sur, Bahía Blanca, Argentina.
16.30 - 17.00 hs
Café
14
PROGRAMA
_____________________________________________________
17.00 -19.00 hs
SIMPOSIO 5: ESTRATEGIAS
BIOFÍSICAS Y
COMPUTACIONALES PARA EL
ESTUDIO DE LA ESTRUCTURA DE
PROTEÍNAS
Coordinador Dr. Marcelo Costabel
“How do Sco Proteins Score the
COX?”
Dr. Alejandro Vila
Instituto de Biología molecular y Celular de
Rosario y Depto. de Qca. Biol, Fac. de Cs.
Bioq. y Farm, Universidad Nacional de
Rosario (UNR), Argentina
“On
the
Search
of
a
Comprehensive Representation of
Human Frataxin”
Dr. Javier Santos
Fundación Instituto Leloir and IIBBACONICET, Argentina
“Ligand binding mechanisms of
bitter taste receptors: Insights from
multiscale simulation approaches”
Dr. Alejandro Giorgetti
Department of Biotechnology, University of
Verona, Verona, Italy and Computational
Biophysics, German Research School for
Simulation Sciences, Juelich, Germany
15
PROGRAMA
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“A simple model to simulate
protein-membrane interactions with
inclusion of electrostatics”
Dr. Marcos Villarreal
INFIQC-Dpto Matemática y Física.
CONICET-Universidad Nacional de
Córdoba. Argentina.
19.00 - 20.30 hs
ASAMBLEA ANUAL DE LA
SOCIEDAD ARGENTINA DE
BIOFISICA
20.30 – 22:30 hs
SESIÓN DE POSTERS Y CENA
SNACK
16
PROGRAMA
_____________________________________________________
Viernes 5 de diciembre
8.30 - 10.30 hs
SIMPOSIO 6: MICROSCOPÍA
Coordinadora Dra. Lía Pietrasanta
“Mechanical and thermodynamic
properties of lipid membranes using
optical microscopy”
Dra. Natalia Wilke
CIQUIBIC, Departamento de Química
Biológica, Facultad de Ciencias Químicas,
Universidad Nacional de Córdoba,
Argentina
“Dynamics and interactions of
transcription factors in the cell
nucleus through fluorescence
fluctuation spectroscopy”
Dra. Valeria Levi
Departamento de Química BiológicaIQUIBICEN. Facultad de Ciencias Exactas y
Naturales-Universidad de Buenos AiresArgentina
“Far-field fluorescence nanoscopy
of neurons”
Dr. Fernando Stefani
Centro de Investigaciones en
Bionanociencias - CIBION, CONICET, y.
Departamento de Física, Facultad de
Ciencias Exactas y Naturales, Universidad
de Buenos Aires, Argentina
17
PROGRAMA
_____________________________________________________
“Sistema Nacional de Microscopía:
resultados y perspectivas”
Secretaría de Articulación Científico
Tecnológica
Ministerio de Ciencia, Tecnología e
Innovación Productiva
10.30 - 11.00 hs
Café
11.00 - 12.00 hs
CONFERENCIA 7
“Use of proteliposomes to study
biomineralization process”
Dr. Pietro Ciancaglini
Department of Chemistry, FFCLRP-USP, Brasil
12.00 - 13.00 hs
CONFERENCIA 8 SACT-MinCyT
Secretaría de Articulación Científico
Tecnológica
Ministerio de Ciencia, Tecnología e
Innovación Productiva
13.00 - 14.30 hs
Almuerzo
14.30 - 16.30 hs
SIMPOSIO 7: TRANSPORTADORES
Y CANALES
Coordinadora Dra. Cecilia Bouzat
“Two signaling pathways mediate
presynaptic voltage gated calcium
channels inhibition by two ghrelin
receptor activation modes”
Dra. Jesica Raingo
Multidisciplinary Institute of Cell Biology
(IMBICE) La Plata Argentina
18
PROGRAMA
_____________________________________________________
“Redox modulation of the
GABAergic neurotransmission in the
retina and the hippocampus”
Dr. Daniel Calvo
INGEBI- CONICET, Buenos Aires, Argentina
“Proton permeation in Ci-Hv1
voltage-gated proton channels
occurs through a proton wire
involving residues D160 and D222
and it is modulated by N264”
Dr. Carlos González
C. Interdisc. de Neurociencias de
Valparaíso, Universidad de Valparaíso,
Chile.
“The role of auxiliary β1 subunit of
the BKCa channel as a tissue
selective target for endogenous
and exogenous substances”
Dr. Verónica Milesi
Laboratorio de Canales Iónicos, IIFP
CONICET-UNLP, La Plata, Argentina.
16.30 - 17.30 hs
CONFERENCIA 9
“Structure-based design of new
inhibitors against aggregation of
amyloid β-peptide and BACE-1
enzyme”
Dr. Ricardo Daniel Enriz
Departamento de Farmacia, Universidad
Nacional de San Luis, y JIMIBIO-SL
(CONICET), San Luis, Argentina.
17.30 - 18.00 hs
Café
19
PROGRAMA
_____________________________________________________
18.00 - 19.00 hs
CONFERENCIA 10 –
CONFERENCIA DE CLAUSURA
“Control of the levels of PIP3:
Structure and Function of the lipid
kinase PI3Kalpha”
Dr. Mario Amzel
Johns Hopkins University School of
Medicine, Baltimore, Estados Unidos
19.00 - 20.30 hs
CIERRE DE LA REUNIÓN
21.00 hs
CENA DE CLAUSURA
20
CONFERENCIAS Y SIMPOSIOS
_____________________________________________________
CONFERENCIAS
21
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CONFERENCIA 1
Insertion of a transmembrane helix, and its uses in
targeting tumors
Donald M. Engelman
Department of Molecular Biophysics and Biochemistry, Yale,
The discovery that the C helix of Bacteriorhodopsin exhibits
spontaneous, pH-dependent insertion to form a helix across lipid
bilayers has led to the use of related peptides, pHLIPs (pH (Low)
Insertion Peptides), to study peptide insertion across bilayers, to
selectively target cargoes to tumors and other acidic tissues in
vivo, and to deliver molecules across the plasma membranes of
living cells.
Because pHLIP is unfolded on the surface of a bilayer and folding
is pH-triggered, we are able to begin to observe and understand
the molecular events that accompany the insertion and folding of a
peptide entering a bilayer. When the pH is dropped, it is found that
a helix forms rapidly on the bilayer surface, followed by a slow
insertion across it in several kinetically distinct steps. The exit
pathway when the pH is suddenly raised is more rapid, and
includes partial unfolding of the helix while still in the bilayer. Lipid
composition and sequence features affect the insertion pK and
pathway intermediates.
pHLIPs target acidic tissues in vivo, where the transmembrane
insertion stabilizes them in cells, enabling targeting of imaging and
therapeutic agents. We have shown targeting of dyes,
radioisotopes, nanogold and Peptide Nucleic Acids (PNAs) to
tumors in mice, and will report on the use of PNAs to target the
suppression of onco-micro RNAs, resulting in tumor growth
inhibition in vivo
22
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CONFERENCIA 2
Dynamic Aspects of Peptide/Protein-DNA Interaction
Ritzefeld, M.; Wollschläger, K.; Walhorn, V.; Anselmetti, D.; Sewald,
N.
Bielefeld University, Organic and Bioorganic Chemistry,
Universitätsstr. 25, 33615 Bielefeld, Germany,
[email protected]
Protein-DNA interactions are involved in many biochemical
processes like replication, gene regulation and transcription. The
dynamics of DNA recognition by the transcription factor PhoB from
E. coli was investigated as a model system. PhoB recognizes
cognate DNA sequences containing a TGTCA consensus
sequence and an A/T-rich minor groove. A combined approach
based on isothermal titration calorimetry (ITC), fluorescence
resonance energy transfer (FRET) experiments, circular dichroism
spectroscopy (CD), atomic force microscopy – dynamic force
spectroscopy (AFM-DFS) and surface plasmon resonance (SPR)
was applied to elucidate the mechanism of protein-DNA complex
formation and the impact of protein dimerization of the DNAbinding domain of PhoB (PhoBDBD). The ITC, SPR, FRET, and CD
results indicate a positive cooperative binding mechanism and a
decisive contribution of dimerization on the complex stability. With
a combination of dynamic force spectroscopy and total internal
reflection fluorescence microscopy (TIRF), we were able to identify
specific bond rupture events.
1.
2.
3.
4.
5.
R. Eckel et al., Biophys. J. 2003, 85, 1968.
R. Eckel et al., Angew. Chem. Int. Ed. 2005, 4, 3921.
K. Wollschläger et al., Small 2009, 5, 484.
M. Ritzefeld et al., Mol. Biosyst. 2011, 7, 3132.
M. Ritzefeld et al., Biochemistry 2013, 52, 8177.
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CONFERENCIA 3
Solvent accessibility profiling in proteins
1
1
2
1
Gómez, G.E. ; Bernar, E.M. ; Arán, M. , Sabeckis, L. , González
1
1
Lebrero, M.C. and Delfino, J.M.
1
IQUIFIB (UBA-CONICET), Junín 956, (C1113AAD), Buenos Aires,
Argentina. 2FIL, Av Patricias Argentinas 435, (C1405BWE),
Buenos Aires, Argentina.
Topography of proteins and their interactions can be investigated
through photochemical mimicry of the aqueous solvent, an
approach aimed at estimating the size and nature of the solvent
accessible surface area (SASA). After reacting diazirine (DZN, the
smallest CNN heterocycle) with proteins, it is possible to measure
quantitatively the extent of modification (methylation) by the use of
radiotracers (tritiated DZN), by metrics derived from modern mass
spectrometry techniques (MALDI-TOF and ESI-MS) or by
multidimensional NMR. Maximal resolution of the labeled site is
achieved after fragmentation into small peptides or individual
amino acids. Interestingly, the NMR approach does not demand
cleavage and is potentially rich in conformational information.
Predictably, methylation of amino acid side chains rules the DZN
modification event. Molecular dynamics simulations highlight the
distribution of the reagent onto surface components prior to
photolysis. Thus, the probability of reaction at individual sites along
the polypeptide reveals the map of solvent accessibility.
Conformations can be distinguished corresponding to native or
intermediate states, or the unfolded ensemble. Moreover, a
paradigm of a peptide-protein complex (calmodulin-melittin)
illustrates the value of this approach as a foot-printing technique
able to pinpoint the area of interaction. One cannot overemphasize
the worth of these new methods for the benefit of structural
proteomics and interactomics.
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CONFERENCIA 5
Triatoma virus as viral model and biotechnological tool:
A simple machine able to do complex tasks
Guérin D. M. A.
Unidad de Biofisica (CSIC-UPV/EHU), Universidad del País Vasco
(EHU), Leioa, Vizcaya, Spain
As many other small spherical and non-enveloped viruses,
Triatoma virus (TrV; Dicistroviridae: Cripavirus) is built only with
four capsid peptides, one single +ssRNA molecule, and several
water molecules and ions. This simple macromolecular aggregate
performs astonishing functions: i) assembles by itself
spontaneously: ii) destroys the host membrane cell to escape; iii)
its capsid supports very harsh environmental conditions during the
journey to the target cell; iv) specifically binds to its receptor; v)
disrupt the membrane cell to enter; vi) safely releases its genome
into the cytoplasm, and finally, vii) sequesters the cell machinery to
make its own progeny. Decades of study gave some clues to
unravel these mechanisms, but still a great effort will be needed to
understand how viruses work at atomic level. For instance, we will
explain our hypothesis on how genome release may work in TrV.
On the other hand, this insect virus naturally infects triatomines,
the haematophagous insect vectors of Chagas disease. Also, TrV
is considered as potential biological agent to control the disease's
vectors. We explored in several countries some biological aspects
of TrV and our results seem to support its potential as biopesticide.
Moreover, TrV capsid can be used as platform to make 'fifthgeneration' chimeric-VLP vaccines, as a nano carrier, and more....
25
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CONFERENCIA 6
TRPC channels and Ca2+ entry into cells.
Lutz Birnbaumer,
Instituto de Investigaciones Biotecnológicas (IIBINTECH,CONICET), Universidad Nacional de San Martin, Prov. de
TRPCs, of which there are 7, were cloned in the mid 1990’s with
the expectation that they would encode channels responsble for
replenishment of Ca2+, not only after agonist stimulation, but also
after mere depletion of stores without generation of IP3 or DAG,
the latter generating a Ca2+ reléase activated Ca current (Icrac),
which is exquisitey Ca2+ selective.. But when expressed, TRPCs
generated non-selective catión currenst with minimal selectivity of
Ca2+ over Na+. In 2005 and 2006 two misisng players were
discovered: STIM, resident in the ER membrane, sensing loss of
Ca2+ from stores, and ORAI, resident in the plasma membrane,
which upon co-expression with STIM leads to expression of large
Icrac currents. Yet, store depletion also activates catión currents
requiring TRPC1, and expression of low levels of ORAI in cells
expressing TRPCs in stable form leads to increases in Ca2+ entry
and of Icrac. Given that ORAI function had not been tested in a
TRPC-null cell, we hypothesized that ORAI might be a regulator of
TRPCs changing their Ca2+ selectivity. To resolve the conundrum,
we set out to generate a TRPC-null mouse in which all Trpc alleles
have been inactivated by classical or conditional gene targetting.
Surpisingly, in May 2013 a female mouse with only disrupted Trpc
genes was born and was fertile. Upon single inactivation, all our
KO alleles have given interesting phenotypes, includng loss of
Icrac in endotelial cell devoid of TRPC4, proving that they are
disrupted. We found that MEFs from TRPC-null mice have
unaltered store depletion activated Ca2+ entry, proving that ORAIs
are the molecular substrate of Icrac currents. ORAI molecules in
resting cells are dimers. Upon activation the ORAI dimers trimerize
to form hexameric channels. It remains to be determined if ORAI
dimers are regulators of TRPC channels. I thank the invaluable
collaboartion of C. Dulac (Harvard, Trpc2 KO), M. Freichel/V
Flockerzi (Saarland, Trpc4 KO) and Joel Abramowitz (NIEHS).
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CONFERENCIA 7
Use of proteliposomes to study biomineralization
process
Ciancaglini Pa, Simão AMSa, Bolean Ma, Hoylaerts MFb and Millán
JLc
a
Department of Chemistry, FFCLRP-USP, Brazil; bCenter for
Molecular and Vascular Biology, University of Leuven, Leuven,
Belgium; cSanford Children’s Health Research Center, SanfordBurnham Medical Research Institute, La Jolla, CA, USA
During endochondral bone formation, chondrocytes and
osteoblasts are responsible for the synthesis and mineralization of
the extracellular matrix through a carefully orchestrated process,
believed to initiate within membrane-invested matrix vesicles (MVs)
at the sites of initiation of hydroxyapatite (HA) deposition and end
with bone mineral propagation onto the collagenous scaffold.
Acidic phospholipids and other MV components are thought to
nucleate these intravesicular nanocrystals1. As we strive to
understand the physiological interplay between TNAP,
PHOSPHO1, NPP1, and other important MV-associated enzymes
and channeling proteins in the initiation of biomineralization, we
must keep in mind the microenvironment in which these proteins
function, which can have a profound effect on their biological
properties, since phospholipids play an important role in the
initiation of the biomineralization process. The ability of synthetic or
natural vesicles to mimic the organizational structure and function
of biomembranes makes these structures an advantageous and
convenient experimental model to help us advance our
understanding of MV-mediated calcification2. The proteoliposome
system provides a means of reconstituting lipid vesicles that will
function like MVs3-5. Proteoliposomes can be manufactured using
different methods and with controlled lipid and protein composition,
electrolytes and sizes, representing a convenient experimental
model to mimic the organizational structure and function of natural
biomembranes and to reproduce some essential features of the
biomineralization process2,6
1. Millán, Calcif. Tissue Int. 93(4):299-306. (2013).
2. Ciancaglini et al., Biophys. Rev. 4: 67-81
(2012).
3. Simão et al., J. Biol. Chem.285(10):7598-7609
(2010).
4. Bolean et al., Biophy. Chem. 152:74-79
(2010).
5. Bolean et al., Biophy. Chem. 158:111-118
(2011).
6. Ciancaglini et al., J. Bone Miner. Res.
25(4):716-723 (2010)
27
_____________________________________________________
CONFERENCIA 8
Control of the levels of PIP3: Structure and Function of
the lipid kinase PI3Kalpha
b
a
a
a
S.B. Gabelli , Michelle Miller , Ignacia Echeverria , Yunlong Liu ,
B. Vogelstein c, and L M. Amzel a
a
Department of Biophysics and Biophysical Chemistr.,bDepartment
c
of Medicine and Ludwig Center for Cancer Genetics and
Therapeutics and Howard Hughes Medical Institutions, Johns
Hopkins University School of Medicine, Baltimore, MD 21205,
USA.
Phosphatidylinositide-3-kinase-α (PI3Kα) is a lipid kinase that
catalyzes the phosphorylation of PIP2 to produce PIP3 in response
to phosphorylated receptor tyrosine kinases (RTK) or their
substrates. The increased levels of PIP3 initiate a number of
signaling pathways by recruiting other kinases, such as Akt, to the
plasma membrane. The enzyme is composed of two subunits,
p110 and p85, each comprising five domains. PI3Kα is frequently
mutated in many cancer types and the mutations increase PI3K
kinase activity leading to increased tumor cell survival, cell motility,
cell metabolism, and cell cycle progression. Several atomic
resolution structures of the enzyme reveal that the enzyme has a
complex architecture in which each domain interacts with several
domains of the same or the other subunit. The structural and other
data show that physiological activation, as well as activation by
some oncogenic mutations, involves relief of autoinhibition by
dislodging the inhibitory nSH2 domain of the regulatory subunit p85
from its inhibitory position. Computational studies show that most
of these effects involve, in addition to structural changes,
modifications of the dynamics of the protein that alter the relative
stabilities of the different states accessible to the enzyme. Recent
progress toward determining the mechanism of activation benefited
from two developments: the determination of the structure PI3K
bound to short chain phosphoinositides, and the characterization of
the conformations accessible to the activation loop in molecular
dynamics simulations.
28
_____________________________________________________
SIMPOSIOS
29
_____________________________________________________
SIMPOSIO 2
Regulation of Voltage gated sodium channels by
Calmodulin
Gabelli SB, Boto A, Halperin Kuhns V, Bianchet MA, Farinelli F,
Aripirala S. Yodder J, Jakoncic J, Tomaselli GF, Amzel LM.
a
Structural Enzymology and Thermodynamics Group. Department
of Biophysics and Biophysical Chemistry, Johns Hopkins University
School of Medicine, 725 N Wolfe St, WBSB 608, Baltimore,
Maryland 21205, USA; bDivision of Cardiology, Department of
Medicine, Johns Hopkins University School of Medicine, Baltimore,
MD 21205, USA. cDepartment of Oncology, Johns Hopkins
University School of Medicine, Baltimore, MD 21287, USA.
d
Department of Neurology, Johns Hopkins University School of
Medicine, Baltimore, MD 21287, USA. eBrookhaven National
Laboratory, National Synchrotron Light Source, Upton, NY 11973
Voltage gated sodium channel are responsible for the rapid
upstroke of action potential in tissues such as the heart, skeletal
muscle and the brain (VGSC). VGSC fast and long inactivation are
regulated by channel interacting proteins (CIP). We determined the
structure of the complex of the cytoplasmic portion of a VGSC
2+
(Nav1.5; CTNav1.5) with a CIP, calmodulin (CaM), and Mg and
show that both CaM lobes interact with the CTNav1.5. Based on
the conformational differences between this structure and that of
an inactivated complex, we propose that this structure represents a
non-inactivated state of the CTNav1.5, i.e., the state that is poised
for activation. Site-specific mutagenesis and patch clamp
recordings further support the importance of the interactions
identified. The dimerization of the Nav1.5 elucidates the effect of
some dominant negative disease mutations and provides unique
insights into the physiological activation and the pathophysiology of
VGSC channels
30
_____________________________________________________
SIMPOSIO 2
Sterols in membranes: structural features that rule the
interaction with phospholipids
Wenz, JJ
Instituto de Investigaciones Bioquímicas de Bahía Blanca,
Universidad Nacional del Sur, Camino “La Carrindanga” Km 7,
B8000FWB Bahía Blanca, Argentina. Tel +54(291)4861201.
[email protected]
The relationship between sterol structure and the effects on
membrane properties is still controversial. This study introduces a
multivariate analysis that relates the molecular structure with the
activity of sterols on phospholipid bilayers. Chemical structures
were encoded by using binary variables reporting on the
presence/absence
of
substituents
in
the
pentaneperhydrophenanthrene. The sterol activity was encoded
regarding physical properties affected in sterol-containing
membranes. By means of Principal Coordinates Analysis, the
sterol population was grouped into five well-defined clusters
according to structural similarities/differences. An examination of
the sterol distribution between clusters revealed that a hydroxyl
group at C3 and an 8-10 carbon chain at C17 are common in
sterols having rigidifying, molecular ordering/condensing effects
and/or a raft promoting ability. In contrast, sterol containing a keto
group at C3, a C4-C5-double bond, and polar groups or a short
alkyl side-chain at C17 (3 to 7 atoms) are mostly present in sterols
having opposite effects. Combined with Logistic Regression, these
approaches conclude that the most important structural features
affecting the physical properties of sterol-containing mixtures were
the presence of an 8-10 carbon C17 isoalkyl side-chain, followed
by a hydroxyl group at C3 and a C5-C6 double bond. Finally, a
simple structure-based Logistic Regression model that predicts the
activity of sterols is proposed.
31
_____________________________________________________
SIMPOSIO 2
Lipid and protein aggregates activate receptors of the
innate system.
J-M Ruysschaert and C. Lonez –
Free University of Brussels-Belgium- [email protected]
Toll-like receptors are major members of the Pattern Recognition
Receptors (PRRs) from the innate immune system, which
recognize bacterial or viral components. It was suggested that
those receptors, that usually recognized molecular patterns
characteristic of pathogens, are activated by lipid and protein
ligands aggregated into particles and structurally different from the
natural ligands. We will illustrate this aspect with two examples
related to nanoparticles and neurodegenerative diseases (1,2)
It is hard to believe that molecules which are so different from
natural ligands do activate receptors the same way natural ligands
do. How lipid and protein aggregates made of a large number of
molecules activate pattern recognition receptors is still unknown
but it is very likely that it proceeds via a mechanism quite different
from what has been described so far for monomeric natural
ligands. Implications in nanotechnologies(3) and nanomedicine will
be briefly discussed.
1. Lonez C, Vandenbranden M, Ruysschaert JM.-Adv Drug
Deliv Rev. 2012,64,1749-58
2. Gustot A, Raussens V, Dehousse M, Dumoulin M, Bryant
CE, Ruysschaert JM, Lonez C.-Cell Mol Life Sci. 2013
Aug;70(16):2999-3012
3. Lonez C, Bessodes M, Scherman D, Vandenbranden M,
Escriou V, Ruysschaert JM.
Nanomedicine. 2014 10(4):775-82
32
_____________________________________________________
SIMPOSIO 2
Structural Characterization of Heparin-induced GAPDH
Protofibrils Preventing α-synuclein Oligomeric Species
Toxicity
César Ávila1, Clarisa Torres-Bugeau1, Leandro Barbosa2, Elisa
Morandé Sales2, Mohand Ouidja3,4, Sergio Socías3, M. Soledad
Celej5, Rita Raisman-Vozari3, Dulce Papy-Garcia4, Rosangela Itri2,
and Rosana N. Chehín1,
1
INSIBIO-CONICET-UNT(Tucumán) Argentine.2IFUSP (São Paulo)
Brazil.3INSERM-CRICM, ICM, Thérapeutique Expérimentale de la
neurodégénérescence (Paris) France.4CRRET, Université Paris
Est Créteil, France.5 CIQUIBIC, CONICET-UNT (Córdoba)
Argentine.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a
multifunctional enzyme associated to neurodegenerative diseases.
In a previous work, we showed that glycosaminoglycans-induced
GAPDH prefibrilar species accelerates the conversion of αsynuclein to fibrils. However, it remained to be determined whether
the interplay among glycosaminoglycans, GAPDH and α-synuclein
has a role in pathological states. Here we demonstrate that the
toxic effect exerted by α-synuclein oligomers in dopaminergic cell
culture is abolished in the presence of GAPDH prefibrilar species.
Structural analysis of prefibrilar GAPDH performed by Small angle
X-ray scattering, showed a particle compatible with a protofibril.
Using biocomputational techniques we obtained the first all-atom
model of the GAPDH protofibril, which was validated by crosslinking coupled to mass spectrometry experiments. Since GAPDH
can be secreted outside the cell where glycosaminoglycans are
present, it seems plausible that GAPDH protofibrils could be
assembled in the extracellular space kidnapping α-synuclein toxic
oligomers. Thus, the role of GAPDH protofibrils in neuronal
proteostasis must be considered. The data reported herein could
open alternative ways in development of therapeutic strategies
against synucleinopathies like Parkinson’s disease.
33
_____________________________________________________
SIMPOSIO 3
Synaptic gain-of-function effects of mutant CaV2.1
channels in a mouse model of familial hemiplegic
migraine are due to increased basal [Ca2+]i.
Di Guilmi MN1; González Inchauspe C1; Forsythe I2; van den
Maagdenberg A3; Borst JG4; Uchitel OD1.
1
IFIBYNE, UBA- CONICET, Buenos Aires, Arg.; 2MRC Tox. Unit,
U.Leicester, Leicester. UK.; 3 Dep. of Hum Gen & Neurolog and
4
Dep. of Neurol, Leiden U., Leiden, The Netherlands, Dep. of
Neurosc, Erasmus MC, Rotterdam, The Netherlands.
Specific missense mutations in the CACNA1A gene, which
encodes a subunit of voltage-gated CaV2.1 channels, are
associated with familial hemiplegic migraine type 1 (FHM1), a rare
monogenic subtype of common migraine with aura. We used
transgenic knock-in (KI) mice harboring the human pathogenic
FHM1 mutation S218L to study presynaptic Ca2+ currents and
EPSCs in the MNTB-calyx of Held synapse. Whole-cell patchclamp recordings of presynaptic terminals from S218L KI mice
showed a strong shift of the calcium current I-V curve to more
negative potentials, leading to an increase in basal [Ca2+]i,
increased levels of spontaneous transmitter release, faster
recovery from synaptic depression, and enhanced synaptic
strength despite smaller action-potential-elicited Ca2+ currents. This
synaptic phenotype may explain the misbalance between
excitation and inhibition in neuronal circuits resulting in a persistent
hyperexcitability state and other migraine-relevant mechanisms
such as an increased susceptibility to cortical spreading
depression.
34
_____________________________________________________
SIMPOSIO 3
ABA-1A: a Nematode Polyprotein Allergen (NPA) of
Ascaris suum. Structure and binding properties.
1
1
2
2
Franchini,GR ; Bélgamo JA , Kennedy, MW ; Smith, BO and
Córsico, B1
1
2
INIBIOLP-CONICET, Fac.de Ciencias Médicas, Universidad de La Plata, Argentina .
Institute of Biomedical & Life Sciences, University of Glasgow, United Kingdom
The acquisition and transport of lipids from their hosts is crucial to
parasitic helminths, being the proteins and receptors involved in
lipid transport and exchange potential targets for chemo- and
immunotherapy. Among helminth lipid binding proteins (LBPs), the
polyprotein allergens/antigens of nematodes (NPAs) represent a
novel class of lipid binding proteins which has been described
exclusively in nematodes. NPAs are small, helix-rich proteins, and
have no known structural counterparts in other phyla. The
biochemical activity of the NPA of A. suum was first described as a
binding protein for small lipids such as fatty acids and retinoids.
Recently, the structure of a single unit of the polyprotein array
(ABA-1A) has been solved in the presence of saturating
concentration of oleic acid describing two binding sites. In the
present project we are working with ABA-1A in the absence of the
ligand (apo- form) and its atomic structure is under analysis
employing NMR spectroscopy, for which high quality data have
already been obtained and full structure calculation is in progress.
In order to obtain more information about the structural
perturbations due to ligand binding; an oleic acid titration of ABA1A monitored by NMR spectroscopy was performed. Briefly, it was
possible to distinguish two binding events according to the nature
of the perturbations observed. Additionally, as a first attempt to
determine the natural ligands bound by this protein a lipidomic
analysis was done using recombinant ABA-1A without the
delipidation step.
35
_____________________________________________________
SIMPOSIO 3
Membrane role in the rational design of HIV fusion
inhibitors
1,2
1
1
Axel Hollmann , Marcelo T. Augusto , Susana Gregório , Pedro
M. Matos1, Matteo Porotto3, Antonello Pessi4, Miguel A. R. B.
Castanho1 and Nuno C. Santos1
1
Instituto de Medicina Molecular, Faculdade de Medicina,
Universidade de Lisboa, Portugal; 2Laboratory of Biointerfaces
and Biomimetic Systems- CITSE- CONICET, Argentina. 3 Weill
Medical College, Cornell University, New York, USA;
4
PeptiPharma, Rome, Italy.
The Human Immunodeficiency Virus type 1 (HIV-1) is a highly
pathogenic and evasive virus, for which no cure has yet been
achieved. Fusion of viral and host cell membranes is a crucial step
in virus infectivity, therefore the development of viral entry inhibitors
has great advantages since they prevent the release of the viral
content into the host cell. In this work, we found that boosting
membrane affinity of the established HIV fusion inhibitors C34 and
enfuvirtide, by conjugation with lipid moieties, results in a dramatic
increase of their antiviral activity. We demonstrated, by fluorescent
partition, dipole potential assays and surface pressure assays, that
these novel fusion inhibitors have membranotropic behavior
towards biomembrane model systems and human blood cells
membranes. The ability that these peptides have to bind to cell
membranes facilitates the delivery of the peptides to this confined
environment, as some peptide is already locally present. Beside
the ability of membranes to concentrate the inhibitors locally, their
role in the presentation of the peptide in the proper orientation for
gp41 binding should also be considered. In this context, we also
evaluated the location of the different peptides in the membrane,
using aqueous and lipophilics quenchers. Overall, the results of
this study offer a rational basis for the design of improved viral
fusion inhibitors, since they suggest that maximizing antiviral
activity requires finding the proper balance of membrane affinity
and exposure of the peptide moiety. Moreover, we offer an
experimental strategy to guide the development of the structureactivity relationship (SAR) towards this goal.
36
_____________________________________________________
SIMPOSIO 3
Effects of nitric oxide on heart mitochondrial calcium
handling
1
2
2
Zaobornyj T. , Sivakumaran V. , O’Rourke B.
Institute of Biochemistry and Molecular Medicine (IBIMOL, UBACONICET), School of Pharmacy and Biochemistry, University of
2
Buenos Aires, Buenos Aires, Argentina; Institute of Molecular
Cardiobiology, School of Medicine, The Johns Hopkins University,
Baltimore, USA
1
Mitochondria provide the cells with both the energy and with the
signals that command cell death and survival. Indeed,
mitochondria are involved in the production of reactive oxygen
species and in Ca2+ handling. The aim of this work was to evaluate
heart mitochondrial function, in order to establish the effects of NO
and Ca2+ in energy metabolism. Guinea pig heart mitochondria
were exposed to NO released from GSNO and SNAP.
Mitochondrial NO production, ∆Ψ and Ca2+ uptake were followed
simultaneously using a spectrofluorometer. Isolated mitochondria
O2 consumption was assessed using an extracellular flux analyzer.
Energized mitochondria were submitted to Ca2+ pulses (10 µM) up
to a final concentration of 80-100 µM (200-450 nmol/mg protein),
showing no significant alterations in matrix volume and ∆Ψ. In the
presence of NO donors (25 to 100 µM), Ca2+ uptake was slower
and extramitochondrial Ca2+ concentration increased. When single
50 µM Ca2+ pulses were added, mitochondria treated with NO
donors showed a decreased Ca2+ accumulation rate (40-50%) with
an IC50 of ≅ 400 µM (180 nM NO). The addition of L-arginine or
NOS inhibitors to control mitochondria produced changes in Ca2+
uptake and in DAF-FM signal. State 4 O2 uptake was not modified
by NO. The addition of Ca2+ to the medium produced a 20%
enhancement in state 4-O2 consumption and this effect was
abolished by NO. These results suggest that Ca2+ and NO act as
signals that coordinate cytosolic workload and mitochondrial
energy metabolism.
37
_____________________________________________________
SIMPOSIO 4
Mathematical models for cytoskeletal transport and
cellular viscoelasticity
Bouzat, S.
Grupo de Física Estadística e Interdisciplinaria, Centro Atómico
Bariloche (CNEA). Argentina.
CONICET. Argentina
Molecular motors use the energy from ATP hydrolysis to move
cargoes of radius 100-1000 nm along the cytoeskeletal filaments.
For instance, the motor proteins kinesin and dynein drive cargo
transport along microtubules, while myosin motors move on actin
filaments. Due to the fact that the cytoplasm is a complex
environment which is crowded by polymers and macromolecules,
the motors have to overcome viscous and elastic responses from
the medium. In this contribution we present a general model for
active cargo transport inside cells [1,2]. It combines a Langevin
description of the cargo motion with a Monte Carlo dynamics ruling
the motor stepping, binding and unbinding processes. We also
show the way in which the elastic responses of the medium can be
taken into account [3]. This is done by considering a Generalized
Langevin equation for the cargo motion or, equivalently, by
introducing virtual environmental particles which interact
harmonically with the cargo [3,4]. We discuss some of the
modeling results in connexion with recent experiments in the
literature.
1- A. Kunwar and A. Mogilner. Phys. Biol. 7 (2010) 016012.
2- S. Bouzat and F. Falo. Phys.Biol. 8 (2011) 066010 .
3- S. Bouzat. Phys. Rev. E. 89 (2014) 062707.
4- I. Goychuk, V. O. Kharchenko and R. Metzler. PLoS ONE 9
(2014), e91700.
38
_____________________________________________________
SIMPOSIO 4
Using Machine Learning for Predictive Modeling on
Systems Biology and Molecular Informatics
Ponzoni, I
Instituto de Ciencias e Ingeniería de la Computación (Universidad
Nacional del Sur, CONICET)
The use of machine learning methods for predictive modeling in life
sciences is growing during last decades. In this lecture, we will
present the development of different computational strategies
based in these approaches in the context of Molecular Informatics
and Systems Biology. The first part of this lecture is related to the
development of quantitative structure–activity relationship (QSAR)
models. These models play a central role in several industrial
applications, such as drug discovery and design of new materials.
The design of QSAR/QSPR models requires to solve several
mathematical problems. For dealing with these issues, we are
working in the design of computational methodologies based on
machine learning and visual analytics for assisting experts in the
design of new QSAR/QSPR models. The second application
presented in this talk is related with the field of Systems Biology.
We will focus on gene regulation and the ways that transcriptome
data can be used to unravel the complex relationships between
genes and pathways. In particular, we will describes the main
topics that must be considered in the field of association rule
mining for reverse engineering of biological networks and some
techniques currently available in the literature.
1. Ponzoni I., Nueda M.J., Tarazona S., Götz S., Montaner
D., Dussaut J.S., Dopazo J., Conesa A. “Pathway network
inference from gene expression data”, BMC Systems
Biology, 8(S2):S7, 2014.
2. Soto, A.J., Vazquez, G.E., Strickert, M., Ponzoni, I.
“Target-driven subspace mapping methods and their
applicability domain estimation”, Molecular Informatics,
30:779–789, 2011.
39
_____________________________________________________
SIMPOSIO 4
Water at the nanoscale: Towards new principles and
design elements for biophysics
Appignanesi, G. A.
INQUISUR-UNS-CONICET and Depart.de Química, Universidad
Nacional del Sur, Bahía Blanca.
A full comprehension of the behavior of water, the matrix of life, at
the nanoscale would be essential in order to understand biology at
a molecular level. In biological organization, water acts as a
mediator between complex surfaces that associate by means of
non-covalent interactions, producing nanoconfined environments
where descriptions borrowed from the bulk might be useless and,
thus, the development of a new intuition is demanded. For
instance, as two hydrophobic surfaces in aqueous solution
approach each other to a nanometric separation, the
thermodynamic properties of the hydration water are affected to
the point to produce the "drying" that triggers hydrophobic collapse.
Also, a ligand is expected to replace easily removable hydration
water in order to bind to a protein. Thus, the knowledge of how
different
nanoconfinement
conditions
affect
the
local
hydrophobicity and modulate non-covalent interactions is of great
significance both to understand and predict the behavior of
biological systems and to emulate them in bioengineering efforts
based on rational design. In this talk we shall present some
preliminary studies aimed at identifying generalizable principles
towards a picture of nanoconfined water of interest in biophysics.
First, we will focus on model systems studying a number of
properties such as water density fluctuations. We shall investigate
generic contexts with controlled chemistry and geometry in order to
determine how chemical topology and topography define the local
hydrophobicity. We also aim at determining the contextdependence and the (possibly non-additive) mutual interplay
between different non-covalent interactions, attentive to the
detection of design elements of general validity. Finally, we shall
also tackle more specific contexts such as protein binding, the
rational design of small molecules predecessors of disruptive drugs
and membrane hydration.
40
_____________________________________________________
SIMPOSIO 5
How do Sco Proteins Score the COX?
1
2
1
Morgada MN , Abriata LA and Vila AJ
1
Laboratorio de Metaloproteínas, Instituto de Biología molecular y
Celular de Rosario (IBR-CONICET), Depto. de Qca. Biol, Fac. de
Cs. Bioq. y Farm, Universidad Nacional de Rosario (UNR),
Argentina. 2Swiss Federal Institute of Technology, École
Polytechnique Fédérale de Lausanne (EPFL), Switzerland
The dinuclear copper center CuA is the electron entry point of the
cytochrome c oxidase (COX) and the correct assembly of this
metal center is essential for the function of the complex and thus
for the survival of the cell.
Following the copper transfer reactions using NMR, our group
proposed a mechanism for the insertion of the copper ions to the
CuA center of bacterial COX. Bacterial Sco, despite its ability to
bind copper ions, keeps the cysteines from the CuA center in the
reduced state thus the copper ions can be transferred from a
periplasmic metallochaperone (PCuAC).
In the case of humans, biochemical studies shown several proteins
involved in the assembly of COX. Two of the identified proteins,
Sco1 and Sco2 are directly involved in the formation of the CuA
center in subunit II (COX II) and structural studies showed that
both proteins can act as metallochaperones or as thiol-disulphur
oxidoreductases since both proteins has two Cys residues in their
copper biding site.
The unavailability of the soluble domain of an eukaryotic COX II
has stalled further investigation but using a model of the eukaryotic
oxidase (COX II*), we have been able to assess the function of
these Sco proteins finding that Sco1 act as copper transfer protein
while Sco2 is crucial in the maintenance of the cysteines redox
state homeostasis.
41
_____________________________________________________
SIMPOSIO 5
On the Search of a Comprehensive Representation of
Human Frataxin
1
1
2
1
Aran M , Faraj SE, Gallo M , Noguera ME , González Lebrero R ,
Roman EA1 and Javier Santos1
1
Fundación Instituto Leloir and IIBBA-CONICET, Av. Patricias
Argentinas 435, 1405, 2IQUIFIB, Universidad de Buenos Aires,
Junín 956, 1113AAD, Buenos Aires.
Friedreich’s ataxia (FRDA) is a neurodegenerative disease linked to a
deficiency of frataxin (FXN), a protein involved in iron-sulfur cluster
biosynthesis. Human FXN contains a folded C―terminal domain starting
at residue 90 with α/β topology followed by a C-terminal region (CTR) that
packs against the protein’s core. We have investigated the impact of the
alteration of the CTR on the stability, internal dynamics and folding
dynamics of FXN. The pathological mutation L198R yields a global
destabilization and a significant and highly localized alteration of
dynamics, mainly involving residues that are in contact with L198 in wildtype FXN. Variant FXN90―195, which is closely related to the
FRDA―associated mutant FXN81―193, preserves its native-like
structure. However, the truncation of the CTR results in an extreme
decrease of global stability and alteration of protein dynamics over a vast
range of timescales, including regions far from the CTR. Moreover, both
mutants exhibit an important deficiency in iron-binding, suggesting
coupling dynamics between the CTR and the acidic ridge (helix α1, loop1,
strand β1) involved in iron binding. The increased sensitivity to proteolysis
observed in vitro, the reduced ability to bind iron, and the enhanced
tendency to aggregate exhibited by the truncated variant may explain why
the alteration of the CTR causes FRDA. The alteration of the dynamics
and stability of FXNL198R is in line with the rapid disease progression
observed in patients carrying this mutation. Folding kinetic experiments
firmly suggest that the change in global stability observed in CTR
mutations is most probably caused by a native-state destabilization than
by a change in the stability of the transition state ensembles. These results
contribute to understanding how stability and activity are linked to protein
motions, and might be valuable for the design of target-specific binders to
control local protein motions for stability and activity enhancement.
Authors
are
listed
in
alphabetical
order.
Correspondence:
[email protected]. Thanks to UBACyT, CONICET and ANPCyT.
42
_____________________________________________________
SIMPOSIO 5
Ligand binding mechanisms of bitter taste receptors:
Insights from multiscale simulation approaches
Giorgetti, Alejandro
Computational Biophysics, German Research School for
Simulation Sciences, Juelich, Germany and Department of
Biotechnology, University of Verona, Verona, Italy
Sensing chemicals present in the food is of fundamental
importance for the survival of the species. Life evolved a host of
mechanisms to communicate chemical information from the
environment to elicit cellular responses that provide an advantage
in avoiding or seeking the chemical signatures of foods, mates,
toxins, etc. In particular, mammals during evolution have been
prevented from ingesting toxic compounds because of their strong
bitter taste. This protection mechanism has been carried out by a
family of 25 bitter taste receptors (TAS2Rs). A characterization of
the mechanisms underlying their function is lacking. Here I will
present our studies on the interaction of TAS2R38 [1] with two of
its agonists. Indeed, by using a combination of homology models
together with docking [2], molecular mechanics/coarse-grained
(MM/CG) simulations [3] and experimental data, we were able to
provide a detailed description of the ligand-binding site in the
receptors, satisfying site-directed mutagenesis experiments [4].
Our approach could be used for other GPCRs with direct
applications in drug design.
1.
2.
3.
4.
Biarnés X.;et al., PLoS ONE 5(8): e12394 (2010).
Sandal, M et al. Plos ONE. 8(9): e74092 (2013)
Leguèbe M, et al.. PLoS ONE 7(10): e47332 (2012).
Marchiori et al. PLoS ONE 8(5): e64675. (2013)
43
_____________________________________________________
SIMPOSIO 5
A simple model to simulate protein-membrane
interactions with inclusion of electrostatics.
1
2,3
2,3
1
Villarreal MA , Emperador A , Sfriso P , Leiva EPM , Orozco
2,3,4,5
M
.
1
INFIQC-Dpto Matemática y Física. CONICET-Universidad
Nacional de Córdoba. Argentina. 2Institute for Research in
Biomedicine, Barcelona, Spain. 3Joint IRB-BSC Program in
Computational Biology, Barcelona, Spain. 4Barcelona
Supercomputing Center, Barcelona, Spain. 5Department of
Biochemistry and Molecular Biology, University of Barcelona,
Spain.
We present a minimalistic model to describe protein-membrane
interactions with inclusion of electrostatics. One bead per
aminoacid is used to describe protein structure, centered in the
alpha carbon position. Structure-based potentials were used to
model particle-particle interactions within the protein. The lipid
bilayer is represented as five slabs, which account for the water
phase, the lipid headgroup, and the membrane interior. The
interaction of the protein with the membrane is modeled with two
terms. One is based on lipid-water partition coefficients and the
exposed surface area of each amino acid, while the second term
accounts for the electrostatic interaction with charged residues
only. Both interaction potentials are modeled with discrete changes
in energy, and therefore can be used in discrete molecular
dynamics simulations (DMD). Here we present the calibration of
the model against experimental data of binding constants, and also
selected applications of the model.
44
_____________________________________________________
SIMPOSIO 6
Mechanical and thermodynamic properties of lipid
membranes using optical microscopy
Wilke, N
Centro de Investigaciones en Química Biológica de Córdoba
(CIQUIBIC). Departamento de Química Biológica, Facultad de
Ciencias Químicas, Universidad Nacional de Córdoba. E-mail:
[email protected]
Depending of the conditions and of the mixture, lipid membranes
may show phase segregation with a fluid phase and a denser
phase coexisting in the film. The distribution of the phases (texture
of the membrane) depends of the line tension, the electrostatic
repulsion and of curvature effects of each phase, while the amount
of each phase depends of the energy of mixing of the lipids and of
the proportions of them in the mixture. The relative amount of the
phases and the manner in which they distribute modulates the
compressibility and the apparent viscosity of the membrane.
In this presentation, we will discuss how the line tension,
electrostatic interaction and phase diagram of lipid mixtures can
be determined using optical microscopy. The membrane was
studied by pasive two-particle rheology and manipulating the film
with electrical fields.
Results found with different models of biomembranes are
compared and the effect of two phases on the mechanical
properties of the film is also described. The molecular determinants
for the line tension in a system are discussed along with the
possibility of its modulation with linactants.
Acknowledgements: SecyT-UNC, CONICET and FonCyT (PICT
2012-0344)
45
_____________________________________________________
SIMPOSIO 6
Dynamics and interactions of transcription factors in the
cell nucleus through fluorescence fluctuation
spectroscopy
Levi, V.
Departamento de Química Biológica- IQUIBICEN. Facultad de
Ciencias Exactas y Naturales-Universidad de Buenos AiresArgentina
Fluorescence fluctuation spectroscopy (FFS) methods are powerful
techniques to explore the dynamical organization of cells. These
methods are based on analyzing the fluorescence intensity
fluctuations caused by molecules moving through the observation
volume of confocal or two-photon excitation microscopes. The
quantitative analysis of these fluctuations provides important clues
regarding the mobility of the molecules and their interactions with
other species. In this talk, we show applications of different FFSbased methods to explore relevant aspects of the mechanisms of
action of transcription factors (TFs). First, we demonstrate the
utility of
fluorescence correlation spectroscopy (FCS) for
measuring the dynamics of the TFs Oct4, Sox2 and Cdx2 involved
in embryonic development at the level of a single cell in early
mouse embryos. The combination of Monte Carlo simulations with
photoactivatable FCS allowed us to distinguish specific and nonspecific DNA binding of TFs and track their dynamics during
embryonic development. In another example of application of FFS
methods, we studied the mechanism of action of the glucocorticoid
receptor (GR) though number and brightness analysis (N&B).
Glucocorticoids bind to GR, a ligand-dependent transcription factor,
and regulate gene expression directly by binding to DNA or
indirectly by modulating the activity of other transcription factors. It
is currently accepted that the direct pathway is mostly responsible
for glucocorticoids side-effects and that the oligomerization state of
the GR determines which pathway (direct or indirect) will prevail.
Our analyses allowed us to directly measure the oligomerization
state of GR and thus to test this established model.
46
_____________________________________________________
SIMPOSIO 6
Far-field fluorescence nanoscopy of neurons
Bordenave, M
Centro de Investigaciones en Bionanociencias - CIBION,
CONICET, Godoy Cruz 2390, C1425FQD Buenos Aires.
Departamento de Física, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires.
Far-field fluorescence nanoscopy methodologies, also known as
super-resolution fluorescence microscopy are the future of
fluorescence based visualization. This family of techniques include
wide-field and scanning methods, such as PALM/STORM and
STED, respectively, and provide spatial resolutions of 20-80 nm
maintaining the low invasivity of fluorescence microscopy. In this
contribution I will first present the fundamental concepts of
fluorescence nanoscopy and describe the most popular
methodologies, remarking their comparative advantages and
limitations. Secondly, I will present the fluorescence nanoscopy
facilities at CIBION, which are available through the Sistema
Nacional de Microscopía and finally, I will give some examples of
the biological questions being currently addressed with nanoscopy
in our group: i) Restructuring of citoesqueleton of growth cones
and initial axon segments during the neuronal polarization process
(in collaboration with the group of Alfredo Cáceres - INIMEC,
CONICET), and ii) restructuring of pre- and post-synapse regulated
by Nedd8 (in collabotation with the group of Damián Refojo IBIOBA, CONICET).
47
_____________________________________________________
SIMPOSIO 7
Two signaling pathways mediate presynaptic voltage
gated calcium channels inhibition by two ghrelin
receptor activation modes.
Jesica Raingo.
Electrophysiology Laboratory. Multidisciplinary Institute of Cell
Biology (IMBICE)
La Plata Buenos Aires
Argentina
[email protected]
Growth hormone secretagogue receptor type 1a (GHSR1a) has
the highest constitutive activity of any G protein coupled receptor
(GPCR). GHSR1a mediates the orexigenic effects of the gutderived hormone ghrelin. It is a therapeutic target of anti-obesity
drugs and of several eating disorder treatments. GHSR1a is
present at presynaptic terminals in the hypothalamus and it
regulates neuronal activity, but the mechanism of its actions
remains poorly understood. Presynaptic voltage gated calcium
channels, CaV2.1 and CaV2.2, control neurotransmitter release and
their activities are modulated by GPCRs. Here we show that
constitutive as well as agonist-dependent GHSR1a activation
triggers a strong impairment of both CaV2.1 and CaV2.2 currents.
Constitutive GHSR1a activity reduces CaV2 currents by a Gi/odependent mechanism that involves persistent reduction in
channel numbers in the plasma membrane, whereas, ghrelindependent GHSR1a inhibition is reversible and involves altered
CaV2 current gating via a Gq-dependent pathway. Thus we show
that GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq
pathways depending on whether GHSR1a activation is constitutive
or ghrelin-dependent respectively, and probably impacting on
neuronal activity.
48
_____________________________________________________
SIMPOSIO 7
Redox modulation of the GABAergic neurotransmission
in the retina and the hippocampus.
Calvo, D.J.
INGEBI CONICET
y-aminobutyric acid (GABA) is the major inhibitory neurotransmitter
in the central nervous system and its actions are mediated by two
receptor classes: ionotropic (GABAA) and metabotropic (GABAB).
GABAA receptors are ligand-gated chloride channels that belong to
the superfamily of “Cys-loop” receptors which also include the
nicotinic acetylcholine, glycine and serotonin 5HT3A receptors.
Heteromeric and homomeric GABAA receptor variants can be
assembled from a great diversity of subunit subtypes (α1-6, β1-3,
γ1-3, δ, ε, θ, π, ρ1-3).
Studies from our lab revealed that the extra and intracellular levels
of ascorbic acid in the retina regulate the function of tonic
(GABAAρ1) and phasic (GABAAα1β2γ2) receptors expressed in
bipolar cells. We have also demonstrated that tonic GABAAρ1
receptors are sensitive to endogenous redox agents such as
ascorbic acid, glutathione, reactive oxygen species and reactive
nitrogen species. The aminoacidic residues which act as targets for
redox agents were identified using site directed mutagenesis and
receptor expression in oocytes. Preliminary results indicate that
responses mediated by tonic and phasic GABAergic receptors in
the hippocampus are also subject to redox modulation. Taken
together our results suggest that inhibitory neurotransmission
mediated by ionotropic GABA receptors is modulated by multiple
pathways involving redox signaling.
49
_____________________________________________________
SIMPOSIO 7
Proton permeation in Ci-Hv1 voltage-gated proton
channels occurs through a proton wire involving
residues D160 and D222 and it is modulated by N264.
Amaury Pupo1,2, David Baez-Nieto1, Ester Otarola1, Osvaldo
Yañez3, Ariela Vergara-Jaque3, Wendy Gonzalez3, Karen Castillo1,
Gustavo Contreras1, Ramón Latorre1, Carlos Gonzalez1.
1
Centro Interdisciplinario de Neurociencia de Valparaiso.
2
Universidad de Valparaiso, Chile. Doctorado en Ciencias,
mención Neurociencias. Universidad de Valparaiso. 3Universidad
de Talca, Chile.
Hv1 channels are integral membrane proteins with the capacity to
selectively permeate protons in a voltage and pH dependent
manner. As Hv1 lacks a pore domain, permeation must occur
through the voltage-sensing domain. Previous reports propose a
permeation pathway consisting in an stable water wire which
allows proton to permeate by a Grotthuss mechanism. Our
molecular dynamics simulations do not support the formation of
such stable water wires throughout all the channel pore, existing a
dry zone around residue N264. Mutations in position 222 and 264
affects single channel conductance (determined by non-stationary
noise analysis) and selectivity, suggesting that both residues are
involved in the permeation pathway. Quantum dynamics
simulations performed in our models of Ci-Hv1 wt and mutants
suggest that permeation occur through a proton wire involving
residues D160 and D222, a process modulated by N264.
Supported by Beca de Doctorado Nacional para Extranjeros de
Conicyt (A.P), FONDECYT Grants 1110430 (R.L.), 1120802 (C.G.);
ANILLO Grant ACT1104 (C.G.); Postdoctoral Fellowships 3140590
(G.F.C.). CINV is a Millennium Institute.
50
_____________________________________________________
SIMPOSIO 7
The role of auxiliary β1 subunit of the BKCa channel as
a tissue selective target for endogenous and exogenous
substances
Milesi V.
Laboratorio de Canales Iónicos, IIFP CONICET-UNLP, La Plata,
Argentina.
BKCa channel is activated by membrane depolarization and
intracellular Ca2+ concentration. It is a tetramer of pore-forming αsubunits (encoded by KCNMA1 gene, also named Slo1) and can
be associated with one of four auxiliary β-subunits codified by
KCNMB1-4 genes. Each one of these subunits confers distinctive
functional and pharmacological properties of the resulting α–β
channel complex. Differential expression of β-subunits could
explain the diversity of functions and regulations on BKCa channel
among diverse tissues and cells, and could be used to obtain a
tissue-selective BKCa channel activation, using the auxiliary
subunit as a specific molecular target. An example of this is the
activation of BKCa channels by lithocholate, which is only
observed in presence of β1-subunit. Using patch-clamp technique
on isolated human vascular smooth muscle cells (VSMCs), we
showed that arachidonic acid (AA), a physiologically relevant 20carbon omega-6 polyunsaturated fatty acid, increases the open
probability (Po) of BKCa channel tenfold, mainly by a reduction of
closed dwell times and also induces a left-shift in Po versus
voltage curves without modifying their steepness. Furthermore, AA
accelerates the kinetics of the voltage channel activation by a
fourfold reduction in latencies to first channel opening. When AA
was tested on BKCa channel expressed in HEK cells with or
without the β1-subunit, activation only occurs in the presence of
the β1-subunit. Understanding the molecular basis of the
interactions of the α and β subunits in relation to the selective
action of endogenous and exogenous substances define another
novel physiological modulation point to the role that the BKCa
channel plays in each tissue.
51
POSTERS
_____________________________________________________
POSTERS
52
BIOENERGETICS AND ELECTRONIC TRANSFER
_____________________________________________________
BIOENERGETICS
AND ELECTRONIC
TRANSFER
53
_____________________________________________________
BET-1] Inhibition of mitochondrial complex III by NO
involves ubisemiquinone formation
Iglesias, D.E; Bombicino, SS; Valdez, L.B; Boveris, A
Institute of Biochemistry and Molecular Medicine (IBIMOL; UBACONICET), Physical Chemistry Division, School of Pharmacy and
Biochemistry, University of Buenos Aires, Junín 956, C1113AAD,
The effect of NO on mitochondrial complex III was studied using
bovine heart-submitochondrial particles (SMP). GSNO (25-500 µM)
and SPER-NO (2.5-30 µM) inhibited complex II-III activity (222 ± 4
nmol/min.mg protein) in a concentration dependent manner, and
produced a hyperbolic increase in O2•- production rate (up to 1.3 ±
0.1 nmol/min.mg protein). Considering that complex III produces
O2•- by autoxidation of ubisemiquinone (UQH•), the aim of this work
was to evaluate through EPR the formation of UQH• as a result of
NO interaction with complex III. In the presence of succinate, SMP
showed an EPR signal at g~1.99 that could be attributed to UQH•
implicated in the Q cycle. This signal was 42% increased by
antimycin addition. The incubation of SMP with antimycin plus
myxothiazol avoided the UQH• formation. The UQH• signal was
completely absent if the SMP were previously denatured. In the
presence of 500 µM GSNO (~1.3 µM NO) or 20 µM SPER-NO
(~1.0 µM NO), the EPR signal was increased by 33% and 34%,
respectively. When GSNO plus mixothiazol were simultaneously
added to the reaction medium, the signal was not observed,
similarly to the effect observed by the addition of antimycin plus
myxothiazol. The EPR spectra obtained under N2 atmosphere
were similar to the ones obtained in air saturated conditions,
suggesting that UQH• signal is not caused by NOx species on
complex III area. In conclusion, NO inhibits the ubiquinonecytochrome b area leading to an UQH• steady-state concentration
enhancement which, in turn, increases O2•- production rate
54
_____________________________________________________
BET-2] Complex
I
functionally
interacts with
mitochondrial nitric oxide synthase (mtNOS)
Bombicino, S.S; Iglesias, D.E; Zaobornyj, T; Boveris, A; Valdez
L.B.
Institute of Biochemistry and Molecular Medicine (IBIMOL; UBACONICET), Physical Chemistry Division, School of Pharmacy and
Biochemistry, University of Buenos Aires, Junín 956, C1113AAD,
The functional association between complex I and mitochondrial
nitric oxide synthase (mtNOS) was studied. Bovine heart
2+
phosphorylating electron transfer particles (ETPH-Mg ) showed a
+
NAD reductase activity of 13.6 ± 0.7 nmol/min.mg protein,
sustained by reversed electron flow of the respiratory chain, at
expenses of ATP when succinate was added. This activity was
inhibited by rotenone (88%), oligomycin (98%) and m-CCCP
(93%). ETPH-Mg2+ produced NO at a rate of 0.79 ± 0.06 nmol
NO/min.mg protein by the classic NOS reaction. In the presence of
0.5 mM MgCl2 and 0.3 mM KCN and of the compounds needed to
carry out the reverse electron flow, ETPH-Mg2+ produced 0.46 ±
0.03 nmol NO/min.mg protein by electron transfer from complex I
to mtNOS. Rotenone inhibited (80%) mtNOS activity supported by
reversed electron flow, but that inhibitor did not reduce the activity
of isolated nNOS, indicating that the inhibitory effect of rotenone on
NO production by ETPH-Mg2+ is due to an electron flow blockage
and not to a direct action on mtNOS structure. A heart
mitochondrial fraction enriched in complex I was recognized by
anti-nNOS antibodies. Altogether, the data obtained in ETPH-Mg2+
suggest that complex I interacts physically and functionally with
mtNOS. Electrons from reversed electron flow or from complex I
reductants support NO production, in agreement with the
dependences of mtNOS activity on mitochondrial metabolic state
and on membrane potential.
55
_____________________________________________________
BET-3] Effects of nitric oxide on heart mitochondrial
calcium handling
1
2
2
Zaobornyj T. , Sivakumaran V. , O’Rourke B.
Institute of Biochemistry and Molecular Medicine (IBIMOL, UBACONICET), School of Pharmacy and Biochemistry, University of
Buenos Aires, Buenos Aires, Argentina; 2Institute of Molecular
Cardiobiology, School of Medicine, The Johns Hopkins University,
Baltimore, USA.
1
Mitochondria provide the cells with both the energy and with the
signals that command cell death and survival. Indeed,
mitochondria are involved in the production of reactive oxygen
species and in Ca2+ handling. The aim of this work was to evaluate
heart mitochondrial function, in order to establish the effects of NO
2+
and Ca in energy metabolism. Guinea pig heart mitochondria
were exposed to NO released from GSNO and SNAP.
Mitochondrial NO production, ∆Ψ and Ca2+ uptake were followed
simultaneously using a spectrofluorometer. Isolated mitochondria
O2 consumption was assessed using an extracellular flux analyzer.
Energized mitochondria were submitted to Ca2+ pulses (10 µM) up
to a final concentration of 80-100 µM (200-450 nmol/mg protein),
showing no significant alterations in matrix volume and ∆Ψ. In the
presence of NO donors (25 to 100 µM), Ca2+ uptake was slower
and extramitochondrial Ca2+ concentration increased. When single
50 µM Ca2+ pulses were added, mitochondria treated with NO
donors showed a decreased Ca2+ accumulation rate (40-50%) with
an IC50 of ≅ 400 µM (180 nM NO). The addition of L-arginine or
NOS inhibitors to control mitochondria produced changes in Ca2+
uptake and in DAF-FM signal. State 4 O2 uptake was not modified
2+
by NO. The addition of Ca to the medium produced a 20%
enhancement in state 4-O2 consumption and this effect was
abolished by NO. These results suggest that Ca2+ and NO act as
signals that coordinate cytosolic workload and mitochondrial
energy metabolism.
56
ENZYMOLOGY
_____________________________________________________
ENZYMOLOGY
57
ENZYMOLOGY
_____________________________________________________
EZ-1] Effect of CHAPS and Tween-20 on the
measurement of enzymatic activity of dengue virus's
NS3
1
1
2
2
Cababie LA , Incicco JJ , Gebhard LG , Gamarnik AV , GonzálezLebrero RM1, Kaufman SB1
1
Instituto de Quimica y Fisicoquimica Biologicas y Deperatemento
de Quimica Biologicas, Facultad de Farmacia y Bioquimica,
Universidad de Buenos Aires, Argentina
2
Fundacion Instituto Leloir-Consejo Nacional de investigaciones
Cientificas y Tecnicas, Ciudad de Buenos Aires, Argentina
The dengue virus's NS3 protein is a helicase that catalyzes the
hydrolysis of ATP and couples the free energy of this reaction to
drive translocation on single strands and unwinding double strands
of RNA. A frequent experimental problem encountered working
with enzymes is the loss of specific catalytic activity when this
measurement is performed at low enzyme concentrations.
Therefore we examined whether the presence of detergents in the
reaction medium during activity measurements is able to overcome
this difficulty. We will present experimental results which show that
two detergents of different chemical nature such as CHAPS (3 [(3-Cholamidopropyl) dimethylammonio] -1- propanesulfonate) and
Tween 20 (Polyoxyethylene (20) sorbitan monolaurate) keep the
catalytic activity constant regardless of the concentration enzyme
used.
With grants from Universidad de Buenos Aires (UBACyT), CONICET,
and ANPCyT
58
ENZYMOLOGY
_____________________________________________________
EZ-2] Productive Induced Metastability (PIM): a new
concept compatible with 50 years of history in the study
of the allosteric regulation mechanisms.
1
1
1
Montes de Oca J.M. , Rodriguez-Fris A ., Appignanesi G.A .
Sección Fisicoquímica, INQUISUR-UNS-CONICET-Departamento
de Química, Universidad Nacional del Sur, Avda. Alem 1253, 8000
Bahía Blanca, Argentina
1
Already in the middle of the last century, Lumry R., Eyring, Biltonen
and others reported that allosteric signaling is an activated process
that requires the presence of structural or mobile defects to change
the free energy of the protein and promote the ligated state. In
particular, Lumry refers to the distortions of the H-bonds and local
1,2
structures as the driving forces of "fluctuating enzymes" .In this
study, we elucidate the allosteric binding modality by unraveling a
local structural motif that promotes association with the ligand. We
specifically show that allosteric modulators promote a local
metastable state that is stabilized upon association. The induced
conformational change generates a local enrichment of the protein
in the so-called dehydrons3, which are solvent exposed backbone
hydrogen bonds. These structural deficiencies that are inherently
sticky are not present in the Apo form and constitute a local
metastable state that promotes the association with the ligand. In
other words, we find that the allosterically activated conformation of
the enzyme could not prevail in the ensemble of conformations of
the Apo form of the enzyme because of the inherent instability of
the packing defects (the Lumry "mobile defects"). Therefore the
allosteric ligand acts protecting these exposed Interaction from
water, thus turning the active state into the most stable
configuration. This productive induced metastability4 (PIM) is likely
to translate into a general molecular design concept.
1.
2.
3.
4.
Lumry R & Eyring H (1954) Conformational changes in proteins, J. Phys.
Chem. 58, 110-120.
Lumry, R., The new paradigm for protein research, in Gregory R B (ed.),
Protein-solvent interactions, Marcel Dekker, Inc. New York (1995).
Fernández A, Transformative Concepts for Drug Design: Target
Wrapping (Springer, Heidelberg, 2010).
Montes de Oca J., Rodriguez-Fris A., Appignanesi G. and Fernández A.
Productive induced metastability in allosteric modulation of kinase
function,
FEBS
J
,281,
3079–3091
(2014).
59
LIPIDS AND BIOMEMBRANES
_____________________________________________________
LIPIDS AND
BIOMEMBRANES
60
_____________________________________________________
LBM-1] Surface Properties of Insulin-DPPC/POPC mixed
Langmuir monolayers.
Grasso E.J, Oliveira R.G, Maggio B.
Centro de Investigaciones en Química Biológica de Córdoba
(CIQUIBIC-CONICET), Dpto. de Química Biológica, Facultad de
Ciencias Químicas, Universidad Nacional de Córdoba. Pabellón
Argentina, Ciudad Universitaria, X5000HUA, Córdoba, Argentina.
The surface properties of the binary mixed Langmuir monolayers of
Insulin and DPPC (dipalmitoylphosphatidylcholine) and POCP
(palmitoyloleylphosphatidylcholine) spread at the air-water
interface was studied. Pure and mixed monolayers were spread on
Zn2+ containing solutions. Miscibility and interactions between the
components were studied on the basis of the analysis of the
surface pressure-mean molecular area isotherms, surface
compressional modulus and plots of mean molecular area and
surface potential versus mole fraction of Insulin. Our results
indicate that the intermolecular interactions between Insulin and
DPPC/POPC depend on both the monolayer state and the
structural characteristics of Insulin at the interface, which is
strongly influenced by the Zn2+ ions in the subphase (1). Brewster
angle microscopy was applied to describe the surface topography
of the monolayers. We concluded that Insulin forms mixed surfaces
with DPPC/POPC at all the mole fraction studied.
1
Grasso EJ et al. Colloids and Surfaces B. 2014, 219.
61
_____________________________________________________
LBM-2] Detection of lipid bilayer formation in
macroporous Silicon by EPR spectroscopy
a
b
b
d
b
León X. , Forzani L. , Garcés F. , Rodi P.M. , Osorio E. ,
b,c
b,d
Koropecki R.R. , Gennaro A.M.
a
Centro de Investigación en Dispositivos Semiconductores,
Universidad Autónoma de Puebla CIDS-ICUAP, 14 sur y Av. San
Claudio, San Manuel, 72570 Puebla, México. b Instituto de Física
del Litoral (CONICET-UNL), Güemes 3450, 3000 Santa Fe,
c
Argentina. Facultad de Ingeniería Química, UNL, Santiago del
Estero 2829, 3000 Santa Fe, Argentina. d Facultad de Bioquímica y
Ciencias Biológicas, UNL, Ciudad Universitaria, 3000 Santa Fe,
Argentina
Spin label EPR spectroscopy was used to study the pore filling of
macroporous silicon with DMPC liposomes. Macroporous samples
having a thickness of around 13 µm, and nearly cylindric pores of
1.0-1.2 µm diameter were obtained by electrochemical anodization
of p-type silicon wafers, in darkness. The samples were oxidized in
a KOH solution, in order to turn hydrophilic the inner surface of the
pore network. Then, the pore structure was filled with an aqueous
solution of 100 nm unilamellar DMPC liposomes including 2 mole
percent of the spin label 5-SASL. EPR spectra were acquired in X
band at 30ºC after different incubation procedures. While
liposomes have isotropic EPR spectra, the spectra of lipids inside
porous samples when B is parallel to the pore axis were different
from those with B perpendicular to the axis. Simulations of the
spectra indicate that cylindrical lipid bilayers were formed covering
the inner surface of the pores, although some liposomes remain.
Diverse protocols were explored in order to achieve an adequate
lipid hydration. We propose the use of a 2D photonic crystal made
of macroporous silicon, which can sense changes in the dielectric
function of the filling material, to study the effects of confinement
on DMPC phase transition.
62
_____________________________________________________
LBM-3] The incorporation of amphiphilic derivates of L ascorbic acid induce stiffness of phospholipid bilayers
Giudice F.; Mottola M.; Fanani M.L.
Centro de Investigaciones en Qca. Biológica de Córdoba
(CIQUIBIC), Depto. Química Biológica, Fac. Ciencias Químicas,
Univ. Nac. Córdoba, Ciudad Universitaria, X5000HUA,Córdoba,
Argentina.
We investigated the interaction of three amphiphilic derivatives of
L-ascorbic acid (ASCn), ascorbyl palmitate (ASC16), ascorbyl
myristate (ASC14) and ascorbyl laurate (ASC12) with model
membranes. The membrane system chosen was phospholipid
bilayers organized in nanoscale vesicles. Since ASCn have an
acidic group with a pKa of 4.2, the insertion of these drugs to
originally neutral phospholipid vesicles, was monitored by
measurements of changes in zeta potential. Our results show a
large change in the hydration of the polar head group of
phospholipids by measuring changes in the Generalized
Polarization of laurdan, consistent with a rigidifying effect for all the
ASCn studied in a magnitude ASC14 > ASC12 > ASC16. Similar
effect was observed by measurement of the anisotropy of DPH
(diphenylhexatriene), which reported an increase in the
microviscosity of the hydrophobic core of the membranes. Those
rheological changes in the membrane occur with the maintaining of
the structural integrity of the membrane: the low amount of soluble
phosphorus in ASCn-treated vesicles along with a negligent
release of vesicles fluorescent content leads to the conclusion that
none of the three drugs have a detergent effect. These studies
show that the ASCn interact strongly with model lipid membranes
and alter their rheological properties, likely by increasing the
stiffness of the membranes. This work is a first attempt to
characterize the biophysical properties of ASCn-phospholipids
systems, which is aimed to get a better understanding of their
pharmacological and immunological effects.
63
_____________________________________________________
LBM-4] Fluctuation patterns of voltage-induced ionic
currents in black lipid membranes. Effect of αHemolysin.
a
b
a
a
Colmano, G.N , Vazquez, R.F , Mottola, M , Corvalán, N.A , Clop
PDa, Bakas, LRb, Perillo, M.Aa.
a
IIBYT (CONICET- Unviersidad Nacional de Córdoba), bINIBIOLP
(CONICET-UNLP).
Previously1 we reported that the rise in holding potential (∆V) and
cholesterol (CHO) content in planar lipid bilayers (BLM), increased
the number of conductance states, the magnitude of conductance
levels and favored the development of long-range autocorrelated
(fractal) processes. This was explained as a CHO induced
modulation of the ∆V threshold that allowed a percolation of
channel behavior, where isolated channels become an
interconnected network. Moreover, α-Hemolysin (HlyA) is a protein
with a known capability of forming lytic pores in biomembranes2.
Thus, in the present work we investigated the fluctuation patterns
of ion currents through DOPC/CHO (0-30 mole%) BLMs, using
symmetric bathing solution (10 mM HEPES-KOH pH 7,4, 150 mM
NaCl, +/- 10 mM CaCl2) with or without 0-20 nM HlyA. Electrical
current intensities (I) were measured in voltage clamped conditions
between 0 and ± 200mV. The autocorrelation parameter α, derived
from detrended fluctuation analysis (DFA) on I temporal fluctuation,
showed that in BLMDOPC HlyA induced a transition from noncorrelated (α=0.5, white noise) to a long range correlated
(0.5<α<1) behavior at ∆V>100 mV. In CHO containing BLMs, the
effect of the steroid prevailed.
Acknowledgements: Foncyt, Conicet, SeCyT-UNC.
1. Corvalán NA et al. BBA Biomembranes 2013, 1828, 1754.
2. Bakás et al. Biophys J. 2006, 91, 3748.
64
_____________________________________________________
LBM-5] Structure and dynamics of stearic acid spin label
(n-SASL) on CHAPS micelles, studied by molecular
dynamics simulations
a
a
a
A. Sergio Garay , Fernando E. Herrera , Paula Pruvost , Daiana
a
a,b
Barcarolo , Daniel E. Rodrigues
a
Departamento de Física, Facultad de Bioquímica y Ciencias
Biológicas, Universidad Nacional del Litoral (UNL), Ciudad
Universitaria, 3000 Santa Fe, Argentina. bINTEC (CONICET-UNL),
Güemes 3450, 3000 Santa Fe, Argentina.
Detergents are essential tools in the study of biological
membranes. In particular, the nondenaturing zwiterionic detergent
CHAPS
(3-[(3-cholamidopropyl)-dimethylammonio]-1propanesulfonate) was originaly designed for membrane
biochemistry. In this context, EPR experiments with stearic acid
spin labels (n-SASL) are also used to probe the ordering and
molecular mobility at different depths of membranes or micelles. In
this work, molecular dynamics simulations were used to study the
structure and dynamics of different stearic acid spin labels (nSASL), labeled at different positions of their doxyl group (n=5, 12
and 16), in contact with micelles of 14 CHAPS. The obtained
results show differences in the interaction of the three spin labels
with the micelles. The 5-SASL is anchored to the micelle by
introducing its hydrophobic tail into a hydrophobic pocket,
maintaining the corboxylate group almost in solution. The 12-SASL
instead, is anchored similar to the 5-SASL but with and additional
stabilization of the carboxylate group interacting with the
hydrophilic moieties of the CHAPS molecules. Finally, the 16-SASL
is stabilized in the micelle only by passing through it, with the
carboxylate and doxyl groups located almost on the surface of the
micelle. All these differences make the doxyl group behave
dynamically different depending on its position. The 12-SASL was
the most stabilized molecule and therefore the less mobile, with an
increasing on their motilities in the 16 and 5-SASL. The results of
this work will help in the understanding and interpretation of the
EPR spectra of similar systems.
65
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LBM-6] Membrane role in the rational design of HIV
fusion inhibitors
1,2
1
1
Axel Hollmann , Marcelo T. Augusto , Susana Gregório , Pedro
1
3
4
M. Matos , Matteo Porotto , Antonello Pessi , Miguel A. R. B.
1
1
Castanho and Nuno C. Santos
1
Instituto de Medicina Molecular, Faculdade de Medicina,
2
Universidade de Lisboa, Portugal; Laboratory of Biointerfaces
and Biomimetic Systems- CITSE- CONICET, Argentina. 3 Weill
Medical College, Cornell University, New York, USA.
4
PeptiPharma, Rome, Italy.
The Human Immunodeficiency Virus type 1 (HIV-1) is a highly
pathogenic and evasive virus, for which no cure has yet been
achieved. Fusion of viral and host cell membranes is a crucial step
in virus infectivity, therefore the development of viral entry inhibitors
has great advantages since they prevent the release of the viral
content into the host cell. In this work, we found that boosting
membrane affinity of the established HIV fusion inhibitors C34 and
enfuvirtide, by conjugation with lipid moieties, results in a dramatic
increase of their antiviral activity. We demonstrated, by fluorescent
partition, dipole potential assays and surface pressure assays, that
these novel fusion inhibitors have membranotropic behavior
towards biomembrane model systems and human blood cells
membranes. The ability that these peptides have to bind to cell
membranes facilitates the delivery of the peptides to this confined
environment, as some peptide is already locally present. Beside
the ability of membranes to concentrate the inhibitors locally, their
role in the presentation of the peptide in the proper orientation for
gp41 binding should also be considered. In this context, we also
evaluated the location of the different peptides in the membrane,
using aqueous and lipophilics quenchers. Overall, the results of
this study offer a rational basis for the design of improved viral
fusion inhibitors, since they suggest that maximizing antiviral
activity requires finding the proper balance of membrane affinity
and exposure of the peptide moiety. Moreover, we offer an
experimental strategy to guide the development of the structureactivity relationship (SAR) towards this goal.
66
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LBM-7] Effect of polyelectrolytes on the rheological
properties of lipid monolayers.
Cámara C. I. and Wilke N.
CIQUIBIC-CONICET. Departamento de Química Biológica.
Facultad de Ciencias Químicas. Universidad Nacional de Córdoba.
Ciudad Universitaria. Córdoba. Argentina.
Glycosaminoglycans (GAGs) are important constituents of the
extracellular matrix of microorganisms, animals and plants. They
are large polymers formed by mono, di or oligosaccharides
bounded by glycosidic linkages and may have a variety of
functional groups along their chains. Some biological functions
have been associated to the presence of polysaccharides in the
extracellular matrix, however, their specific role is not known. It has
been reported that its presence affects the dynamics of lipids and
proteins and therefore, the aim of this work was to study how the
presence of different polysaccharides modifies the rheological
properties of model membranes. We studied the effect of the
presence of different polysacarides on lipid monolayers of
dimyristoyl phosphatidyl glycerol (DMPG) and dipalmitoyl
phosphatidyl choline (DPPC). The polysacharides were dextran
sulfate of high (DSA) and low (DSB) sulphate content, and di-ethylamino-ethyl dextran (DEAE). Both dextrane sulphates are
negatively charged while DEAE is a cationic polymer. Both lipid
monolayers were prepared on subphases composed of CaCl2 (10
mM) and NaCl (145mm) at 24±1ºC. Under these conditions both
lipids show a phase transition from liquid expanded to liquid
condensed phase (at about 15mN/m for DMPG and 7,5 mN/m for
DPPC). When the monolayers were formed in the presence of
DSB, no change in the isotherms or in the surface viscosity was
observed. For DSA, the effect on the film depends of the lipid while
in the presence of DEAE the expanded phase of the lipids resulted
stabilized and the viscosity changed.
Acknowledgements: SecyT-UNC, CONICET and FonCyT (PICT
2012-0344)
67
_____________________________________________________
LBM-8] Equilibrium spreading pressure,
overcompression and metastability of C10-CER
Marzari, G., Fanani, L., Maggio, B.
CIQUIBIC, UNC-CONICET, Departamento de Química Biológica,
Facultad de Ciencias Químicas, Universidad Nacional de Córdoba,
Haya de la Torre y Medina Allende, Ciudad Universitaria,
X5000HUA, Córdoba, Argentina.
Ceramide is one of the simplest sphingolipids, consisting of a
sphingosine base N-linked to a fatty acid, and is a mediator for cellsignaling events. In this work, the dependence of the interfacial
organization and topography of films of ceramide N-acylated with a
10 carbon fatty acid (C10-Cer) with the compression-expansion
kinetics were studied in Lagmuir monolayers. The molecular
packing, the bidimensional transition, its cooperativity and the
morphology of condensed domains under cycles of compressionexpansion at different rates indicates that the interfacial structure is
kinetically limited at the molecular and supra-molecular levels.
Recent experiments indicate that at 21 °C these monolayers have
an "equilibrium spreading pressure" lower than 0.5 mN/m and
monolayer formation was not detected when compressed at very
slow rates. Thus, at the usual compression rates, the ceramide film
is in overcompressed state at all surface pressures. At these
relatively faster rates isotherm having different compressionsexpansion hysteresis were observed depending on the scan rate.
The hysteresis thermodynamic functions point to entropic-enthalpic
compensations that result in favorable enthalpic contributions
derived from a more condensed packing which overcomes the
unfavorable diminution of configurational entropy. These results
suggest formation of kinetically limited and metastable condensed
states.
Suported by CONICET, FONCyT, SECyT-UNC
68
_____________________________________________________
LBM-9] Liposomes and L-cysteine: a potential
antioxidant therapy
Perrotta, R; Siri, M; Alonso, S. del V; Chiaramoni, N.S.
Laboratorio de Biomembranas (LBM), GEByB, UNQ-IMBICECONICET, Quilmes, Buenos Aires.
Glutathione is the main mechanism of eliminating reactive oxygen
species (ROS) in cells. GSH is synthesized de novo in a two-step
enzymatic process in which glutamine and cysteine are covalently
linked. In this process cysteine is the rate limiting reactant and the
component that provides GSH with antioxidant activity, as
cysteine's sulfhydryl bond is oxidized during the reduction of ROS
(1). With the main goal to induce glutathione in cells and reduce
ROS damage in several diseases such as cystic fibrosis or viral
infections, we developed lipid transporters that can encapsulate Lcysteine. These transporters were formulated with three different
acyl chain length lipids (DMPC-DPPC-DSPC) mixed with Lcysteine in a molar ratio 1:0.5 respectively. In order to characterize
these systems we studied the hydrophobic defects in the bilayer
surface and the lipid-aminoacid interaction by FTIR. For further
characterization we evaluated the particle size, surface charge and
release profile. We found that L-cysteine interacts with the
liposome surface increasing their negative charge and stabilizing
them. Finally, cytotoxicity was evaluated in Caco2 cell line. No toxic
effects were observed.
In conclusion, lipid transporters studied herein are good candidates
to deliver L-cysteine and therefore could reduce ROS damage.
1. D.Morris,etal.,Glutathioneandinfection,Biochim.Biophys.Act
a(2012),http://dx.doi.org/10.1016/j.bbagen.2012.10.012
69
_____________________________________________________
LBM-10] Proposal about zeta potentials origin in
liposomes prepared with zwitterionic lipids
1
1
1
1
1
Morini,M. , Sierra,M.B. , Pedroni,V. , Alarcón,L. , Appignanesi,G. ,
2
Disalvo,E.A.
1
Laboratorio de Fisicoquímica. Dpto. de Química. INQUISUR.
2
Universidad Nacional del Sur Laboratorio de Biointerfases.
CITSE. Universidad Nacional de Santiago del Estero
We measured the zeta potentials of MLVs and LUVs liposomes of
DMPC, DPPC and DMPE dispersed in water, in KCl (1 mM) and in
KClO4 (1mM) at pH 7.4 and temperatures according to its solidcrystalline and liquid-crystalline states. Liposomes used are
composed of neutral lipids, each having one phosphatidyl group
and one choline group in their molecules. In spite of this fact, the
liposomes exhibit non-zero mobility in an external electric field,
thus these liposomes have non-zero surface electric potentials. In
this work we chose water as dispersing medium of liposomes to
exclude the possibility of ion adsorption onto the liposome surfaces
in order to investigate possible changes in the zeta potential
caused by increases of ionic strength, being low but comparatively
higher than that of water. Two main models can be found in
literature about surface charge. Our observations in presence and
absence of ions are in better agreement with the orientation of the
lipid head group in the liposome surface region (1,2) than with the
proposal about adsorption of ions onto liposomes as responsible of
this charge (3).
Acknowledgements: Dr. Marcelo Avena for Zetasizer availability.
Funds from CONICET, UNS.
1. Makino K. et al. Biophysical Chemistry 41 (1991) 175
2. Lairion F and Disalvo E.A. J.Phys.Chem.B 113 (2009)
1607
3. Tatulian S. Biochimica et Biophysica Acta, 736 (1983) 189
70
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LBM-11] PHENYLALANINE INTERACTION WITH MEMBRANES
IN DIFFERENT STATES OF HYDRATION.
Cutró, Andrea C, E. A. Disalvo.
Laboratorio de Biointerfases y Sistemas Biomiméticos,
Laboratorios Centrales, CITSE- UNSE. Santiago del Estero. Email: [email protected]
It has been reported that Phenylalanine (Phe) produces leakage
and fusion on the thylakoid and liposome membranes during
freezing process. Apparently, the damage is related with
membranes under stress conditions, i.e. partial dehydration [1]. On
the other hand, Phe is able to interact with monolayers of stearic
acid producing a disappearance of its liquid-crystalline phase and a
premature collapse [2]. Studies carried out in DPPC monolayers
(Maturana et al, SAB 2014) showed that the Phe-lipid interaction
depends on the water content.
In order to correlate the Phe interaction with the hydration state in
bilayers we study the interaction of Phe in DPPC liposomes in gel
(25°C) and liquid-crystalline phases (50°C) and in liposomes in the
gel state subjected to osmotic dehydration. The effect was
measured using Merocyanine (MC 540) as a fluorescent marker.
The presence of Phe in DPPC liposomes at 50°C, does not
produce significant change in MC 540 monomer concentration. On
the contrary, in DPPC liposomes at 25°C Phe decrease
significantly the monomer adsorption. This behavior was also
observed, when DPPC liposomes were subjected to a hypertonic
media. These results indicate that phenylalanine interaction
depends on the membrane hydration states.
1. Popova, A. V. et al. Biochimica et Biophysica Acta (2002),
109.
2. Griffith, E. C et al. The Journal of Physical Chemistry
(2012), 7849.
71
_____________________________________________________
LBM-12] Hidratation of lipid membranes and
hydrophobic and hydrophilic monolayers.
1
1
1
1
Alarcón, L.M , Sierra, M.B , Morini, M.A , Pedroni, V , Appignanesi,
1
2
G.A and Disalvo, E.A .
1
Instituto de Química del Sur - Universidad Nacional del Sur, Bahía
Blanca. 2 Centro de investigaciones y Transferencia de Santiago
del Estero, Universidad Nacional de Santiago del Estero.
By molecular dynamics simulations we studied the hydration of
membranes composed of dipalmitoylphosphatidylcholine (DPPC)
lipid molecules, revealing that the water molecules do not only
moisturize the polar groups of the lipids, but also penetrate through
alkane chains of such molecules. To characterize these lipid
systems and compare them with other systems as hydrophobic
and hydrophilic monolayers we study different parameters, such as
density fluctuation, residence times and orientation of the water
molecules on the surface of the monolayer, in addition to typical
parameters in lipid membranes as area per lipid and number of
water molecules per lipid.
72
_____________________________________________________
LBM-13] Membrane surface charge in yeast depends on
physiological state.
1,2
1
2
1
Lavaisse, L. M. , Hollmann, A. , Nazareno, M.A. , Disalvo, E.A.
Laboratorio de Biointerfases y Sistemas Biomiméticos;
2
Laboratorio de Antioxidantes y Procesos Oxidativos - CITSE –
CONICET - UNSE
1
The wall that surrounds yeast cells is responsible of its shape and
integrity. It has a complex structure, which bears a net negative
charge due the phosphate groups of the phosphomannan that
compose its outer layer. The net charge can be determined by zeta
potential measurements, which indicates the electric potential
difference between the fixed stationary layer at the cell and the
aqueous solution. Based on the behavior of other microorganisms,
the goal is to determine a relationship between the different stages
of growth of yeast with zeta potential measurements in order to
demonstrate that the physicochemical properties of the surface are
dependent of the physiological state of the cell. For this purpose,
yeasts from a commercial dry powder were isolated and grown in
standard media. At different incubation times, the medium was
collected in order to determine yeast growth, performing CFU,
OD600nm and following variations in the electrophoretic mobility of
cells in an electric field. In each sample we found distributions of
cells with different zeta potentials. This suggests that different
metabolic stages converge at each time of culture. This was also
observed by fluorescent microscopy.
In this work, we obtain growth curves by zeta potential. Data allow
to conclude that zeta potential provides information about the
different metabolic state that coexist in a culture, because this
methodology enables us to measure the zeta potential of individual
cells at different stages of growth. Further experiment should be
done in order to relate internal cell variables such as pH, ion
distribution and membrane potential during the different growth
phases of cell with zeta potential
73
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LBM-14] EFFECT OF WATER STRESS ON ERUCA
SATIVA Mill PROTOPLASTS: STUDY ON A MODEL
SYSTEM ASSOCIATED WITH POSTHARVEST STAGES.
1,2
2
1
Sain, P. Rodriguez, S. y Disalvo, A.
Laboratorio de Biointerfases y Sistemas Biomiméticos 2
Laboratorio de Ciencia y Tecnología de Alimentos. Centro de
Investigaciones y Transferencia de Santiago del Estero (UNSECONICET).
1
One abiotic stress factor in post harvest stages of plants is related
to water availability which affects the quality and suitability for
commercialization. Therefore, we study in an experimental model
the influence of water stress on the surface properties of
protoplasts isolated from stems of arugula, in their post-harvest
stages, comparing its evolution with a stock suspension of
protoplasts isolated on harvest. They were subjected to different
osmotic gradients varying sucrose in the external solution At each
concentration the protoplast diameter was determined by optical
microscopy. Under these conditions two stages in the volume
decrease were detected. At low concentrations, a steep decrease
of volume was found at concentrations below 0.18M. However,
above 0.18M sucrose, the volume changes in a very slight way. In
correlation with the response of volume the changes in the plasma
membrane surface was determined using Merocyanine 540
(MC540) as a membrane marker. The maximum signal of MC540
appears at the inflection point of diameter versus concentration
indicating surface changes in response to osmotic stress. Based
on these results, we can relate the evolution of Eruca Sativa Mill
postharvest stages, understanding the effects at the cellular level.
74
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LBM-15] Interaction of Cholesterol with Sphingomyelins:
kinetics of cholesterol extraction by ciclodextrin from
natural and model membranes.
(1)
(2)
(1)
(3)
Daza Millone, M.A. ; Herlax, V. ; Vela, M. E. ; Bakás, L. and
Maté, S. (1)
1
Instituto de Investigaciones Físicoquímicas Teóricas y Aplicadas
(INIFTA), CCT-La Plata, Universidad Nacional de La Plata. 2
Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP),
CCT- La Plata, CONICET. Facultad de Ciencias Médicas.
3
Universidad Nacional de La Plata Departamento de Ciencias
Biológicas. Facultad de Ciencias Exactas. Universidad Nacional de
La Plata, La Plata, Argentina.
Preferential interaction between sphingomyelin (SM) and
cholesterol (Chol) in both cell and model membranes has been
proposed as central for the formation of Chol and SM-rich domains
in membranes. However, Chol does not interact equally with
different SMs; in fact, we have recently found that when an
unsaturated sphingolipid, N-nervonoyl sphingomyelin (24:1∆15), that
is abundant in mammalian erythrocytes is included in the bilayers,
no lateral phase separation is detected (1). The desorption kinetics
of Chol from membranes can be used as a sensitive indicator of
Chol/phospholipid interactions. In this concern, it was reported that
lateral organization and SM content of the membrane affects Chol
extraction. The aim of this work was to explore the degree of
interaction between Chol and different SMs in model (SUVs) and
natural membranes (erythrocytes), by measuring Chol efflux using
cyclodextrin. We employed a Surface Plasmon Resonance
approach, which allows monitoring the extraction process in real
time, to address the question of kinetics of Chol extraction from
membranes of different lateral organization. Finally, we studied the
effects of Chol-depletion on the packing properties of erythrocyte
membranes by determining the GP values of Laurdan by two
photon microscopy.
1. Sabina Maté. Biophys. J. 2014, Vol 106; 2606
75
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LBM-16] Nanoberries: further characterization of
liposomal polyphenols and in vitro photoprotection
against UV attack
Bucci, P.L.; Montanari, J.A.M.; Alonso, S. del V.
GBEyB- IMBICE CONICET, LBM, Dept.de Ciencia y Tecnología,
Universidad Nacional de Quilmes,
Nanoberries1 are ultradeformable liposomes loaded with an
ethanolic extract rich in polyphenols from blueberry (Vaccinium
myrtillus, variety Millenia). They are potentially useful as delivery
systems for photoprotection against UV-mediated skin damage.
We prepared the liposomes with soy phosphatidylcholine and
sodium cholate as a border activator, at 0.223 extract/lipid w/w.
Their phenolic and anthocyanine content, drug to lipid ratio, size
and Zeta potential after manual extrusion were determined, and we
obtained images by Scanning Electron Microscopy after drying the
Nanoberries under different conditions: lyophilization and vacuum
dry2. With the aim of assessing their photoprotection capacity
against UV attack, we performed experiments on HaCaT cells. A
calibration curve of phototoxicity was obtained by viability
determination after exposure to different amounts of energy from
an UV source. Then, the protection against phototoxicity by
Nanoberries and free ethanolic extracts of blueberry were
assessed.
Acknowledgements: We thank “The Berry Store” for kindly
providing blueberries for this research work, and Dr. Rebeca
Oliveira de Souza (Fcfrp, USP, Brazil) for her help with
phototoxicity assays.
1. Montanari et al. Journal of Cosmetic Sciences. 2013. 469.
2. Montanari et al. International Journal of Pharmaceutics.
2009. 184
76
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LBM-17] Effects of Glucocorticoids on the
microstructure of an exogenous pulmonary surfactant
Cimato, A; Hoyos Obando, A; Zurdo, F; Piehl, L; Facorro, G;
Martínez Sarrasague, M.
Cátedra de Física, Facultad de Farmacia y Bioquímica, UBA
Pulmonary surfactant could be used as a carrier of corticosteroids
drugs in therapy for respiratory diseases. It is vital that
corticosteroids delivered via the lungs not interfere with surface
activity of the pulmonary surfactant lining layer. In our previous
work we have found that cholesterol change surfactant structure
and consequently altered its activity. As corticoids have a similar
molecular structure, could be expected to have similar behavoir.
The aim of this study is to evaluate the effects of budesonide,
beclametasone and fluticasone on structural properties of an
exogenous
pulmonary
surfactant
(EPS).
Bovine
EPS
(PL=10mg/ml) added with each corticoid (1 mg/ml) was labeled
with 5 or 16 doxyl stearic acid. Order parameter S, correlation time,
and S/W ratio, calculated from Electron Spin Resonance (ESR)
spectrawere used to evaluate the conformational and fluidity
changes. EPS fractions were separated by centrifugation
(12000rpm). The active/inactive subtype ratio was evaluated by PL
determination. Corticoid incorporated into surfactant was evaluated
by UV absorption. Surface tension was measure with a pulsating
bubble
surfactometer.
Budesonide,
beclametasone
and
Cholesterol increased the order parameter. Fluticasone and
betametasone increased the S/W ratio. No significant changes
were found in the correlation time or in the active / inactive ratio
subtype. All samples showed appropriate surfactant activity. We
have demonstrated that glucocorticoids change the structure of the
bilayer on the polar region without altering surfactant activity.
1. María Martínez Sarrasague, et al. Respiratory Physiology
& Neurobiology 189 (2013) 581.
77
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LBM-18] Phenyalanine lipid membrane interaction
assessed by surface pressure studies.
Maturana, P., Cejas, J., Frías, M.A., Cutro, A.C., Hollmann, A.,
Disalvo, E.A.
Laboratory of Biointerphases and Biomimetic Systems - CITSE
(University of Santiago del Estero-CONICET), 4200 Santiago del
Estero, Argentina
Small amphyphilic molecules may partition in lipid membranes,
among them, aromatic amino acids such as Tyr, Trp and Phe have
an amphiphilic character due to the hydrophobicity of the indol and
phenyl rings (1). In particular, Phe has been shown to damage
thylakoid membranes at very low concentrations during freezing.
Also, in liposomes, it also induces leakage and membrane fusion
(2). Apparently, at relatively low concentrations the damage is
produced on membranes under stress conditions, i.e. partial
dehydration. Many amphiphilic compounds may protect
membranes from oxidative stress under conditions of low water
availability. Therefore, the influence of aminoacids such as Phe on
the stability of membranes can be regulated by the water stress. It
is well known that water/lipid ratio can be modified in lipid
monolayers by changing the surface pressure. Thus, in order to
evaluate the effects of Phe on the water-lipid interphase, the
changes in the surface pressure of DPPC monolayers at different
initial surface pressure (14mN/m; 26 mN/m and 40 mN/m) were
evaluated in a Langmuir balance. The results suggest that in
condensed membranes (40 mN/m), i.e. with less hydration, Phe
are able to induce a higher change of the initial surface pressure.
Also with the aim to evaluate the role of the charges on the Phemembrane interactions, the experiments were carried out at two
different pHs, 5 close to Ip of Phe and 7.3 were higher changes
were observed.
1. Petelska, AD et al., Cell Biochem Biophys. (2011), 289.
2. Popova, AV et al., Biochimica et Biophysica Acta (2002), 109.
78
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LBM-19] Effect of polymerics dendrons on membranes
models.
1
2
1
1
Sassi, M. , Cámara, C. , Yudi, L.M. , Juarez A.V.
INFIQC (CONICET-Universidad Nacional de Córdoba).
2
Departamento de Fisicoquímica. CIQUIBIC-CONICET.
Departamento de Química Biológica. Facultad de Ciencias
Químicas. Universidad Nacional de Córdoba. Ciudad Universitaria.
Córdoba. Argentina
1
Dendritic molecules are extensively used as nanoscopic auxiliaries
for the constructions of dendronized polymers, macromonomers or
for the dendronization of other materials or surfaces. With the
increasing interest in dendritic chemistry, it is important to develop
suitable methods to characterize their properties. Our particular
interest is the characterization of dendronized polymers. The
dendron employed in this work, aminotriester (AT), was used to
modify chitosan and this change in the chemical structure confers
new properties to the polymer. The main interest in this kind of
macromolecules is based on the multiplicity of functional groups
which enhance the bio-recognition when they are employed as
pharmaceutical drugs carriers for controlled delivery.
The main objective of this work is to study the effect of
aminotriester in the compactness and permeability of lipids
monolayer. This study can contribute to the characterization of the
behaviors of these molecules in biological environments. In this
work we study the interaction of 1,2-dilauroyl-sn-glycero-3phosphate (sodium salt) (DLPA) and distearoyl phosphatidic acid
(DSPA) with the dendritic molecule AT present at different
concentrations. For both phospholipids monolayers analyzed, an
expansion effect in the isotherm was observed due to the presence
of AT between the lipids molecules.
Acknowledgements: Dra. M. Martinelli, Dra. A. Aldana. SecyTUNC, CONICET for financial support.
79
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LBM-20] MOLECULAR DYNAMIC SIMULATION OF THE
TRANSMEMBRANE PORTION OF THE THERMOSENSOR
DESK CHIMERA IN DMPC MEMBRANES
Rodrigues D.E. [a,b], Garay A.S. [a], Cybulski L.E. [c],
[a] Área de Modelado Molecular, Lab. de Biomembranas, Dpto. de
Física, Fac. de Bioquímica y Ciencias Biológicas, Universidad Nac.
del Litoral e [b] INTEC (UNL+CONICET). Argentina.
[c] Instituto de Biología Molecular y Celular de Rosario (IBRCONICET) y Dpto. de Microbiología, Fac. de Ciencias Bioquímicas
y Farmacéuticas, Universidad Nacional de Rosario. Argentina.
The Bacillus subtilis histidine kinase DesK is an example of
thermosensor integral enzyme that promotes the membrane
fluidity when temperature drops below ~30°C. A chimera of 31aa of
the transmembrane region has been probed to reproduce the
thermosensor ability in native and synthetic membranes. We had
previously characterized the structural behavior of that chimera by
Molecular Dynamics simulations (MD) at 25 ºC (kinase active) and
37ºC (fosfatase active) and found that at low temperature the Cterminal portion between aa 7:27 present an alpha helix secondary
structure. From experimental evidences it is known that the
following polar residues that links the chimera to the cytoplasmatic
portion of the enzyme plays an important role. We performed MD
simulation of this extended peptide in DMPC membrane at both
relevant temperatures. We found that the transmembrane portion
of the peptide breaks in 3 alpha helix sectors at 25°C and results in
a coil structure at 37°C. The linker portion of the peptide protrude
from the membrane surface at 25°C more that at 37°C. We have
proposed a dimeric structure of the chimera based on these
results, experimental information and performed MD simulations to
characterize its stability and properties at both temperatures.
80
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LBM-21] Structural characterization of GAPDH
protofibrils formed in the presence of acidic membranes
1
1
1
Avila, C.L ; Torres-Bugeau, C.M ; Vera, C.C ; González Lizárraga,
1
2
3
4
M.F ; Barbosa, L.R.S. ; Raisman-Vozari, R ; Papy-Garcia, D ;Itri,
2
1
R ; Chehín R .
1
INSIBIO and Inst.de Química Biológica Dr Bernabé Bloj
(CONICET-UNT). 2 Instituto de Física da Universidade de São
Paulo. 3 INSERM. ThérapeutiqueExpérimentale de la
neurodégénérescence. 4 Laboratoire CRRET, Université Paris
EstCréteil.
Parkinson's disease (PD) is characterized by the loss of
dopaminergic neurons. Cell death has been attributed to certain
oligomeric intermediates formed during the aggregation pathway of
α-synuclein, which can alter membrane permeability and promote
mitochondrial, proteasomal and membrane trafficking dysfunction.
Experiments on SH-SY5Y cells showed that the toxicity exerted by
α-synuclein could be abolished by protofibrilar species of
Glyceraldehide-3-phosphate dehydrogenase (GAPDH) formed in
the presence of glycosamineglycanes. This mechanism for the
mitigation of α-synuclein toxicity might be relevant at the
extracellular space, where both GAPDH and glycosamineglycanes
are present, inhibiting the spreading of the disease. During aging,
certain changes could affect the normal functioning of this
protective pathway, facilitating the onset of Parkinson’s disease.
We described how GAPDH aggregation can also be triggered by
the presence of negatively charged lipidic membranes. This data
acquires relevance in the light of recent reports associating the
overexpression of phospholipase D to neurodegenerative disease
during aging. In this workwe show that the increase of phosphatidic
acid in the lipid membrane could drive the aggregation of GAPDH
through a different pathway. We show that the protofibrils formed in
this pathway are structurally different from the protective pathway
and incapable of binding α-synuclein. In this way, we propose that
an increase in phospholipase D levels would result in a depletion of
the extracellular GAPDH available to form neuroprotective species
contributing to the onset of the Parkinson disease during aging
81
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LBM-22] Interaction of magnetic nanoparticles with
phospholipid films adsorbed at a liquid/liquid interface.
1
3
3
2
Cámara C. I ., Monzón L. M. A , Coey J. M. D and Yudi L. M .
CIQUIBIC-CONICET. Departamento de Química Biológica.
2
INFIQC
(CONICET-Universidad
Nacional
de
Córdoba).
Departamento de Fisicoquímica. Facultad de Ciencias Químicas.
Universidad Nacional de Córdoba. Ciudad Universitaria. Córdoba.
Argentina.3School of Physics, SNIAMS building, Trinity College
Dublin, Dublin 2, Ireland
Magnetic nanoparticles (MNPs) are widely used in biomedicine
due to their versatility. The most common applications are in
biosensor platforms, in drug or radio isotopes delivery, as a
magnetic spacers of labeled cells, in tissue engineering, as a
contrast agents in magnetic resonance studies and in hyperthermia
based- therapies among others. Most applications are related with
the interactions between MNPs and membranes models and, for
this reason, the investigation of these interactions using different
methodologies has gained great importance in the last years.
In the present work we evaluate the effect of Co MNPs on the
interfacial structure and permeability of distearoyl phosphatidic acid
(DSPA) and disteroyl phosphatidyl glycerol (DPSG) films adsorbed
at a water / 1,2-dichloroethane interface. The study is carried out
employing cyclic voltametry (CV), electrochemical impedance
spectroscopy (EIS), capacity curves and interfacial pressure – area
isotherms. DSPA and DSPG adsorb at the interface forming
homogenous films and producing a blocking effect on the transfer
process of tetra ethyl ammonium (TEA+), used as probe cation. In
presence of Co MNPs this effect is reversed and the reversible
+
transfer process for TEA is reestablished, in greater or lesser
extent depending on the structuration of the film. Co-DSPA hybrid
films have a homogeneous structure while Co-DSPG films present
different domains. Moreover, the presence of Co on DSPA film
modifies the partition coefficient of the organic electrolyte into the
hydrocarbon layer.
1
82
_____________________________________________________
LBM-23] INTERACTION OF PHENYLALANINE WITH
DPPC MEMBRANES BY FTIR-ATR: INFLUENCE OF
HYDRATION
Rosa, A.S.; Disalvo, E.A.; Frías, M.A.
Laboratory of Biointerphases and Biomimetic Systems - CITSE
(University of Santiago del Estero-CONICET), 4200 Santiago del
Estero, Argentina. e-mail: [email protected]
Amino acids are ubiquitously found in all living cells [1, 2].
Therefore, although they are usually only present in low
concentrations in cells, amino acids represent interesting model
substances to examining the interaction between amino acids and
bilayer lipid membranes. Aromatic amino acids such as
phenylalanine (Phe), has an aromatic ring of different characteristic
sizes and hydrophilicity, which is expected to influence molecular
arrangements, therefore, the polycondensation monolayers.
According to results obtained with DPPC monolayers, Phe
produces an association with the PC molecules which suggests a
molecular interaction of the head groups with Phe residues at
different hydration states .FTIR results indicate that the symmetric
stretching of the phosphate group, which is quite sensible to
hydration, is affected in a great extent by Phe. Also, the water
bands of the phospholipids are significantly modified. Topological
changes may modify the exposure to water of different residues of
the lipid molecules such as CH2 and carbonyl groups, to water
affecting the Phe-PC interaction.
1. Popova A.V., Heyer A.G., Hincha D.K. Biochimica et
Biophysica Acta. 2002;1561:109–118.
2. Zarandi M. Amino acids. Amino Acids, Peptides and
Proteins. 2007;36:19–81.
83
NEW TECHNIQUES AND APPLICATIONS IN BIOPHYSICS
_____________________________________________________
NEW TECHNIQUES
AND
APPLICATIONS IN
BIOPHYSICS
84
_____________________________________________________
NTA-1] An NMR approach to scanning protein surface
1
1
2
1
Gómez, G.E. ; Bernar, E.M. ; Arán, M. and Delfino, J.M.
IQUIFIB (UBA-CONICET), Junín 956, (C1113AAD), Buenos Aires,
2
Argentina. FIL, Av Patricias Argentinas 435, (C1405BWE),
1
The solvent accessible surface area (SASA) is a key parameter to
understand protein conformation and interactions. The
photoreaction of diazirine (DZN) with the polypeptide chain serves
to estimate the size and nature of SASA. DZN, similar to water in
size and shape, generates methylene carbene, an intermediate
species that reacts unselectively with its molecular cage. We used
DZN for investigating protein folding and for mapping interfaces in
protein complexes (1-3). Mass spectrometry techniques allowed us
to derive a quantitative signal proportional to the extent of
modification of the sample (4). Here, we study the feasibility to
detect methylated products by multidimensional NMR, an approach
that does not demand cleavage and is potentially rich in
conformational information. We used E. coli thioredoxin (TRX) as a
protein model. Expectedly, on the basis of the larger extent of
SASA, the dominant modification phenomenon is the methylation
of amino acid side chains, giving rise predominantly to insertions
into C-H bonds. A solvent accessibility profile of the protein can be
derived from 1H13C and 1H15N-HSQC spectra.
References
5- Ureta DB et al. Biochemistry. 2007. p 14567.
6- Craig PO et al. J Mol Biol. 2009. p 982.
7- Gómez GE et al. Protein Sci. 2006. p 744.
8- Gómez GE et al. J Am Soc Mass Spectrom. 2012 p 30.
85
_____________________________________________________
NTA-2] Interaction of dopamine with graphene
Rossi Fernández, A.C., Castellani, N.J
IFISUR (UNS-CONICET), Bahía Blanca, Argentina
Dopamine (DA) is an important neurotransmitter that plays a
significant role in the function of the central nervous, renal and
hormonal systems. Ascorbic acid (AA) usually coexists with DA in
biological systems with concentrations that are much higher than
those of DA. Development of an efficient method for the
determination of DA with high selectivity and sensitivity is a
desirable objective for diagnostic uses. Because DA and AA are
electroactive compounds electrochemical techniques for their
detection have received large attention. Unfortunately, they shear a
similar oxidation potential in electrochemical detection. Recently,
various carbon materials have been proposed as electrodes to
obtain a better response to DA molecules, including carbon
nanotubes, carbon nanofibers, graphite and graphene (G). The
latter in particular showed excellent performance, attributed to the
π-π stacking interaction between DA and G surface. In this
communication the interaction of DA with G was theoretically
studied within the formalism of Density Functional Theory. The G
system was represented by using a slab model where the G layer
is replicated in the perpendicular direction with a gap of 10 Å
between layers. The 2D supercell has 50 carbon atoms. KohnSham eigenstates were obtained with the VASP code. Dispersive
interactions were included according to the scheme of Grimme.
Several DA/G geometrical configurations were considered,
analyzing in each case the presence of non-covalent interactions.
CONICET, ANCyT and UNS are acknowledged
86
_____________________________________________________
NTA-3] Supported lipid bilayers for molecular interaction
studies by SPR
1*
1
2
M. Antonieta Daza Millone , M. Elena Vela , Vanesa S. Herlax y
2
Sabina Maté
1
2
INIFTA (CONICET-UNLP), La Plata, ARGENTINA. INIBIOLP
(CONICET-UNLP), La Plata, ARGENTINA.
Lipid vesicles can adsorb and become planar bilayers from solution
onto hydrophilic surfaces like mica and silica [1]. Supported lipid
bilayers (SLBs) are suitable as model cellular membranes for
biophysical studies and medical applications. Nevertheless, there
is a technological challenge: to be able to succeed preparing SLBs
the influence of variables such surface modification, lipid
composition of vesicles and buffer composition must be separately
studied [2]. SPR (surface plasmon resonance) is a technique that
allows following molecular interactions in real time through
changes in the media surrounding a thin gold film without need of
labeling the lipids [3]. In this work, we prepared small unilamelar
vesicles (SUVs) with single composition (DMPC, POPC, DPPC
and DOPC) and ternary composition (DOPC/16:0 SM/Cho and
DOPC/24:1/Cho) to attempt fusion at a constant temperature
(23 °C). The thin gold surfaces were modified with self-assembled
monolayers (SAMs) of two different alkanethiols (dithiothreitol, DTT
and mercaptoundecanol, MUOH). Buffer composition PBS or Tris
was assayed with or without Ca2+ 1mM. According to the lipid
composition, conditions to immobilize vesicles or allow fusion was
optimized. In general, DTT SAMs allow higher number of
immobilized vesicles and Ca2+ was required to induce fusion.
MUOH SAMs allows direct fusion but the adhesion is poorer than
to DTT SAMs.
1 Richter, R. P., R. Berat, and A. R. Brisson. Langmuir.2006.
22:3497–3505
2 Nollert, P., H. Kiefer, and F. Jähnig. Biophys. J. 1995. 69:
1447–1455.
3 Morigaki, K and Tawa, K, Vesicle Fusion Studied by
Surface Plasmon Resonance and Surface Plasmon
Fluorescence Spectroscopy. Biophys. J. 2006. 91 1380–
1387
87
_____________________________________________________
NTA-4] Quantitative Mechanical Property Mapping of
bacteria with PeakForce QNM I
1
2,1,3
2
2,3
Bianchi, M. , Diaz, C. , Miñán, A. , Schilardi, P.L. , Pietrasanta,
2,3,4
L.I.
1
2
Centro de Microscopías Avanzadas, FCEN, UBA; Instituto de
Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA)
CONICET-UNLP; 3Consejo de Investigaciones Científicas y
Técnicas; 4Departamento de Física, FCEyN, UBA. Argentina
Since its development, AFM has proven itself to be a tool of choice
to image soft biological samples, especially with the emergence of
different operation modes and force spectroscopy and the fact that
it is one of the few microscopy techniques that allows observation
of cells under near-physiological conditions. PeakForce QNM is a
new mode developed that acquires and analyzes the individual
force curves from each tap that occurs during the imaging process.
The curves are then analyzed to obtain the properties of the
sample (adhesion, elasticity, modulus, deformation, and
dissipation) and the result is images that contain maps of material
properties [1]. In this study, we analyzed the effect of different
treatments on the mechanical properties of the cell membrane of
two types of bacteria: Pseudomona aeruginosa (Gram -) and
Staphylococcus aureus (Gram +). The microorganisms were
subjected to different treatments: silver ions (Ag +) and antibiotics.
Single cells topographic images and spatially resolved forced maps
reveal local significant variations of elasticity and nanomechanical
properties across the cell surface due to complex, anisotropic
composition of their walls.
Acknowledgements: CONICET, ANPCyT and UBA. M. B. has a
fellowship from CONICET.
1 Pittenger, B. et al., Bruker Application Note #128 (2011).
88
_____________________________________________________
NTA-5] Design of sensors to measure macromolecular
crowding in vivo
1
2
1
Labanda, M. S. , Craig, P. O. , Caramelo, J. J.
Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBACONICET) y Fundación Instituto Leloir, Av. Patricias Argentinas 435,
C.A.B.A. C1405BWE 2 Instituto de Química y Fisicoquímica Biológicas
(IQUIFIB/CONICET),Junin 956,C.A.B.A. 1113
1
The interior of cells is characterized by a high content of
macromolecules which occupy between 20 and 40% of the total
volume. Due to the mutual impenetrability of particles, this volume
fraction is unavailable to other molecules, producing a steric
repulsion that generates important kinetic and thermodynamic
consequences on processes occurring in vivo1. This makes
macromolecular crowding a physiological parameter of great
relevance that should be considered during in vitro experiments.
The aim of this work is to develop a probe to measure
macromolecular crowding in vivo. We started from a chimeric
protein consisting of a FRET pair, CFP and YFP2, linked by a
natively unfolded linker (CtCRT), which has a high content of acidic
amino acids. We made two variants using monomeric or dimeric
variants of CFP and YFP. Fluorescence spectra of the proteins in
media with increasing concentrations of PEG 8000 show that
FRET efficiency increases as PEG concentration increases,
suggesting that in crowded conditions the protein adopts compact
conformations. In order to understand these observations, we
performed coarse grained molecular dynamics simulations of the
protein at various fractions of volume occupied by inert spheres.
The analysis of the trajectories shows that the average distance
between chromophores decreases as crowding level increases.
Since the length and chemical identity of the unstructured linker
may influence the behavior of the sensors, we are working on the
replacement of the charged CtCRT linker by an inert and
unstructured poly-glycine segment.
1. Zimmerman S.B. J. Mol. Biol. (1991) 599.
2. Miyawaki A.Nature(1997)882.
89
_____________________________________________________
NTA-6] Quantitative mechanical property mapping of
bacteria with PeakForce QNM II: effect of Ag
nanoparticles
1,3
2,
1,3
1
Diaz, C. ; Bianchi, M. ; Miñan, A. ; Pissinis, D. ; Schilardi,
P.L.1,3; Pietrasanta, L.I.2,3,4
1
Instituto de Investigaciones Fisicoquímicas y Técnicas Aplicadas
(INIFTA), CONICET-UNLP. 2Centro de Microscopías Avanzadas
(CMA), FCEyN-UBA. 3Consejo Nacional de Investigaciones
Científicas Técnicas (CONICET). 4Departamento de Física,
FCEyN, UBA.
Bacterial resistance to antibiotics is considered one of the primary
global risks facing the modern medicine. Ag nanoparticles are the
most popular inorganic nanoparticles used as antimicrobial
promoter. Nevertheless, it has not been yet fully elucidated a
mechanism that correctly explains the antimicrobial action of Ag
nanoparticles. Some researchers suppose that, Ag nanoparticles
can induce pits and gaps in the bacterial membrane and then
fragment the cell. The investigation of the effect of silver on
bacterial cell wall is important since it can be useful to explain
some aspects of the action mechanism of Ag nanoparticles and to
further developed better antimicrobial therapies. The determination
of the mechanical properties of the bacteria membrane in situ,
without any immobilization treatment, has been a longtime
challenge. Since its development, AFM is a powerful diagnostic
and investigation tool. Nowadays, Peak Force QNM® allows
quantitative nanomechanical mapping of material properties,
including modulus, adhesion and dissipation, while simultaneously
imaging sample topography at high resolution [1]. In the present
work, we have studied the effect of Ag nanoparticles on
Pseudomona aeruginosa (Gram(-)) and Staphylococcus aureus
(Gram(+)) since they have structural differences in their cell walls.
Acknowledgements: CONICET, UNLP, ANCyPT, FCEyN, UBA
1 Pittenger, B. et al., Bruker Application Note #128 (2011).
90
_____________________________________________________
NTA-7] Structural study of silicate matrix for enzyme
based bionanosensor applications.
1
2
1
Burgos, M.I ., Oliveira, R.G. , Perillo, M.A.
2
IIBYT and CIQUIBIQ (CONICET- Universidad Nacional de
Córdoba)
1
Protein encapsulation in solid matrixes is of interest for
biotechnological purposes and it also serves as a model of
molecular crowding. We successfully entrapped the enzyme βgalactosidase (Eβ-Gal) in silicate gels via a sol-gel reaction and
obtained comparable levels of hydrolytic activity with those
obtained for soluble β-gal (Sβ-Gal). From the Michaelis-Menten
kinetic analysis employing 2-nitrophenyl-β-D-galactopyranoside
(ONPG) as substrate it was observed that both kcat and KM were
higher for Eβ-Gal than for Sβ-Gal, and they increased with the gel
aging time (At). In order to understand the enzymatic modulation of
β-Gal upon encapsulation in the silicate gels we performed several
structural studies of the silicate matrix. A qualitative analysis of the
topological structure was performed from SEM images. For this
study it was necessary to dry the gels with CO2 in supercritical
conditions obtaining aerogels, in order to preserve the original
porous structure. The SEM images showed that the gels consisted
in the agglomeration of ∼32 nm diameter particles (ranging
between 3 nm and 180 nm). From the BET isotherms obtained by
dynamic water vapor sorption it was observed that the aerogels
have an remarkably large surface-to-volume ratio and a
7
2
corresponding high specific surface area (1.5x10 cm /g). Wet gels
at At = 0 days, submitted to SAXS analysis, exhibited a ∼20 nm
gyration radius (Rg) which was consistent with mean particles sized
estimated by SEM for dried gels. The fractal dimension exponent
was D≈2. Neither drying, aging time nor the presence of β-Gal
affected significantly the porous structure of the gel.
Acknowledgements: Foncyt, Conicet, SeCyT-UNC (Argentina) and
SAXS1 beam line of LNLS (Brazil), for financial support. BMI, ORG
and PMA are career members of CONICET
91
_____________________________________________________
NTA-8] Three dimensional orbital tracking in a modified
two-photon microscope: an application to the tracking
of single gold nanoparticles inside living cells
1
3
1, 2
Gabriel, M. Gratton, E. Estrada, L.
Laboratorio de Electrónica Cuántica, Departamento de Física,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires; 2 Instituto de Física de Buenos Aires, Facultad de Ciencias
Exactas y Naturales. CONICET – UBA; 3 Laboratory for
fluorescence dynamics, Biomedical Engineering Department,
Universoty of California, Irvine, USA
1
We have developed a three dimensional (3D) single-particletracking microscope based on a two-photon raster scanning
microscope that allows us to localize and follow the 3D
displacement of a moving particle with high spatial (nm) and
temporal (ms) resolutions The orbital tracking technique is based
on a feedback algorithm that follows the particle position by: first,
scanning a circular orbit around the particle and then changing the
coordinates of the center of the orbit in such a way to keep the
particle always at the center. In this work, we have built a twophoton microscope and modified the scanning module in order to
allow orbital scanning. As an application, we have track single gold
nanoparticles (AuNPs) within the nucleus of CHO-K1 and NIHT3T
cells. Since there is electrostatic interaction between AuNPs and
the chromatin, while they move, they follow the chromatin structure
building up a 3D trajectory that contains high-resolution information
of the chromatin organization. The possibility to follow the NP's
position allows us to explore the chromatin architecture on the
nanometer scale and reveal some aspects of chromatin dynamics.
Our results suggest that the NPs undergo two different motion
regimes throughout the nucleus. We observed regions of random
diffusion connected by segment-like regions of active motion as
shown by the local time dependent mean square displacement
analysis.
92
_____________________________________________________
NTA-9] Fluorescence microscopy analysis in prostate
cancer cells reveals morphological and organizational
differences between hemin-treated and control cells.
1
2
2
2
2
C. Pallavicini , A. Paez , V. Levi , E. Vazquez , G. Gueron and L.
1
Bruno
1
Departamento de Física, FCEN, UBA and IFIBA, CONICET.
2
Departamento de Química Biológica, FCEN,UBA and IQUIBICENCONICET
Cellular motility is the basis for cancer cell invasion and
metastasis. It has been demonstrated that Heme Oxygenase-1
(HO-1) (rate-limiting enzyme in heme degradation) regulates the
adhesive properties and morphology of prostate cancer cells (PC3)
cell proliferation, migration and invasion. In order to characterize
the role of HO-1 in PC3 we further explored the influence of hemin,
a pharmacological inducer of HO-1, on the organization of the
cytoskeleton. With this aim, we labeled the microtubules with
Alexa-Fluor647 and studied their effective persistence length in
fixed cells, yet no significant differences were obtained. On the
other hand we explored the organization of phalloidin-rhodamine
labeled F-actin and we observed that the treatment affected the
contacts (in the form of filopodia-like protrusions (FLP)) among
cells and the distances between cell pairs. By combining confocal
microscopy with computational tools we were able to quantify the
contacts among cells, the filopodias per cell and the first-neighbor
distances for both hemin-treated and control cells. Our results
revealed a higher amount of cell contacts in hemin-treated cells.
These cells also contained a significantly larger amount of FLP.
Finally, by analyzing the first-neighbor ensembles in fluorescence
wide-field images we concluded that hemin treated-cells are nearer
from each other than control cells. Our approach allowed us to
quantify morphological and organizational differences between
both conditions which are probably related with the influence of
HO-1 in the remodeling of the actin filament architecture at
filopodia, altering cellular morphology, towards a more adhesive
and less invasive phenotype.
93
PROTEINS AND NUCLEIC ACIDS
_____________________________________________________
PROTEINS AND
NUCLEIC ACIDS
94
_____________________________________________________
PNA-1] Thioredoxin-like reactivity of the cysteine-rich
35-131 segment of ICA512
Ríos, A. S., Toledo, P. L., Llovera, R., Ermácora, M. R.
Laboratorio de Expresión y Plegado de Proteínas, Univ. Nac. de
Quilmes.
ICA512, also known as IA2, is a tyrosine phosphatase
transmembrane protein present in secretory granules of
neuroendocrine cells. The extracellular domain of ICA512
comprises a cysteine-rich fragment (CRF ICA512, residues 35-60,
containing four cysteine residues), a segment homologous to
RESP18 (residues 61-131), and the mature, membrane proximal
ectodomain (MPE ICA512, residues 469-557). We previously
characterized MPE ICA512 by x-ray crystallography, however, very
little is known about CRF ICA512 and the RESP18-like domain. To
gain further insight into the structure and function of the two latter,
recombinant ICA512 35-131 was prepared and subjected to
biophysical studies. ICA512 35-131 is unstable under neutral and
alkaline conditions. However, at pH 4.5, it acquires a metastable,
partially-folded structure which tends to form aggregates stabilized
by disulfide bonds. In the soluble sate, between three and four
cysteine residues are highly reactive in disulfide-exchange
reactions, in a way that is reminiscent to the thioredoxin activity. A
detailed kinetic analysis of ICA512 35-131 was performed using
two 5,5'-dithiobis-(2-nitrobenzoic) acid and 4,4'-dithiodipyridine,
comparing several disulfide-containing proteins. Second order rate
constants were derived from the analysis and a thioredoxin-like
activity was demonstrated. In parallel with this assays were tested
binding of insulin to ICA 521 35-131 by ELISA and Surface
Plasmon Resonance. The biological significance of the unusual
reactivity of the cysteine residues of ICA512 35-131 and its relation
with the insulin binding capacity of the protein are being analyzed.
95
_____________________________________________________
PNA-2] Auto catalytic ROS dependent proteolysis of
ICA512
Toledo, P, Llovera, R, Rasia, R y Ermácora, M.
Grupo Vinculado de Biología Estructural y Biotecnología, Imbice,
Univ. Nac. de Quilmes-Conicet
Instituto de Biología Molecular y Celular de Rosario, CONICETUniv. Nac. de Rosario.
ICA512/IA-2 is a receptor-type protein-tyrosine phosphatase
(RPTP) that localize in secretory granules of various
neuroendocrine cells. In pancreatic islet β-cells, it participate in the
regulation of insulin secretion, ensuring proper granulogenesis,
and β-cell proliferation. In previous works we reported the auto
catalytic, ROS dependent, proteolysis of the membrane proximal
ectodomain of ICA512 (MPE ICA512; residues 448-576). The
cleavage observed in vitro removes about 20 amino acids at both
the N- and C-termini. To gain further insight in this topic, two
shorter version of MPE ICA512, including residues 469 to 576 and
469 to 557, were prepared. The three MPE ICA512 variants were
compare with regard to their auto proteolytic properties. It was
found that the activity depends of sequential determinants located
at residues 557 to 576. The residues in that region presumably
bind a redox active metal required for catalysis. The removed tails
may be spatially close and for that reason undergo cleavage
concomitantly. Since this auto proteolytic activity may be an
important physiological feature leading to the shedding of the
ICA512 ectodomain in vivo, we further investigated the structural
properties of the termini. To that end, NMR studies were conducted
which showed that the N- and C-terminal region behaves in
solution as unfolded segments. The results contribute to our long
term effort to prepare a 3D model of the receptor embedded in the
membrane.
96
_____________________________________________________
PNA-3] Implications of Galectin-1 binding to lactose in
the dimerization equilibrium
1
2
3
1,3
Romero J. M. , Estrin D. A. , Rabinovich G. A. , Di Lella S.
1
2
INQUIBICEN-CONICET, FCEN-UBA;
INQUIMAE-CONICET,
3
DQIAyQF, FCEN-UBA, Laboratorio de Inmunopatología, IBYMECONICET.
Galectin-1 is a β-Galatoside binding protein involved in cell
communication and differentiation processes.1 It has been proved
to form a non-covalent homodimer at physiological conditions,2
presumably responsible for the formation of lattices structures on
the surface of target cells.3,4 However, little is known about the way
these structures are formed and organized. In this work, we
employ stopped-flow experiments to measure the rate constants
associated with the ligand binding and dimerization processes of
recombinant human Galectin-1. Interestingly, we find that dimer
dissociation process is kinetically affected by the presence of
ligand. From a computational perspective, we analyze the
molecular determinants of the kinetic barrier to the occupation of
water sites as a function of the reaction coordinate. A discussion
about the biophysical and biochemical implications of these
findings in the formation of the lattices and Galectin-1 function is
presented.
Acknowledgements: Conicet, ANPCyT, UBA, Fundación Sales,
CIN, and Dr. Madia Trujillo
1. Rabinovich GA and Toscano MA, (2009) Nat. Rev.
Immunol; 9(5):338-52.
2. López-Lucendo, et al, (2004) J Mol Biol; 343:957–970
3. Garner OB and Baum LG, (2008) Biochem Soc Trans;
36(6): 1472–1477.
4. Brewer et al, (2002) Curr Opin Struct Biol;12(5):616-23.
97
_____________________________________________________
PNA-4] Bovine Serum Albumin Nanoparticle (BNp):
Structure/Function
1
1
1
Macarena Siri , Gabriela Torchio , Ramiro Llovera , Juan
4
1
1
Francisco Delgado , Mariano Grasselli , Silvia del V.Alonso
1
2
. GBEyB- IMBICE CONICET, LBM, LaMaBio, LEPP – UNQ. .
Laboratorio de Obtención, Modificación, Caracterización y Estudio
de Materiales (LOMCEM)- UNQ
A new nanoparticle made from bovine serum albumin (BSA) was
characterized as a possible carrier for a drug delivery system. The
nanoparticle was obtained according to Soto Espinoza et al., 2012.
BNp spectroscopic studies were carried out by UV-Visible,
Fluorescence, FTIR, DLS and Z potential, microscopy (TEM) and
surface characterization (amino groups, thiols and carbonyls
detection). The stability of the BNp was also studied by pH,
different detergents and temperature. Freeze-drying was studied
as a possible way of BNp storage. The product was studied by
SEM, UV-Visible, 4th derivative and fluorescence. The
lyophilisation process gave particles of larger size (40 nm, 130 nm
y 350 nm). Lastly, a drug of choice (Emodin) was carrierencapsulated in order to test its structure/function as a drug
delivery system. Assays on encapsulation under different
conditions, release kinetic profile and saturation curve by
fluorescence were carried out. Results showed the existence of the
nanoparticle (with a slightly elliptic shape), which has a size
between 20 – 70 nm. By spectroscopy assays BSA molecules
forming the BNp, conserved a similar protein structure and stability.
Differences appeared in the surface characterization where the
free thiols on the BNp differed in quantity from those in the
molecule, c.a. doubling it. As regards the encapsulation process,
room temperature and 15 minutes of incubation was the most
efficient condition (79%). The release kinetic profile was similar to
that of the BSA. The saturation curve showed similar results
between both systems tested.
1. Silvia L. Soto Espinoza, Mirna L. Sánchez, Valeria Risso,
Eduardo E. Smolko, Mariano Grasselli Radiation synthesis
of seroalbumin nanoparticles J. Rad.Phys.Chem. (2012),
1417.
98
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PNA-5] Quaternary Structure and Activity Modulation of
HPRT of Trypanosoma cruzi
Valsecchi, WM; Santos, J; Delfino, JM.
Departamento de Química Biológica, IQUIFIB (UBA-CONICET),
FFyB, UBA.
Hypoxanthine/guanine phosphoribosyl transferase (HPRT) is a
globular α/β protein of 221 amino acid residues that catalyzes the
transfer of a ribose-1P from phosphoribosyl pyrophosphate (PRPP)
to hypoxanthine or guanine bases yielding IMP or GMP,
respectively. HPRT has been proposed as a potential target for
drugs useful for treating diseases caused by protozoan parasites,
given that, while in humans purine nucleotides may be obtained
both through the salvage pathway or by de novo synthesis, the
salvage pathway is the only one operational in trypanosomatids,
therefore becoming essential for their survival. Contrary to the
long-standing claim that TcHPRT is a dimer, we have previously
shown that this enzyme behaves as a tetramer in solution. In
addition, we have crystallized and solved its structure, which
allowed us to infer that the C-terminal region (CTR) is flexible and
might be involved in the stabilization of its quaternary structure.
Here we present evidence on the role of the CTR in the
consolidation of the quaternary structure and on the effect of
tetramerization on enzymatic activity. We found that proteolytic
removal of the CTR yields a defined dimeric species with increased
activity. We also show preliminary data on the inhibitory activity of
a set of several bisphosphonates -which resemble PRPP, and are
currently used to treat osteoporosis- and on the co-crystallization of
these ligands with the protein. A concrete picture of the molecular
features determining the inhibitory power emerges from this data.
All in all, our results suggest that the CTR represents an important
structural element bearing a significant role in both structure and
function. Besides, the assay of new inhibitory compounds bears
promise for the development of more effective treatments for
protozoan diseases.
With grants from ANPCyT, UBACyT and CONICET.
99
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PNA-6] Using chimeras to explore protein activity: A
case study of neuroglobin and myoglobin
1, 2
1
1, 3
Ignacio Boron
, Lucía Chisari , Luciana Capece , Diana E.
1, 2
1, 4
3, 4
Wetzler , Marcelo A. Marti , Dario A. Estrin and Alejandro D.
1, 2
Nadra .
1
Departamento de Química Biologica Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires. 2IQUIBICENCONICET, Argentina. 3Departamento de Química Inorgánica
Analítica y Química Física, Facultad de Ciencias Exactas.
4
INQUIMAE-CONICET, Argentina.
Globins are well-known proteins that generally exhibit two histidine
residues close to the iron, so it could be expected them to
coordinate the heme. Nevertheless, typical globins such as
myoglobin (Mb) and hemoglobin are pentacoordinated (5c) with
only one His binidng to the iron. In the last decade, however, many
hexacoordinated (6c) globins were discovered and several roles for
hexacoordination in protein function were proposed. We are
interested in neuroglobin (Ngb), which is expressed in the nervous
system of vertebrates and whose function is still under debate. Our
goal is to shed light on the key determinants for heme 6c⇆5c
equilibrium that regulates globin reactivity. For this purpose we
engineered Mb and Ngb by swapping their CD region with the goal
of exchanging the 6c⇆5c equilibrium behavior. Further, a point
mutant was designed to enhance coordination shift. Results
Residue contact map and dynamic properties of parent proteins
are generally transferred to the chimeras. Stopped flow, laser flash
photolysis and autoxidation experiments support this behavior
transfer. Altogether, our results confirm a significant role of the CD
region in the modulation of the 5c⇆6c equilibrium and
consequently in globins fuction, but also suggest contributions of
other protein regions.
100
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PNA-7] Mimicking the initial steps of aggregation using
low concentrations of TFE: the conformational
coalescence of the IFABP abridged family.
1
1
2
1
1
CR Angelani , M Poncino , JJ Caramelo , JM Delfino , LM Curto
1
Instituto de Química y Fisicoquímica Biológicas “Prof. Alejandro
Paladini” (UBA-CONICET), CABA, Argentina. 2Instituto de
investigaciones Bioquímicas de Buenos Aires, Fundación Instituto
Leloir. CABA, Argentina
∆98∆ and ∆78∆ are two all-β sheet variants of IFABP (intestinal
fatty acid binding protein). These frameworks became useful to
study the molecular determinants related to aggregation of β-barrel
proteins. Albeit displaying increased conformational plasticity,
these variants exhibit a native-like topology and are able to support
a cooperative folding behavior. At odds with the established notion
that a perturbation of the native fold should necessarily favor the
population of aggregation-prone species, we have demonstrated
that
the
intrinsic
stability
of
these
proteins
(∆G°H2O:IFABP≥∆78∆>∆98∆) does not bear a straightforward
correlation with their aggregation propensity triggered by
trifluoroethanol (TFE: ∆78∆>IFABP>∆98∆). In this scenario, it
might be more insightful to correlate aggregation propensity with
the stability measured in the presence of this co-solvent. With this
in mind, we initially characterize the changes in conformation and
stability of this protein family upon the addition of a subaggregating concentration of TFE (10% v/v). This treatment brings
about the coalescence of all three proteins into conformations
richer in β content and more akin in stability, as shown by thermal
ramps and exposure to chemical denaturants. New biophysical
measurements include high resolution NMR, binding of ANS and
quenching of intrinsic fluorescence. The cumulative evidence
supports the hypothesis that the conformational changes observed
would represent those leading to the aggregation-prone species.
With support from CONICET, ANPCyT and UBACyT.
101
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PNA-8] NMR derived insights into the conformational
changes exerted by TFE on the IFABP abridged family.
1
1
2
2
1
LM Curto , CR Angelani , M Arán , M Gallo , JM Delfino
1
Instituto de Química y Fisicoquímica Biológicas “Prof. Alejandro
Paladini” (UBA-CONICET), CABA, Argentina. 2Instituto de
investigaciones Bioquímicas de Buenos Aires, Fundación Instituto
Leloir. CABA, Argentina
IFABP (intestinal fatty acid binding protein) is a 15 kDa intracellular
lipid binding protein that represents an excellent model of study for
β-barrel proteins. It exhibits a β-clam structure built of 2
perpendicular 5-stranded β-sheets and an intervening helix-turnhelix motif located in between strands A and B. ∆98∆ (fragment
29-126) is a monomeric all-β sheet variant that lacks β-strand A,
most of the helical domain and the last 5 C-terminal amino acids.
By contrast, a further abridged form, ∆78∆ (fragment 29-106)
adopts a stable dimeric structure. Albeit displaying increased
conformational plasticity, these variants exhibit a β-barrel topology
and are able to support a cooperative folding behavior. Despite the
putative exposure of free edges, the constructs are stable in
solution and lack any intrinsic trend to aggregate. We have
postulated that these variants share a compact core decorated by
a loose peripheral region. Here we show preliminary NMR results
on the influence of the co-solvent 2,2,2-trifluoroethanol (TFE, up to
10%v/v) that support a gain of structure. Changes observed go in
line with the global consolidation of a native-like topology, as
evidenced by circular dichroism spectroscopy in both the far and
near UV regions.
With support from CONICET, ANPCyT and UBACyT.
102
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PNA-9] Synthesis of ZnO nanoparticles and conjugation
study with Bovine Serum Albumin
Ledesma, A. E.
Centro de Investigaciones y Transferencia de Santiago del Estero
(CITSE-CONICET). Universidad Nacional de Santiago del Estero
(UNSE).
The interaction of nanoparticles (NPs) with biological fluids may
induce conformational changes in the proteins present in the
medium. Such interaction could induce function loss or important
modification in some proteins. The interaction between Bovine
Serum Albumin (BSA) with semiconductor zinc oxide (ZnO) NPs,
synthetized by aqueous route from zinc nitrate hexahydrate, was
studied using spectroscopy of absorption, fluorescence, Fourier
Transform Infrared and Raman. The addition of ZnO NPs
quenched the intrinsic fluorescence of BSA. The Raman spectra
confirm the interaction of the Trp and Tyr residues on surface of
NP, while that the FTIR results indicate that the metal oxide
surface induce conformational changes where the main
transformation is a change in α-helix (∼ 9 %) and an increasing in
β-sheet structures (∼ 25 %). At high NPs concentration, with the
increment of BSA concentration a blue shift in λmax absorption of
protein was observed, which confirm the conformational changes
in the protein. On the other hand, a decreasing of absorbance and
emition of ZnO NPs was observed by its interaction with BSA. This
protein contributed at the stabilization and bioconjugation of ZnO
NPs reducing the aggregation in solution.
Acknowledgements: Dra. Rosa María Álvarez, Instituto de FísicoQuímica; Facultad de Bioquímica, Química Y Farmacia, UNT.
103
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PNA-10] EXPLORING IN VIVO FUNCTIONS OF Yarrowia
lipolytica STEROL CARRIER PROTEIN 2 (YLSCP-2)
1,2
1,2
1
1
Gianotti A. R. , Ferreyra R. G. , Pérez de Berti F. , Scott C. Y. ,
1
1,2
Burgardt N. I. , Ermácora M. R.
1
Departamento de Ciencia y Tecnología, Universidad Nacional de
2
Quilmes. IMBICE-CONICET
Sterol Carrier Protein 2 (SCP2) is a nonspecific lipid transfer
protein that has been implicated in the transfer, uptake, and
metabolism of cholesterol, branched-chain fatty acids, acyl-CoA
conjugates, and other lipids. SCP2 are present as domains of
multidomain proteins or as single domain polypeptides in all forms
of life. SCP2 structure and function has been studied mostly in
mammals and next in insects. In these organisms, it has been
generally found that the main function of this protein is in the
peroxysomal degradation of lipids. We have previously shown that
Yarrowia lipolytica SCP2 (YLSCP2) is a 128-amino-acid basic
protein inducible by fatty acids, that it is located in the yeast
peroxisomes and able to bind a variety of lipids and transfer them
to membranes by a collision-mediated mechanism. YLSCP2
structure was recently resolved in our lab. X-ray diffraction of the
protein shows the lipid binding site as a large system of
interconnected tunnels and surface pockets partially occupied by
palmitate. However, very little is known about the function of this
protein in plants, yeast and prokaryotes. Intriguingly,
Saccharomyces cerevisiae and S. pombe are the only fungi known
to lack SCP2 or any similar domain. For this reason, we express
the YLSCP-2 gene in S. cerevisiae in order to evaluate the
physiological function of this protein. We found that cells
expressing the protein are more sensitive to oxidative stress
compared to cells lacking the protein. So, we hypothesized that
YLSCP2 may be involved in the oxidative stress response,
kidnapping peroxidized lipids generated by oxidative stress and
disseminating those to different structures in the cells.
104
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PNA-11] Gliadin is able to self-assembly under
physiological pHs. Insights of gliadin intorelance
disorders.
Veuthey, T., Herrera, M. G.; Costabel, M; Dodero, V. I.
Biomolecular Group, Chemistry Department-INQUISUR, UNSCONICET, Bahía Blanca, Argentina Sur.
Gliadin and glutenin are the major component of gluten, a protein
complex present in wheat .It is known that its mean ingestion in a
healthy individual is around 50g per day. Gluten but especially
gliadin is the responsible of a wide range of autoimmune disorders
as celiac disease and gliadin intolerance. Actual statistics have
determined that the first one affects 1% and the second 7% of the
total world population (1, 2). These disorders are caused by the
incomplete proteolysis of gliadin during digestion, however the
reasons of this resistance remains unclear. Taking into account
that change of pH from ≈ 2-3 to 7 is an important step during
digestion (3); we evaluated the supramolecular characteristics of
gliadin at both conditions by combination of UV-Vis and
Fluorescence Spectroscopy, Electron Microscopy and Dynamic
Light Scattering. Based on our results, we hypothesized that the
morphology and nature of gliadin aggregates could led to
proteolytic resistance in vivo.
1. Rubio-Tapia, A. et. al. Curr. Opin. gastroenterol. 2010. 116.
2. Sapone, A.et al. BMC Med. 2012. 13.
3. Kararli, T. T. et. al. Biopharm. Drug Dispos. 1995. 351.
105
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PNA-12] A Supramolecular perspective of the first
stages of gluten intolerance disorders.
a
a
a
b
c
Herrera, M. ; Benedini, L. ; Veuthey, T. , Lonez, C. ; Hellweg, T. ;
b
a
Ruysschaert, J-M. ; Dodero, V. I. Biomolecular Group, Chemistry
Department-INQUISUR, UNS-CONICET, Bahía Blanca, Argentina.
b
Centre de Biologie Structurale et de Bioinformatique (CBSB),
Structure et Fonction des Membranes Biologiques (SFMB),
Universite Libre de Bruxelles, Belgium. c Department of Physical
and Biophysical Chemistry, Faculty of Chemistry, Bielefeld
University, Germany.
Gliadin is a protein present in wheat, rye and barley, which is no
complete degraded during digestion, producing peptides. One of
the most important is 33-mer, which has a rich contempt of proline
and has been identified as one of the major responsible of gluten
autoimmune pathologies, as Celiac Disease (C.D.) and gluten
intolerance (1) The pathological mechanisms involved in these
disorders remain unclear. However it is known that a highly
percentage of the patients that suffer from celiac disease presents
a copy of HLA-DQ2 and a 6% of them have the HLA-DQ8 (2).
Recently our group has demonstrated that 33-mer peptide is
capable of self-assembly in spherical and linear oligomers (3).
Herein, we present a supramolecular perspective of the first stages
of gliadin intolerance disorders by combination of Circular
Dichroism, ATR-FTIR, Dynamic Light Scattering and cells
experiments.
1. Sapone, A. et. al .BMC Medicine. 2012. 10.
2. Liu, J. et. al. Am. J. Hum. Genet. 2002. 51.
3. Herrera, et. al. Biopolymers. 2014. 101
106
_____________________________________________________
PNA-13] Secondary structure determines the rheological
properties of peptide monolayers
Caruso, B; Ambroggio, E.; Wilke, N; Fidelio, G.D.
CIQUIBIC, Depto. Química Biológica, Fac.
CONICET, UNC.
Cs.
Químicas,
Understanding the rheology of interfaces covered with proteins is
of a particular interest for interfacial biophysics. To date, there is no
clear data on how secondary structure modulates the rheological
properties of protein interfaces. Here, we study the surface
rheology of two simple but different peptides: the α-helix Melittin
(Mel) and the β-sheets Beta-amyloids (Aβ1-40 and Aβ1-42) by a)
evaluation of their monolayers response to an oscillatory
anisotropic compressive work and b) tracking of beads diffusing at
the interface (microrheology) which provided higher sensibility for
those monolayers presenting low shear response. Aβ1-40/42,
exhibit marked elastic shear modulus whereas Mel monolayers
exhibit no shear modulus and their microrheological shear was
markedly lower than those for Aβ1-40/42. On the other hand, it has
been proposed that Mel may adopt a β-sheet structure at pH 11 [1]
(verified here by ATR and FT-IR measurements). Interestingly, Mel
monolayers over pH 11 exhibit an increase in their microrheological
shear. In all cases, the rheology scales for a typical polymer and
their analysis suggest that the observed shear response is not due
to steric restrictions (as it is proposed for proteins [2]). The data
suggest that the interactions responsible for the marked shear of
Aβ1-40/42 monolayers are the hydrogen bonds of the β-sheet
structure that can form an infinite planar network at the interface.
Altogether, this study shows a clear-cut difference on the
rheological properties of peptide monolayers that adopt a
differential secondary structure at the air-water interface.
Supported by grants from SeCYT-UNC, CONICET and FONCYT
(Argentina).
1. Fidelio et al. BBA.1986. 49.
2. Cicuta, P.; Terentjev, E. M. Eur Phys J E Soft Matter 2005,
147.
107
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PNA-14] Preliminary Studies on the Folding Mechanism
of Phenylalanine Hydroxylase
Castro, I, Santos, J , Bredeston, L
IQUIFIB (UBA-CONICET)/ Departamento de Química Biológica,
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires,
Argentina.
Phenylalanine hydroxylase from Legionella pneumophila (LpPAH)
catalyzes the hydroxylation of L-Phe to L-Tyr using
2+
tetrahydrobiopterin (BH4) and non-heme Fe as cofactors, and
oxygen as additional substrate with an optimum temperature at
45°C. LpPAH is a 272-residue protein which has a mixed α/β
topology that forms a dimer characterized by a large interface.
Noteworthy, this protein showed high thermostability, with a
reported Tm of 79 °C (1) suggesting that the interface between
monomers may be the source of the high stability observed. Here,
we have ask how coupled the global stability of the dimer and
monomers are. The protein was purified from E. coli (35mg/mL)
and its conformation was studied. Far-UV CD was compatible with
the expected native LpPAH secondary structure. Near-UV CD
exhibits signatures of asymmetric environments for Trp and Phe
residues, suggesting a rigid tertiary/quaternary structure. Unfolding
followed by Trp fluorescence was carried out. We detected an
intermediate (Ieq) state that exhibits a significant decrease in Trp
fluorescence intensity by comparison with the native state,
whereas Trp residues seem to be in average partially exposed to
the solvent as judged by the center of mass of emission spectra
and by comparison to the unfolded LpPAH. Work on the
hydrodynamic behavior of LpPAH by SEC-FPLC and light
scattering is under progress to determine whether the first
transition in the equilibrium unfolding includes loss of quaternary
structure. With financial support of UBA, CONICET and ANPCyT.
1- Flydal MI et al. PLoS One. 2012;7(9):e46209.
108
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PNA-15] Synthesis, purification and characterization of
peptides derived from E. coli alpha hemolysin
a
b
c
Fanny Guzmán , Sabina Maté , Laura Bakás and Vanesa Herlax
Núcleo Biotecnológico de Curauma, Pontificia Universidad
b
Católica de Valparaíso, Chile. Instituto de Investigaciones
Bioquímicas de La Plata (INIBIOLP), CCT- La Plata, CONICET.
Facultad de Ciencias Médicas. Universidad Nacional de La Plata.
c
Departamento de Ciencias Biológicas. Facultad de Ciencias
Exactas. Universidad Nacional de La Plata.
b
a
Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein
which belongs to the family of 'Repeat in toxins'(RTX). On the
basis of experimental data and structural predictions, four peptides
derived from HlyA were synthesized by the solid phase peptide
synthesis method, using the Fmoc strategy. The four peptides
were design as follows: PEP 1: correspond to transmembrane
domain that it was described as hemolytically active; PEP 2: also a
transmembrane domain which sequence correspond to a
cholesterol recognition/interaction amino acid consensus domain
(CARC); PEP3: similar to PEP2 but 6 aminoacids were added in
the amino extreme and PEP4 correspond to a CARC sequence
located near the acylation sites. The aims of this work were to look
for a peptide which present hemolytic activity, and study the
participation of CRAC and CARC in the stabilization of HlyA
monomers in membranes by their interaction with cholesterol.
After peptide synthesis, they were purified by Reverse-phase high
performance liquid chromatography (HPLC), using C-18 column.
The molecular mass of the peptides were determined by mass
spectrometry (MS). Finally, peptide structure was determined by
circular dichroism (CD). It is important to mention that the addition
of 6 aminoacids to PEP 2 (PEP3) gives the peptide a more
organized structure. The hemolytic activity of peptides was
measured using human erythrocytes and inhibition of hemolytic
activity assays were performed pre-incubating erythrocytes with
peptides and then adding them to wild type toxin. Results obtained
for PEP 2 are promising and encourage us to use it in the design of
immunotoxins.
109
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PNA-16] Doxycycline leads to the formation of offpathway non toxic α-Synuclein oligomers
1
1
1
González Lizárraga, MF ; Torres-Bugeau, CM ; Socías, B ; Avila,
1
2
2
3
4
CL ; Barbosa, LRS ; Itri, R ; Papy-Garcia, D ; Raisman-Vozari, R ;
Chehín R1.
1
INSIBIO and Inst. de Química Biológica Dr Bernabé Bloj
(CONICET-UNT). 2Instituto de Física da Universidade de São
Paulo. 3Laboratoire CRRET, Université Paris Est Créteil.
4
INSERM.
Thérapeutique
Expérimentale
de
la
neurodégénérescence.
The dopaminergic neuronal loss observed in Parkinson disease
has been linked to the pathological aggregation of α-synuclein.
Although this protein is mainly found in the cytosol, the presence of
misfolded or aggregated α-synuclein in blood and cerebrospinal
fluid suggests that it might play a role also at extracellular level.
Indeed exposure to extracellular pre-aggreated α-synuclein
induces cytotoxicity in primary glia and human neuroblastoma cell
cultures. Recently, an important number of studies showed that
tetracyclines have remarkable neuroprotective properties in
Parkinson’s disease animal models. In this work we explore the
mechanism by which doxycycline, a semi-synthetic secondgeneration tetracycline, is able to exert such a protection against αsynuclein mediated toxicity. Through small angle x-ray diffraction
and infrared spectroscopy we showed that doxycycline is able to
affect the rate of α-synuclein oligomers formation. Moreover, using
MTT viability assay, we observed that oligomers formed in the
presence of doxycycline show decreased toxicity against
dopaminergic cells. These oligomers seem to be off-pathway since
they are not able to form fibrils detectable by means of ThT
fluorescence assay or electronic microscopy. We propose that
doxycycline is able to modify the aggregation pathway of αsynuclein leading to the formation of a nontoxic oligomer by
binding to tyrosine residues as demonstrated by fluorescence
quenching assays. This study represents a milestone in the
assessment of the feasibility of using doxycycline as a therapeutic
agent in the treatment of Parkinson’s disease.
110
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PNA-17] Conformational analysis of an intrinsically
disordered RNA binding domain
Gauto, D.F, Suarez, I.P, Hails, G, Rasia R.M.
Insituto de Biologia Molecular y Celular de Rosario (IBRCONICET-UNR), Ocampo y Esmeralda, 2000, Rosario, Argentina
A large fraction of the eukaryotic proteome is composed of
Intrinsically disordered proteins (IDPs) and intrinsically disordered
regions within larger proteins. For a subset of IDPs their function is
linked to the acquisition of a folded structure upon partner
recognition. An unresolved issue in this subset of IDPs is whether
the sampling of conformational space is in a way linked to their
ability to recognize different partners. In the present work we
investigate the exploration of the conformational space by the first
dsRBD of DCL1 from A. thaliana. This domain acquires a folded
conformation with a complex topology upon binding dsRNA. We
make use of NMR observables to identify the sampling at residue
level. The protein shows a narrow distribution of chemical shifts,
indicating its disordered nature. However titration with urea leads
to further narrowing, showing that the native state is not fully
unfolded. We find that different regions of the protein show a
varying degree of unfolding with urea, suggesting that partial
structuring is not homogeneous along the protein. Finally with the
help of secondary chemical shifts and residual dipolar couplings
we achieve a physical description of the disordered state in terms
of a ensemble of structures.
This work was financed by grants from ANPCyT (PICT-2012-1702)
and CONICET (Coop Int 2012)
111
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PNA-18] Effect of data-collection temperature in the
radius of gyration of protein X-ray crystal structures
Grille, L., Acierno, J.P., Ermácora, M.R.
Grupo Vinculado de Biología Estructural y Biotecnología, Imbice,
Univ. Nac. de Quilmes-Conicet
The radius of gyration is a useful measure of the atomic distribution
in space and a suitable indicator of protein compactness. This
parameter has a remarkably narrow distribution for each folded
protein. Motivated by the recent report of a hyper compact state of
beta lactamase1, we performed a statistical analysis of the radius
of gyration of a set of circa 1,000 proteins with multiple X-ray
entries in the Protein Data Bank. The analysis showed an effect of
data collection temperature in the the radius of gyration. The X-ray
crystal structures of proteins with data collected at cryotemperatures (below 160 K, most of them collected at 100 K)
showed a radius of gyration significantly smaller than the equvalent
protein structures determined at room temperature. Furthermore,
our analysis showed the existence of a set of structures with a
radius of gyration significantly smaller than the average in the
corresponding subset of cryo-cooled proteins. In these ultra
compact cases the small radius of gyration could not be attributed
to other experimental parameter such as chain length or fold type.
Ultra compaction beyond the temperature effect is isotropic, i.e.
most atoms moves toward the center of mass. This peculiarity
discards rigid body or hinge conformational changes and points to
a more fundamental phenomena, rooted in the thermodynamic of
noncovalent interactions in protein folding.
1. Risso et al. (2012) Protein Science 21, 964–976
112
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PNA-19] On the Folding Mechanism of Human Frataxin
Faraj, S.E; Gonzalez Lebrero, R.M.; Román, E.A.; Santos, J.
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET),
The deficient activity of Frataxin (FXN), a mitochondrial ironbinding protein, is related to Friedreich’s ataxia, a
neurodegenerative disease that severely affects limb motricity and
cardiac function. FXN is an α/β globular protein, which consists of
130 residues in its mature wild-type variant. The C-terminal region
(CTR) of FXN lacks periodic structure and is packed against the
protein’s core. We have previously studied a series of naturally
occurring (pathogenic) and rationally-designed mutants of the
CTR, and found that this stretch is crucial for the consolidation of
tertiary structure, since mutations affect thermodynamic stability
and molecular dynamics over different timescales. In order to
elucidate the role that the CTR plays in the folding mechanism, we
studied the folding and unfolding kinetics of several CTR mutants.
At low urea concentrations the refolding branch of the ln(kobs) vs.
[urea] plot slightly deviates from linearity, and kinetic traces show a
“burst phase”; both these facts support the hypothesis of the
existence of an intermediate state. In addition, we found that the
refolding reaction is unaffected in the different variants—even in
one that has had the CTR completely removed, and both at 15 and
25 °C—while the unfolding mechanism is altered and correlated
with the thermodynamic stability observed in equilibrium unfolding
experiments, including its dependence with the buffer’s ionic
strength. These results imply that the CTR contributes to the
stabilization of the native state, although it may not take a major
part in the transition state for the rate-determining step in the
folding reaction. All in all, our findings allow us to gain further
evidence on the role of the CTR in the stabilization of FXN, as well
as to understand the determinants of kinetic stabilization, which
may help to explain the pathogenicity of some mutations and to
develop stabilizing compounds to be used as drugs to treat the
disease.
113
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PNA-20] Biophysical Characterization of Human
Receptor-Type Protein Tyrosine Phosphatase ICA512
Ectodomain expressed in Pichia pastoris
1
1,2
1,2
Samus, S. I. , Ferreyra, R. G. , Ermácora, M. R.
Departamento de Ciencia y Tecnología, Universidad Nacional de
Quilmes. 2IMBICE-CONICET
1
Human Receptor-Type Protein-Tyrosine Phosphatase ICA512 (or
IA-2) is a transmembrane protein located in secretory granules of
neuroendocrine cells. Identified as one of the main antigens of
autoimmune diabetes, it is associated with protein secretion
processes1. During insulin secretion, the cytoplasmic domain of
ICA512 is cleaved and relocated to the nucleus, where it stimulates
the transcription of the insulin gene. The structures of the
intracellular pseudocatalytic and extracellular mature domains are
known, but the transmembrane domain and several other parts of
the receptor are poorly characterized. We recently solved the
structure of the mature ectodomain ME ICA512 (residues 449 to
575) and identified potential dimerization interfaces involved in the
regulation of the secretion2, 3. ICA512 is a glycosylated protein and
molecular modeling studies suggest that the positioning of the
sugar residues imposes steric restraints on the type of dimerization
interface. To address this question, we previously expressed ME
ICA512 in Pichia pastoris, and the protein secreted to the culture
medium was partially purified and biochemically characterized.
Now, we further characterized ME ICA512 by CD and fluorescence
spectroscopy, to reveal structural features. The implications of
these findings for the biological activity of the protein will be
discussed.
1. Hermel et al. Eur J Neurosci. 1999. 8:2609.
2. Primo et al. J Biol Chem. 2008. 283:4674.
3. Primo et al. PLoS ONE. 2011. 6:e24191.
114
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PNA-21] Arg220 and Thr237 of PER-2 β-Lactamase have
an important role in the stabilization of the active site
and the interaction with β-lactams.
1
2
3
3
1
Ruggiero, M , Curto, L , Galleni, M , Charlier, P , Gutkind, G ,
Sauvage, E3, Power, P1
1
Laboratorio de Resistencia Bacteriana, Facultad de Farmacia y
Bioquímica, UBA, Argentina; 2IQUIFIB, Facultad de Farmacia y
Bioquímica, UBA, Argentina; 3Centre d’Ingénierie des Protéines,
Université de Liège, Belgium
In this study, we analyzed the impact of mutations in Arg220 and
Thr237 towards selected β-lactams and clavulanic acid, by
structural and kinetic analysis. Crystal structure of PER-2 was
refined to 2.20 Å (PDB entry: 4D20). Wild-type blaPER-2 gene and
derived mutants in R220 and T237 were generated by site-directed
mutagenesis. β-Lactamases were purified to homogeneity and
kinetic parameters to selected β-lactams and inhibitors were
determined. Circular dichroism was performed for all enzymes at
near and far UV. Catalytic efficiencies of R220 mutants towards
penicillins and cephalosporins are up to 6- and 100-fold lower than
wild type, respectively, whereas T237A mutant displayed slightly
higher kcat/Km values for some β-lactams, especially penicillins.
Circular dichroism results suggest that none of the mutations have
negative impact in the overall structure. Our results support the
previously suggested role of R220 and T237 in the maintenance
and stabilization of a hydrogen network, necessary for the proper
interaction with β-lactams. In this regard, R220 seems to be
essential for interaction with both clavulanate and cephalosporins,
while T237, though also important, would probably have a
secondary role in this network. The apparent structural stability of
the mutants also suggests that they are prone to be selected in
vivo.
115
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PNA-22] Structural insights into the “ceftazidimase”
behavior of CTX-M-96 β-lactamase.
1
1
2
3
Ghiglione, B , Rodríguez, M.M. , Curto, L , Galleni, M , Charlier,
3
1
3
1
P. , Gutkind, G. , Sauvage, E. , Power, P.
1
Laboratorio de Resistencia Bacteriana, Facultad de Farmacia y
2
Bioquímica, UBA, Argentina; IQUIFIB, Facultad de Farmacia y
3
Bioquímica, UBA, Argentina; Centre d’Ingénierie des Protéines,
Université de Liège, Belgium
Diversification of the CTX-M β-lactamases led to the emergence of
variants responsible for decreased susceptibility to ceftazidime,
being the D240G mutation the most prevalent among the so called
“ceftazidimases”. From the recently solved crystallographic
structure of CTX-M-96 (1.2 Å), we analyzed the organization of the
active site and evaluated the possible role of key amino acid
residues in the overall stabilization of the structure and also in the
interaction with different β-lactams and mechanism-based
inhibitors. Wild-type blaCTX-M-96 and derived mutants in D240 were
generated by site-directed mutagenesis. β-Lactamases were
purified to homogeneity and kinetic parameters to selected βlactams and inhibitors were determined. Circular dichroism was
performed for all enzymes at near and far UV. CTX-M-96 presents
some differences in the disposition of specific amino acids,
although none of them seem to impair the interaction with βlactams. In the absence of oxyimino-cephalosporins (OC), N132,
E166, P167 and N170 seem to be shifted up to 0.7 Å away from
the catalytic cleft, suggesting that the presence of antibiotic
induces the approach of these residues towards the OC through
hydrogen bonds. Circular dichroism results suggest that none of
the mutations have negative impact in the overall structure.
Structural differences do not seem to be conclusive to determine
the “ceftazidimase” behavior, and additional data are still needed to
explain the observed in vivo resistance to OC.
116
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PNA-23] Towards efficient biocatalysts:
photoimmobilization of a lipase on hybrid lysozyme-heparin
amyloid nanofibrils
1
2
3
Silvina Chaves , Cintia M Romero , Ricardo Mignone , Claudio D.
3
2
2
1
Borsarelli , Licia M Pera , Mario Baigori and Rosana Chehín
1
Instituto Superior de Investigaciones Biológicas (INSIBIO), CCTTucumán and Instituto de Química Biológica (CONICET-UNT)
Tucumán, 2PROIMI-CONICET, Av. Tucumán. Fac Bioq, Qca y
Farmacia (UNT), Tucumán. 3 Centro de Investigaciones y
Transferencia de Santiago del Estero (CITSE-CONICET). U.N. de
Santiago del Estero.
Amyloid fibrils have attracted nowadays a growing interest as new
biomaterials due to their special mechanical, chemical, and
structural properties, making them an excellent choice for
development of novel supports for different technological
applications. It is now widely accepted that the ability to form
amyloid aggregates is a common property of any polypeptide
chains. In fact, specific protocols for each protein have been
reported in order to turning them from soluble into highly ordered
amyloid aggregates with the characteristic cross-β structures
among peptide chains. In the present work, we report the
preparation and characterization of a biocatalyst based on the
photo-immobilization of a lipase onto hybrid amyloid nanofibrils of
heparin and lysozyme. The new hybrid nanomaterial lost both the
lysozyme antibiotic activity and its ability to induce changes in
membrane permeability. However, the hybrid nanofibrils present
key reactive amino acids exposed to the solvent, such as tyrosine
residues, which allowed the covalent attach of a lipase molecule
through crosslinking via tyrosyl radicals generated by blue-light
photosensitization of the metal coordination complex ruthenium (II)
tris-bipyridine in the presence of ammonium persulfate. Thus, the
photo-immobilized lipase onto the hybrid nanofibrils showed much
better enzymatic activity under different extreme conditions of
temperature and solvent as compared with the free enzyme. The
procedure reported herein could be useful to design a new
generation of biocatalyst by a single photo-click step in a clean and
faster fashion way than conventional chemical crosslinked
procedure.
117
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PNA-24] Crystallization and preliminary X-ray
characterization of the full-length bacteriophytochrome
from the plant pathogen Xanthomonas campestris pv.
campestris
Klinke, S., Otero, L.H., Rinaldi, J., Sosa, S., Goldbaum, F. A. &
Bonomi, H.R.
Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires,
Argentina
Phytochromes give rise to the largest photosensor family known to
date. However, they are underrepresented in the Protein Data
Bank. Plant, cyanobacterial, fungal and bacterial phytochromes
share a canonical architecture consisting of an N-terminal
photosensory module (PAS2-GAF-PHY domains) and a C-terminal
variable output module. The bacterium Xanthomonas campestris
pv. campestris, a worldwide agricultural pathogen, codes for a
single bacteriophytochrome (XccBphP) that holds this canonical
architecture, bearing a C-terminal PAS9 domain as the output
module. Full-length XccBphP was cloned, expressed and purified
to homogeneity by nickel-NTA affinity and size exclusion
chromatography and then crystallized at room temperature bound
to its cofactor biliverdin. A complete native X-ray diffraction dataset
was collected to a maximum resolution of 3.25 Å. Crystals belong
to the space group P43212 with unit-cell parameters a = b = 103.94,
c = 344.57 Å, and a dimer in the asymmetric unit. Refinement is
underway after solving the structure by molecular replacement [1].
This work was supported by CONICET, ANPCyT, MINCyT and the
SOLEIL Synchrotron (France).
1. Klinke, S. et al. & Bonomi, H.R. Acta Crystallographica
Section F: Structural Biology Communications. 2014. In
press
118
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PNA-25] New insights into antibiotic resistance of
resurrected ancestral β-lactamases.
a
a
b
Martínez-Rodríguez, S. , Risso V.A. , Gavira J.A. , Sánchez-Ruiz
a
J.M.
a
Departamento de Química-Física, Universidad de Granada,
18071, Granada, Spain. bLaboratorio de Estudios Cristalográficos,
Instituto Andaluz Ciencias de la Tierra (IACT-UGR-CSIC), 18100,
Armilla, Granada, Spain.
Laboratory resurrection of ancestral proteins has been shown to
provide insight into the ancient properties of biomolecules as well
as the intracellular and extracellular environments hosting these
proteins [1]. This methodology exploits natural sequence diversity,
and has been used to obtain protein variants with enhanced
characteristics [2,3]. In a previous work, reconstruction of
derivations of statistically probable sequences for Precambrian βlactamases in the Proteobactaria evolutionary track supported the
notions that Precambrian life was thermophilic and that proteins
can evolve from generalists (displaying substrate promiscuity) to
specialists (capable of efficient enzymatic turnover) during the
course of natural evolution [3]. In this work, we report new
biophysical and structural information on the antibiotic resistance of
resurrected ancestral class A β-lactamases.
1. Benner et al., Adv. Enzymol. Relat. Areas Mol. Biol. 2007,
75, 1; (b) Thornton, J. W. Nat. Rev. Genet. 2004, 5, 366;
(c) Carroll et al., PLoS Genet. 2011, 7, No. E1002117; (d)
Gaucher et al., Nature 2008, 451, 704; (e) Perez-Jimenez
et al., Nat. Struct. Mol. Biol. 2011, 18, 592.
2. Cole and Gaucher. Curr Opin Chem Biol. 2011, 15, 399.
3. Risso et al., J. Am. Chem. Soc. 2013, 135, 10580
119
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PNA-26] ABA-1A: a Nematode Polyprotein Allergen
(NPA) of Ascaris suum. Structure and binding
properties.
1
1
2
2
Franchini,G.R ; Bélgamo JA , Kennedy, M.W ; Smith, B.O and
1
Córsico, B
1
INIBIOLP-CONICET, Fac.de Ciencias Médicas, Universidad de La
Plata, Argentina. 2 Institute of Biomedical & Life Sciences, University of
Glasgow, United Kingdom.
The acquisition and transport of lipids from their hosts is crucial to
parasitic helminths, being the proteins and receptors involved in
lipid transport and exchange potential targets for chemo- and
immunotherapy. Among helminth lipid binding proteins (LBPs), the
polyprotein allergens/antigens of nematodes (NPAs) represent a
novel class of lipid binding proteins which has been described
exclusively in nematodes. NPAs are small, helix-rich proteins, and
have no known structural counterparts in other phyla. The
biochemical activity of the NPA of A. suum was first described as a
binding protein for small lipids such as fatty acids and retinoids.
Recently, the structure of a single unit of the polyprotein array
(ABA-1A) has been solved in the presence of saturating
concentration of oleic acid describing two binding sites. In the
present project we are working with ABA-1A in the absence of the
ligand (apo- form) and its atomic structure is under analysis
employing NMR spectroscopy, for which high quality data have
already been obtained and full structure calculation is in progress.
In order to obtain more information about the structural
perturbations due to ligand binding; an oleic acid titration of ABA1A monitored by NMR spectroscopy was performed. Briefly, it was
possible to distinguish two binding events according to the nature
of the perturbations observed. Additionally, as a first attempt to
determine the natural ligands bound by this protein a lipidomic
analysis was done using recombinant ABA-1A without the
delipidation step.
120
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PNA-27] Evaluation of 33-mer gliadin peptide
oligomerization: a fluorescence and microscopy study.
a
b
c
a
Herrera, M.G. ; Celej, M. S. ; Schilardi, P. L. ; Dodero, V. I.
Biomolecular Group, Chemistry Department-INQUISUR, UNSb
CONICET, Bahía Blanca, Argentina. Department of Biological
Chemistry, School of Chemical Science.CIQUIBIC, UNCCONICET, Córdoba, Argentina. c Department of Chemistry,
INIFTA, UNLP-CONICET, La Plata , Argentina.
a
33-mer peptide, LQLQPF(PQPQLPY)3PQPQPF, is a proteolytic
resistance fragment of gliadin protein present in wheat, rye and
barley (1,2). It is known that 33-mer is able to cross the gut
mucosa and trigger an immune response in sensible individuals.
However, the mechanism involved in these processes remain
unclear (2, 3). Previously, we reported that 33-mer oligomerizes
depending on peptide concentration (4). Here, we took advantage
of the presence of three tyrosines (Y) in the 33-mer peptide
sequence to perform anisotropy and time-resolved fluorescence
studies in order to obtain information about the mechanism of
peptide self-assembly in solution.. The different oligomerization
stages were also appraised by complementary atomic force
microscopy experiments.
Acknowledgements:
We thank Dr. G. Montich for allowing us to use the TCSPC lifetime
equipment.
1. Shan, L. et. al. Science.2002. 2275.
2. Shan, L. et. al. J. Proteome. Res. 2005. 1732.
3. Hadjivassiliou, M. et. al. Trends Immunol. 2004. 578.
4. Herrera, M. et.; al. Biopolymers. 2013. 96.
121
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PNA-28] Comparative analysis of the effect of
conservative mutations on resurrected ancestral
proteins
a
a
a
Fadia Manssour-Triedo , Valeria A. Risso , Alvaro Inglés-Prieto ,
Raquel Godoy-Ruizb, Jose A. Gavirac, Beatriz Ibarra-Moleroa and
Jose M. Sanchez-Ruiza.
a
Departamento de Quimica Fisica, Facultad de Ciencias,
Universidad de Granada, 18071- Granada, Spain. b Department of
Biochemistry and Molecular Biology, University of Maryland School
of Medicine, Baltimore, Maryland 21201, United States.
c
Laboratorio de Estudios Cristalográficos, Instituto Andaluz de
Ciencias de la Tierra (Consejo Superior de Investigaciones
Científicas- Universidad de Granada), Avenida de las Palmeras 4,
18100- Armilla, Granada, Spain.
We have carried out an extensive mutational analysis of two
thioredoxins: a laboratory resurrection of a protein of the last
common bacterial ancestor (LBCA thioredoxin), an organism
estimated to have lived about 4 billion years ago, and one of its
modern descendant (E. coli thioredoxin). Both proteins share the
same overall 3D-structure but show only 55% sequence identity (1).
A total of 21 variants involving exchanges between highly similar
amino acids (E/D, I/V)(3-4) were purified and their thermal stabilities
were investigated by Differential Scanning Calorimetry (DSC).
Surprisingly, our results indicate a significant correlation between
the mutation effects on the ancestral and modern backgrounds
suggesting a conservation of protein mutational energetics over
billions of year
1 Ingles-Prieto A, et al. (2013) Structure 21:1690-1697
2 Perez-Jimenez R, et al. (2011) Nat Struct Mol Biol 18:592596
3 Godoy-Ruiz R, Perez-Jimenez R, Ibarra-Molero B and
Sanchez-Ruiz JM. (2004) J Mol Biol 336:313-318.
4 Godoy-Ruiz R, Perez-Jimenez R, Ibarra-Molero B and
Sanchez-Ruiz JM. (2005) Biophys J 89:3320-3331
122
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PNA-29] Comparison between covalent and noncovalent BSA aggregation at acidic and neutral
conditions.
Guauque-Torres M. P., Ledesma, A., Borsarelli C.D.
Laboratorio de Cinética y Fotoquímica (LACIFO), Centro de
Investigaciones y Transferencia de Sgo del Estero (CITSECONICET), Universidad Nacional de Santiago del Estero (UNSE),
RN9, Km 1125. Villa El Zanjón. (CP 4206) - Santiago del Estero.
Argentina
Covalent and non-covalent protein aggregation provides interesting
immobilization strategies to obtain functionalized scaffolds for
biocatalysis and biorecognition. In this report we compared the
formation of non-covalent and covalent BSA aggregates formed in
phosphate buffer solution at pH 3 and 7 by using several
characterization techniques such as fluorescence, infrared
spectroscopy, SEM microscopy and electrophoresis. Non-covalent
BSA aggregation was achieved by heating at 60ºC, while covalent
aggregation was done by formation of di-Tyrosine bridges with blue
light photosensitization of a Ru(bpy)32+/ S2O82- mixture (1:20).
Thermally-induced aggregation kinetics were monitored using ThT
fluorescence, showed that nucleation time was reduced and the
aggregation rate was increased at pH 3 compared to pH 7;
indicating that BSA unfolded under acidic conditions favors the
aggregation process. SEM images indicates that morphology of
aggregates was controlled by pH, as at neutral pH spheroids of ∼
100nm of diameter while at acid pH longer fibril-like shape
aggregates (∼1µm), were formed. In the photosensitized covalent
aggregation of BSA the initial rate of di-tyrosine formation was
similar for acid and neutral pH, indicating that there is not influence
of the conformational state of the protein. Nevertheless, after
prolonged photolysis time there is also contribution to absorbance
by changes in amino acid 1O2-mediated. Both types of BSA
scaffolds will be evaluated for enzyme immobilization.
123
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PNA-30] Thermal unfolding of the PDZ domain of beta2syntrophin characterized by NMR
1
2
2
Gabriela Torchio , Martín Arán , Mariana Gallo , Mario Roberto
1
3
Ermácora y Mauricio Sica
1
Laboratorio de Expresión y Plegado Proteico, UNQ, IMBICECONICET; 2Fundación Instituto Leloir, CONICET; 3Laboratorio de
Bioenergía, Centro Atómico Bariloche, CNEA-CONICET
Beta2 syntrophin regulates insulin interactions of its PDZ domain
(B2S-PDZ). Thermal unfoling of B2S-PDZ do not fit properly to
models with discrete (two- or three) states, and may involve low or
negligible barriers. In this case, known as downhill unfolding, the
protein populates a unique state whose conformational
characteristics varies with temperature, and thus several structure
element can unfold (quasi) independently. Our previous studies
indicate that B2S-PDZ unfolfing better fits to low barriers models.
To get a deeper, we applied NMR spectroscopy the unfolding of
B2S-PDZ. For this purpose, 15N-1H-HSQC of B2S-PDZ were
obtained at different temperatures, from 5 to 50 °C. T1, T2 and
NOE at 20°C indicate that the protein is monomeric and rigid and
except for helix A that suffer slow conformational movements (0.11 ms). Since the intensity of the signals do not decrease with the
temperature, the chemical shift variations are consider to occur in a
fast exchange regime. The highest temperatures allowed by the
technique corresponded to Tm from far-UV Cd and DSC
experiments. Thus, after assigning each the spectra signals the
experiment gave information about the early conformational
behavior, showing that several elements suffer a gradual thermal
unfolding. These elements involve beta strands D,F, both ends of
helix B and C-end of beta strand C. These elements are clusterd in
a region that harbor several residues involved in the PDZ-ligand
binding. Our results indicate that B2S-PDZ is gradually unfolded at
least at temperatures previous to apparent Tm, in agreement with
a downhill model. Furthermore, this behavior could be of relevance
to understand the regulation of the function of this domain.
124
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PNA-31] Design and characterization of specific p38α
peptide inhibitors based on the docking site of its
transcription factor MEF2A
1
2
3
1
Bucci, H.A. , Lopez, E.D. , Iannucci, N.B. , Portela, P. , Turjanski,
A.G.2, Wetzler, D.E.1
1
Departamento de Química Biológica, IQUIBICEN, FCEN – UBA.
2
INQUIMAE, FCEN – UBA. 3Facultad de Farmacia y Bioquímica –
UBA
Mitogen activated protein kinases (MAPKs) are serine/threonine
kinases that play an important role in regulating various cellular
processes including cell growth, differentiation, inflammation and
apoptosis. The development of inhibitors of MAPKs is an important
research area for various diseases such as cancer, diabetes,
arthritis and inflammatory diseases. The kinase domain is highly
abundant in the human genome, therefore it is very important to
develop specific inhibitors for each particular MAPK. The first
inhibitors used the ATP binding site as target (type I inhibitors) but
this gave no specificity for each MAPK, the next generation
compounds targeted a docking site right next to the ATP binding
site (type II inhibitors) but these compounds do not inhibit MAPK
activation. So the next leap was developing compounds that target
the protein-protein docking site. Based on the crystal structure of
p38α - MEF2A peptide complex, we calculated the binding energy
of wild-type and point mutation peptides. We designed potential
inhibitors with higher binding energy than the wt peptide. We
selected 3 peptides, synthesized and tested them in activity
experiments using radioactive ATP. Here, we present in vitro
results of the inhibition effect of the selected peptides on the
phosphorylation of ATF2, a transcription factor target of active
p38α. Using this approach we found a potential inhibitor that not
only has a slightly greater inhibitory effect than the wt peptide but
also does not present the allosteric effect on ATP binding that wt
has at low concentration, making it a good candidate for further
characterization.
125
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PNA-32] Preliminary Structural Characterization of an
Iron-binder Engineered Thioredoxin
1
2
Vazquez D.S , Aran M and Santos J
1§
1
IQUIFIB (UBA-CONICET) and Departamento de Química Biológica,
2
FFyB, UBA, Argentina. Fundación Instituto Leloir and IIBBA-CONICET,
Frataxin (FXN), a member of the CyaY protein family, is
characterized by the presence of a large cluster of acidic residues
on its surface principally involved in iron-homeostasis. We have
designed and characterized, a short peptide (GRAP) that includes
the EExxED motif from the CyaY protein family, using the
sequence of the Ct helix of E. coli thioredoxin (TRX) as scaffold.
GRAP shows certain specificity for Fe2+ and Fe3+ characterized by
an 1:1 stoichiometry and a KD of 1.9±0.2µM. Iron binding is an
entropically driven process that is guided more likely by changes in
hydration. Both binding and peptide folding processes take place
upon metal interaction. To uncouple the coil-to-helix transition and
to study both the iron-binding affinity and the effect on intrinsic
dynamics, we grafted the motif onto the full-length TRX (TRXgrap).
TRXgrap protein is structured and showed a marginal impact on
global stability. Noteworthy, the apo form exhibits a diminished
activity. To characterize at the residue level the TRXgrap/Fe3+
interaction, a preliminary set of NMR and theoretical experiments
were carried out. The evaluation of 1H15N-HSQC spectra of
TRXgrap in the presence or absence of iron shows that the
interaction causes the disappearance of signals of some residues
located in the surrounding of the iron-binding motif, compatible with
the paramagnetic properties of this metal ion. On the other hand,
molecular dynamic simulations of the apo and holo forms shows
significant perturbations at the active site in the reduced state
suggesting effects of mutation and metal binding at medium-large
distances and explaining the observed reduction of enzimatic
activity.
Acknowledgements: This work was supported by grants from
ANPCyT, UBACyT and CONICET.
126
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PNA-33] Stabilizing effects of ATP, Mg2+ and K+ on the
NA+,K+-ATPase thermal inactivation
Placenti, M.A., Kaufman, S.B. González Flecha, F.L. and
González-Lebrero, R.M.
IQUIFIB-Departamento de Química Biológica, Facultad de
Farmacia y Bioquímica, Universidad de
Buenos Aires, Junín 956, 1113, C.A.B.A., Argentina.
Na+,K+ -ATPase is an integral membrane protein which couples
+
ATP hydrolysis to the transport of three Na out and two K into the
cell, cycling between the E1 and E2 conformers. In a previous work
we show that Na+ and K+, which leads the enzyme to E1 and E2
respectively, presented opposite effects on thermal stability of the
pump [1]. In this work, we characterize the effect of some natural
ligands on the protein thermal stability. Thermal inactivation was
performed incubating the enzyme in the presence or absence of
ATP, Mg2+ or K+ for different time periods and temperatures. After
this incubation we measured ATPase activity, Trp fluorescence
and Eosin Y binding. It is known that this fluorescent probe binds to
the ATP binding site, and therefore reflects the structural changes
in it. Ours results showed that in all conditions tested, ATPase
activity decreased following a first-order kinetic, concomitant with
the change in both Trp and eosin fluorescence. Transition state
theory and thermodynamic activation parameters (∆G‡, ∆H‡ y
∆S‡) were used to analyze and interpret the data. A clear
stabilization effect was observed for all three ligands, due to both
enthalpic and entropic contributions. This effect is more important
for K+, which displaces the equilibrium of the enzyme towards E2.
Even though ATP is known to displace the equilibrium to the E1 as
Na+, these two ligands have opposite effects in terms of thermal
stability of the Na ,K -ATPase.
With grants from: UBACyT, CONICET and ANPCyT.
1. Kaufman SB, González-Flecha FL, González-Lebrero RM. J
Phys Chem B. 2012. 116(10):3421-9.
127
SIGNALING AND INTRACELLULAR DYNAMICS
_____________________________________________________
SIGNALING AND
INTRACELLULAR
DYNAMICS
128
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SID-1] Monitoring in real-time focal adhesion protein
dynamics in response to a discrete mechanical stimulus
1,3
2,3
2,5
2,4
von Bilderling C. , Caldarola M. , Masip M. E. , Bragas A. V.
1,4
and Pietrasanta L. I.
1
Centro de Microscopías Avanzadas and Departamento de Física,
FCEN, UBA, Argentina. 2Laboratorio de Electrónica Cuántica,
Departamento de Física, FCEN, UBA, Argentina. 3IFIBACONICET-UBA, Buenos Aires, Argentina. 4Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Buenos Aires,
Argentina. 5present address: Max Planck Institute of Molecular
Physiology, Dortmund, Germany.
The adhesion of cells to the extracellular matrix is a hierarchical,
force-dependent, multistage process that evolves at several
temporal scales [1]. An understanding of this complex process
requires a precise measurement of forces and its correlation with
protein responses in living cells. We present a method to
quantitatively assess live cell responses to a local and specific
mechanical stimulus. Our approach combines atomic force
microscopy (AFM) with fluorescence imaging. Using this approach,
we evaluated the recruitment of adhesion proteins such as vinculin,
FAK and zyxin triggered by applying to live cells forces in the nN
regime. We observed the development of a nascent adhesion site,
which was evident from the accumulation of vinculin at the position
where the force was applied. In addition, we quantified the
recruitment time for FAK in the formation of a new adhesion site,
and analyzed the zyxin spatial distribution remodeling in mature
focal adhesion as a function of the applied force. We have
demonstrated that this method is a useful tool for the study of a
variety of complex biological processes involved in cellular
mechanotransduction.
1. Hoffman B.D, Grashoff C., Schwartz M.A. Nature. 2011.
475.
129
_____________________________________________________
SID-2] Influence of the biophysical properties of
molecular motors on intracellular transport
1
2
1
3
3
De Rossi MC , Bruno L , Wetzler D , Sued M , Rodriguez D , De
4
1
Rossi ME , Levi V
1
Instituto de Química Biológica, IQUIBICEN. FCEN-UBA-CONICET
2
Instituto de Física de Buenos Aires, IFIBA. FCEN-UBA-CONICET
3
Instituto de Cálculo. FCEN-UBA-CONICET 4Instituto de
Astronomía y Física del Espacio, IAFE. UBA-CONICET
Bidirectional transport along microtubules is driven by kinesin and
dynein motors, which transport cargoes toward the plus and minus
end of microtubule, respectively. The overall direction of motion
results from the cooperation and/or competition between these
opposed-polarity motors. In this work, we explore how the
biophysical properties of motors affect the dynamics of fluorescent
peroxisomes in D. melanogatser s2 cells to get insight into the
mechanisms involved in the bidirectional transport of organelles.
Using single particle tracking, we registered trajectories of
peroxisomes and determined the run length and velocity
distributions to get information about the mechanism of transport
and the population of motors involved. We compared the transport
properties in wild type cells with those obtained for cells expressing
the slow kinesin Eg5. While the run lengths and velocities of minus
end directed peroxisomes are not affected by the presence of Eg5,
both run lengths and velocities of plus end directed peroxisomes
decreased. Our results suggest that Eg5 acts as an anchor to
kinesin 1, reducing the forward stepping frequency and increasing
the probability of detachment.
130
_____________________________________________________
SID-3] Calcium signals: experiments and simulations to
make visible the invisible.
Piegari, E., Lopez, L., Perez Ipiña, E. and Ponce Dawson, S.
Departamento de Física (FCEN-UBA) and IFIBA (CONICET-UBA)
Calcium signaling is ubiquitous across cell types. Intracellular
calcium signals are observed in intact cells with minimum
disruption using calcium dyes. The observations, however, are
2+
indirect since they report on the Ca -bound dye rather than the
2+
free Ca distribution. Given an image, it is important to know to
what extent it is affected by the dye properties and whether it
provides an accurate representation of the underlying Ca2+ spatialtemporal dynamics. Determining the artifacts that the imaging
setting introduces are particularly relevant when trying to analyze
the smallest Ca2+ signals. Fluorescent images are noisy. A more
quantitative comparison between experiments and model then
requires that fluctuations be included in the latter. In this work we
present a method that provides a direct quantification of the
fluctuations introduced by the experimental setup which is
applicable to Ca2+ images obtained using single-wavelength dyes.
It involves the performance of a series of experiments and their
subsequent analysis in terms of a fluorescence fluctuation model
with which the model parameters are quantified. The model is
based on the one that underlies the Number and Brightness
technique but it is extended so as to take into account that Ca2+
dyes can fluoresce with or without Ca2+ bound, albeit with different
intensity. Using the model, the signal-to-noise ratio that can be
expected when observing Ca2+ signals is then computed.
Equivalence classes between different experimental conditions that
produce images with similar signal-to-noise ratios can then be
established. We acknowledge useful conversations with Lorena
Sigaut and Hernan Grecco.
1. Piegari E, Lopez L, Perez Ipiña E and Ponce Dawson S.
Plos ONE. 2014. 9(4):e95860.
131
_____________________________________________________
SID-4] Focal adhesion dynamics in response to
mechanical strain in living cells
1,2,3
1,2
1,2,4
1
Sigaut, L. , Bianchi, M. , von Bilderling, C. , Burdisso, J. ,
1
1,2,3
Gastaldi, L.M. and Pietrasanta, L.I. .
1
Centro de Microscopías Avanzadas, FCEN – UBA;
2
Departamento de Física, FCEN – UBA; 3Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET); 4 IFIBA,
CONICET – UBA. Buenos Aires, Argentina.
Focal adhesions (FAs) are macromolecular complexes that provide
a linkage between the cell and its external environment and serve
as points of integration for both mechanical and chemical signaling.
FAs are transient structures that exhibit mechanosensitive
properties: their formation, development and disassembly are
mechanical force-dependent [1]. Characterizing how these
structures dynamically respond in the presence of a mechanical
stimulus is essential for understanding cell migration, motility and
cell proliferation. To this end, in this work we visualized FAs via
molecular scaffold proteins such as vinculin or zyxin tagged with
visible fluorescent proteins and we use a stretching device [2] that
allows both live fluorescence imaging and sustained mechanical
equibiaxial stretching of cells cultured on silicone elastic
membranes. We generate data sets of FAs distribution,
morphology and turnover that can be used to characterize FA
response under mechanical stimulus compared to non-stretched
cells. Mechanical strain resulted to modify the length, area and FAs
dynamics when compared to non-stretched cells.
Acknowledgements: CONICET, ANPCyT and UBA. C. v. B., M. B.
and J. B. have fellowships from CONICET.
1. Shemesh, T. et al., PNAS, vol. 102, No. 35 (2005), p.
12383.
2. Quaglino, A. et al. BMC Cell Biology, vol. 10 (2009) p.55.
132
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SID-5] Estimation of the optimal conditions to perform
point-FCS experiments in Xenopus laevis oocytes.
Villarruel C and Ponce Dawson S.
Departamento de Física and IFIBA (CONICET), FCEyN-UBA,
Ciudad Universitaria, Pabellón I, (1428) Buenos Aires, Argentina.
The versatility of Ca2+ as a signaling agent is based on the great
diversity of spatio-temporal behaviors that its concentration can
display inside cells. Optical techniques and Ca2+ fluorescent dyes
have provided a relatively non-invasive means to study intracellular
calcium signals. Xenopus laevis oocytes are a model system in
which an enormous variety of calcium signals have been observed,
from localized signals to cellular waves. To extract quantitative
information from these optical experiments, it is key to have
reliable estimates of relevant biophysical parameters, in particular,
the Ca2+ diffusion coefficient in cells. Diffusion coefficients can be
estimated with Point Fluorescence Correlation Spectroscopy
(point-FCS) experiments. In this work we explore what are the
optimal experimental conditions to perform point-FCS experiments
Xenopus laevis oocytes. In particular we analyze the range of laser
powers for which the cell is not damaged and the duration over
which the experimental conditions are stationary enough so as to
provide reliable data. To this end we perform experiments using
different concentrations of Tetramethylrhodamine (TMR) and of the
Ca2+ dye Fluo4 either separately or simultaneously in Xenopus
laevis oocytes and in solution.
133
_____________________________________________________
SID-6] Observing the dynamics of luminal and cytosolic
calcium during IP3R-mediated calcium signals.
Lopez, L, Sigaut, L, Ponce Dawson, SP
Departamento de Fisica e IFIBA (CONICET), FCEN-UBA
Ca2+ signaling is ubiquitous across cell types. Ca2+ liberation
through inositol 1,4,5-trisphosphate receptors (IP3R's) is a key
component of the Ca2+ signaling toolkit. The role of cytosolic
calcium on the kinetics of IP3R's and on the dynamics of the
evoked signals has been studied at large both experimentally and
by modeling. The role of luminal calcium has not been investigated
with that much detail although it has been found that it is relevant
2+
for signal termination in the case of Ca release through ryanodine
receptors. In this work we present the results of observing the
dynamics of luminal and cytosolic Ca2+ simultaneously in Xenopus
Laevis oocytes using two dyes that have their peaks of emission at
different wavelengths. Through a continuous photorelease of
caged IP3 we are able to evoke a series of global and localized
signals in this system. The analysis of such signals allows a
characterization of the extent to which distant regions can be
coupled via the dynamics of luminal calcium.
In this way we
expect to advance into answering whether luminal calcium can
generate the global feedback mechanism that is necessary to
explain the robustness of IP3R-mediated calcium spikes as a
signaling tool in spite of the inter-spike interval randomness.
134
THEORY AND MODELLING OF BIOLOGICAL SYSTEMS
_____________________________________________________
THEORY AND
MODELLING OF
BIOLOGICAL
SYSTEMS
135
_____________________________________________________
TMB-1] A coarse grain model for the anti-migraine
prototype sumatriptan
1
1
Wood, I ; Pickholz, M
Inst. NANOBIOTEC (CONICET–UBA),Facultad de Farmacia y
Bioquímica (UBA), Junín 956, 6° Piso, CABA
1
Triptans are drugs for the migraine treatment based on the
serotonin (5-HT) structure, which act as a receptor of 5-HT1B/1D/1F
selective agonists [1]. Here, we propose a coarse grain model for
the prototype sumatriptan, at its prevalent ionization state. The
model was based on the tryptophan model of Martini forcefield [2].
The optimization was conducted comparing with all atom scale
molecular dynamics simulations, at three concentrations, looking to
the distribution of the protonated drug in a fully hydrated bilayer of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline
(POPC).
We were able to reproduce atomistic results with our coarse grain
model: the drug show partition between the lipid head-water
interphase and water phase, preferently at the most relevant high
concentration. Drug molecules show no access to the bilayer
hydrophobic region. The interactions with POPC that stabilize
sumatriptan at the interphase were characterized at the atomic
level [3]. This work represents a first step to the study of
sumatriptan in more complexes systems and processes, as drug
permeation and encapsulation, in presence of co-polymer chains
and micelles.
Acknowledgements: ANPCyT, UBA, CONICET
1 Humphrey, et al. PPA. Ann NY Acad Sci.1990. 587
2 De Jong, et al. DH. J Chem Theory Comput. 2013. 687
136
_____________________________________________________
TMB-2] Triptan partition in model membranes
1
1
Wood, I ; Pickholz, M
1
Inst. NANOBIOTEC (CONICET–UBA), Facultad de Farmacia y
Bioquímica (UBA), Junín 956, 6° Piso, CABA
The antimigraine drugs belonging to the triptan family were
designed based on the structure of neurotransmitter serotonin [1].
In the present work, we have conducted molecular dynamics
simulations of protonated triptans, sumatriptan and naratriptan, in a
fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidyl-choline (POPC). Simulations were carried out at two
concentrations for each of the drugs. Our results show partition
between the lipid head-water interphase and water phase for both
triptans, with increasing access to the water phase with increasing
concentrations. The triptans were stabilized at the interphase
through different specific interactions with the POPC bilayer such
as hydrogen bonds, salt bridges, and cation-π. Besides, at their
protonated state, neither sumatriptan and naratriptan protonated
have access to the hydrophobic region of the bilayer at the studied
conditions. Similar results were obtained for both drugs, however
protonated naratriptan shows slightly higher affinity for the water
phase. This behavior was attributed to the bulky lateral amino
group in its structure under the studied conditions (drugs originally
placed at the water phase). This work represents a first insight to
the comprehensive understanding and comparison of triptan
partition in model membranes.
Acknowledgements: ANPCyT, UBA, CONICET
1- Humphrey PPA. Ann NY Acad Sci. 1990. 587
137
_____________________________________________________
TMB-3] Block co-polymers and their interaction with a
lipid bilayer
Albano,J; Wood, I and Pickholz, M.
Department of Pharmaceutical Technology, Faculty of
Biochemistry and Pharmacy, University of Buenos Aires, C.P.
1113, Buenos Aires, Argentina; and NANOBIOTEC (CONICET)
Poloxamers, also known as Pluronics, are non-ionic triblock copolymers composed of a central hydrophobic poly(propylene oxide)
(PPO) chain capped by two hydrophilic chains of poly(ethylene
oxide) (PEO). These amphiphilic co-polymers can interact strongly
with the cell membranes and modify its mechanical properties [1].
In this work, we aim to investigate the interaction of the poloxamer
L81
with
POPC
(1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidylcholine) phospholipid bilayers through Molecular
Dynamics (MD) simulations. Firstly, in order to refine a
parametrization of pluronic within a CHARMM forcefield, we carried
out quantum chemical calculations of the polymers of PPO and
PEO as function of the monomer numbers, as well as for short copolymers. Lipid bilayers were build up using the packmol program
[2] and the poloxamer was added in 3 different environments,
water, interphase and bilayer core. Molecular dynamics simulations
were carried out and preliminar results show the affinity of the
poloxamer for the interphase, with the PPO blocks anchored in the
bilayer and the PEO tails solvated in water.
Acknowledgements: ANPCyT, UBA, CONICET.
1- Nawaz, S. et al. Soft Matter. 2012. 6744
2- Martínez, L. et al. J. Comput. Chem. 2009. 2157
138
_____________________________________________________
TMB-4] A QM/MM study of the molecular interactions of
different ligands and Sphingosine Kinase1 (SphK1)
ab
ab
c
ab
Vettorazzi, M. ; Andujar, S. ; Gutierrez, L ; Suvire, F. ; Alvarez,
ab
ab
S. ; Enriz, D.
a
Facultad de Química, Bioquímica y Farmacia, Universidad
b
Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina.
IMIBIO-SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina.
c
Facultad de Ciencias Exactas y Naturales y Agrimensura,
Universidad Nacional del Nordeste. Av. Libertad 5470, 3400
Corrientes, Argentina
The potential of the sphingolipid metabolic pathway for the
development of therapeutic targets for cancer has been recognized
for years. Thus, inhibition of SphK1 is considered a novel approach
for the treatment of cancers including metastatic cancer and/or
multi drug resistance. So far, the development of SKIs has been
hampered by the lack of a crystal structure of SphK1, and
therefore, rational drug design was impractical. A recent report
describing the crystal structure of SphK1 is expected to provide a
new direction to SKI design, which may lead to more effective and
specific inhibitors in the near future. The natural substrate and two
synthetic compounds have been recently co-crystallized (PDB
code: 3VZB, 3VZD and 4L02). These inhibitors interact at the
hydrophobic region in the same way but display very different
molecular interactions in the polar region of the binding site. In this
study, we performed first molecular dynamics simulations of the
different molecular systems and in a second step we applied a
hybrid Quantum mechanical-molecular Mechanical (QM/MM)
method followed by a Quantum Theory of Atoms in Molecules
(QTAIM) analysis to investigate the ligand-receptor interactions of
the different complexes. The affinities of the ligands were
evaluated in terms of the electron density (ρ (r)) at the
intermolecular bond critical points.
139
_____________________________________________________
TMB-5] Understanding substrate selectivity in 1-Cys
peroxirredoxin of M. tuberculosis
Pablo Lichtig*, Ari Zeida*, Aníbal M. Reyes*, Martín Hugo**,
Luciana Capece*, Javier Santos***, Luis G. Flecha***, Madia
Trujillo**, Dario A. Estrín*
*DQIAyQF,
INQUIMAE-CONICET,
FCEN-UBA,
Ciudad
Universitaria, C1428EHA Bs As, Argentina. **Depto de Bioquímica,
CEINBIO, Facultad de Medicina, UnLaR, Montevideo, Uruguay.
***Laboratorio de Biofísica Molecular, IQUIFIB, UBA, Bs. As.,
Argentina
Peroxiredoxins (Prxs) are Cys-dependent ubiquitous proteins that
catalyze the reduction of peroxydes. This Cys residue gets
oxidized to its sulfenic form, which may form inter- or
intramolecular disulfide bonds so as to recycle their activity. In
spite of having a highly conserved active site, they have
surprisingly different reactivities towards different peroxides[2].
Alkyl hydroperoxide reductase E (AhpE) is an essential homodimer
of Mycobacterium tuberculosis. It is a Prx with extraordinary
reactivity when reacting with peroxides derived from long-chained
fatty acids (kox=108M-1s-1), yet with hydrogen peroxide it is at least
1000 times less reactive[3]. Using AhpE as a model, we are
studying this system with a series of experimental a theoretical
approaches so as to understand at the molecular level its
selectivity. We postulated that a hydrophobic groove in the dimeric
interface may be an important factor, and we are attempting to test
it with molecular mechanics techniques (such as free energy
calculations, accessible surface area calculations and dynamics
analysis). In addition, we have characterized the interaction of
AhpE with 8-Anilinonaphtalene-1-sulfonic acid via fluorescence
experiments, in order to improve our understanding of this
hydrophobic patch.
1. Ferrer-Sueta et al. Chem. Res. Toxicol. 2011
140
_____________________________________________________
TMB-6] Exploring the dynamics of cell processes
through Monte Carlo simulations of advanced
fluorescence microscopy experiments
a
b
c
Angiolini, Juan ; Plachta, Nicolás ; Mocskos, Esteban ; Levi,
Valeriaa
a
Departamento de Química Biológica-IQUIBICEN, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires,
b
European Molecular Biology Laboratory, Australian Regenerative
Medicine Institute, cDepartamento de Computación, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires
Fluorescence correlation spectroscopy (FCS) and related
techniques are powerful methods to unveil the dynamical
organization of cells. For simple cases such as molecules
passively moving in a homogeneous media, the FCS analysis
yields analytical functions that can be fitted to the experimental
data in order to recover the phenomenological rate parameters.
Unfortunately, many dynamical processes in cells do not follow
these simple models and in many instances it is not possible to
obtain an analytical function through the theoretical analysis of a
more complex model. In those cases, the experimental analysis
can be combined with Monte Carlo simulations to help with the
interpretation of the data. In this work, we present FeRNET, a new
toolkit designed to simulate a variety of FCS-based techniques.
This tool works in combination with MCell-Blender platform which
was designed to treat the reaction-diffusion problem under realistic
scenarios. The new program allows setting complex, user-defined
geometries of the simulation space and distributing the molecules
between different compartments as desired. Also, the platform
includes the possibility of defining reactions among the species
considered in the simulations; the user only needs to set the kinetic
constants, diffusion coefficient and brightness of each species. We
illustrate the possibilities opened by FeRNET showing its
application to the simulation of single and multiple point FCS and
photon counting histogram analysis, raster image correlation
spectroscopy and two-color fluorescence crosscorrelation
spectroscopy. We also demonstrate how these simulations can aid
in the interpretation of FCS data obtained from complex systems,
such as transcription factor dynamics in living mouse embryos.
141
_____________________________________________________
TMB-7] Study of the MDM2- p53 complex and of smallmolecule inhibitory of their interaction
1
1
1
Menéndez C.A , Accordino.S.R , Appignanesi G.A , Gerbino
2
.D.C
1
Sección Fisicoquímica, INQUISUR-UNS-CONICET-Departamento
de Química, Universidad Nacional del Sur, Avda. Alem 1253, 8000
Bahía Blanca, Argentina. 2Sección Química Orgánica, INQUISURUNS-CONICET-Departamento de Química, Universidad Nacional
del Sur, Avda. Alem 1253, 8000 Bahía Blanca, Argentina
The aim of study of this work is the binding site of MDM2 protein
with tumor suppressor p53, as well as the interaction between
MDM2 with disruptive molecules as nutlin and different xanthones,
focusing our interest in principle in the 3,4-dihydro-12-hydroxy-2,2dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one.(1) The latter are part
of a novel family of oxygenated heterocycles, found in nature in
various species of fungi, algae and higher plants. The study of
such compounds is of great interest because they possess
interesting biological activities with potential therapeutic
applications.
Using molecular dynamics studies was possible to establish which
are presumably three fundamental positions in the binding site of
MDM2 is necessary "to cover." In these positions the natural
"partner" (p53) accommodates hydrophobic residues, behavior that
is mimicked by nutlin. The xanthone studied also binds to the same
site or "hot spot", but only has the ability to cover two of these
positions simultaneously,
reason that
adopts different
conformations along time, alternating their interaction with those
positions. From these results it would be possible to rationally
functionalize this prototype and achieve an increase in its activity
as an inhibitor of the MDM2-p53 interaction.
1. Mariana Lea˜o, Biochemical Pharmacology (2013),1234
142
_____________________________________________________
TMB-8] Structural and functional analysis on different
states of the catalytic reaction of the human telomerase
reverse transcriptase protein. A molecular dynamics
study.
Herrera, F.E(1). and Sferco, S.J.(1,2).
Dpto. de Física, Facultad de Bioquímica y Ciencias Biológicas,
Universidad Nacional del Litoral, Santa Fe, Argentina; (2) IFIS
Litoral (UNL-CONICET), Santa Fe, Argentina.
Telomerase is a ribonucleoprotein complex responsible for
maintaining the length and integrity of chromosome ends adding
specific nucleotides to an existing DNA primer having RNA as a
template. It contains a reverse transcriptase domain where the
reaction takes place. Here we present the molecular dynamics
simulations of the structural model of that domain for the human
telomerase (hTERT) in three different states of the reaction
pathway: i) the arriving of the deoxynucleoside triphosphate
(dNTP), ii) the exit of the pyrophosphate (PYR) after the formation
of the phosphodiester bond and iii) the structure with the complete
DNA chain. The simulations were validated by checking the
Watson-Crick base-pairing interactions and the correct
coordination of the magnesium atoms in all simulations. The
results show the role of a conserved key lysine residue for the
entrance of the dNTP and the exit of the PYR. This lysine makes
contacts (direct and water mediated) with the alpha phosphate of
the dNTP in the first state and accompanies the release of the PYR
in the second state. Therefore no mutations in this residue should
be tolerated at least in the hTERT. On the other hand, in the third
state, while the DNA chain is complete, a rearrangement of the
catalytic site occurs involving other conserved key residues of the
primer grip motif. Additionally, these simulations could be helpful in
the comprehension of the translocation mechanism of the
DNA/hTERT after the addition of one nucleotide, a not well
understood process up to date.
143
_____________________________________________________
TMB-9] Molecular insight into conformational transition
of amyloid β-peptide 42 inhibited by Nα,Nε-Di-Z-L-lysine
hydroxysuccinimide ester probed by molecular
simulations
Salcedo, R.ab; Barrera, E.ab ; Andujar, S.ab ; Gutierrez, L.ab ;
Rodríguez, A.M.ab ; Enriz, D. ab
a
Facultad de Quíica Bioquímica y Farmacia, Universidad Nacional
de San Luis, Chacabuco 915, 5700 San Luis, Argentina. bIMIBIOc
SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina
Facultad de Ciencias Exactas y Naturales y Agrimensura,
Universidad Nacional del Nordeste
We recently reported a new series of peptidomimetic compounds
with amyloid beta (Aβ) antiaggregant activity. These compounds
were designed based on a molecular modeling study using a
pentameric Aβ model as molecular target. Nα,Nε-Di-Z-L-lysine
hydroxysuccinimide ester (compoud 7) was one of the
compounds that showed the best antiaggregant properties in the
series. One question which arise is: what effect would this
compound in the Aβ monomer?. To answer this question we
performed molecular dynamic (MD) simulations in both the
monomer alone and the monomer complexed with compound 7.
Simulations were carried out in three steps: 1) Compound 7 was
docked into the monomer using the program Autodock Vina (blind
docking strategy). 2) We performed short MD simulations (10 ns
each) to calculate the relative binding energy of each complex
using Molecular Mechanics/Poisson Boltzman Surface Area
(MM/PBSA) method and 3) To explore the dynamic behaviour of
the preferred complexes, more extensive MD simulations were
carried out in explicit water (300 ns sampling time). Simulations
were compared with those obtained for the monomer alone. Three
distinct binding sites were identified for compound 7 at the
monomer, being the zone located between residues Gln15 and
Asp23 the preferred site for binding. From our simulations it is
possible to observe that compound 7 substantially maintained the
helix forms preventing the conformational transition of the
Aβ monomer necessary for its oligomerization.
144
_____________________________________________________
TMB-10] Insights into the structure of Triatoma Virus
(TrV) capsid. Combining crystallography data with
computational simulations.
Viso, J.F.(a), Zamarreño, F.(a), Amundarain, M.J.(a), Guérin
Aguilar, D.M.(b), Costabel, M.D.(a)
a
Grupo de Biofísica. Departamento de Física, Universidad
Nacional del Sur (UNS), IFISUR, (UNS-CONICET), Bahía Blanca,
Argentina. b Unidad de Biofísica, Universidad del País Vasco
(UPV/EHU) – Fundación Biofísica Bizkaia, Leioa, España.
Triatoma Virus (TrV) is an insect virus that belongs to the
Dicistroviridae family and infects several species of triatomine
insects which are the vectors for human trypanosomiasis,
commonly known as Chagas disease. Because of this, TrV is
proposed as a biological control against this vector. The crystal
structure of TrV was solved recently, but an omitted map of the
structure, in the region of the icosahedral 5-fold axis of the capsid,
shows an interesting electronic density. In this work we study the
5-fold symmetry axis of the icosahedral capsid of TrV, because it
may be responsible for the interaction between the interior and the
exterior of the virus capsid, across a putative pore. Using
molecular dynamics simulations, we have observed that the pore
formed in this axis remains without water molecules in the region
surrounded by a ring of Valines which creates a supposed
hydrophobic gate. Also, we have found certain conditions where
the axis is completely full of water molecules, even in the
hydrophobic region. The complete hydration of the channel may
lead to its “opening”, and the interaction between the interior and
the exterior of the capsid. Combining different results we could
explain the characteristics of the omitted map.
145
_____________________________________________________
TMB-11] Molecular Dynamics of the binding mode of
substrate and inhibitor to CYP125 of Mycobacterium
tuberculosis
1
2
3
Martínez D , Rosi PE *, Arroyo-Máñez P *
1
2
3
FCEN – UBA; DQIAyQF – FCEN – UBA; UMYMFOR – DQO –
FCEN – UBA
One of the most potential candidates for drug targeting in M.
tuberculosis is the cytochrome P450 CYP125, an enzyme that has
been recently demonstrated to catalyze the hydroxylation of
cholesterol in its terminal C27 side chain (Capyk et al, 2009). The
crystal structure revealed a ‘letterbox’-like hydrophobic active site
cavity that narrows in a funnel-like manner on approach to the
heme center (McLean et al, 2010). The catalytic activity of CYP125
is necessary for the parasite surviving in its inner cell phase. Thus,
the understanding of the catalytic mechanism would contribute to
design better CYP125 inhibitors. Looking forward to having a view
of the catalytic cycle as well as the mechanism of the inhibition
activity classical molecular dynamic simulations in presence of its
natural substrate cholesterol and the co-crystallized inhibitor
cholest-4-en-3-one (K2B) (Ouellet et al, 2010) were performed.
Three significant states were modeled: deoxygenated, oxygenated
and ferryle state according to the mechanism proposed by Ouellet
et al, 2010. All the modeled systems remained stable during the
simulation time. Comparative analysis revealed similar binding
modes for both ligands in all systems, showing that the side chain
of cholesterol and K2B remains near the heme group. Also in both
cases, hydrogen bond network between residues, D90 and K196,
and the hydroxyl or keto groups were monitored for the cholesterol
and K2B, respectively. Electrostatic interaction between the
carboxylate and amino groups of D90 and K196, respectively,
seems to be responsible for ligand stabilization as well as for the
opening of the cavity, and therefore, for ligand entrance and exit
from the enzymatic active site.
146
_____________________________________________________
TMB-12] Electrostatic Study of the Interaction between
33-mer Octamer and Biological Membranes
(a)
(a)
(a)
(b)
M.J. Amundarain , F. Zamarreño , J.F. Viso , V.I. Dodero ,
(a)
M.D. Costabel
a
Grupo de Biofísica, Departamento de Física-IFISUR, Universidad
Nacional del Sur (UNS)-CONICET, Bahía Blanca, Argentina.
b
Grupo de Química Biomolecular, Departamento de QuímicaINQUISUR, UNS-CONICET, Bahía Blanca, Argentina.
33-mer is an immunogenic peptide derived from the digestion of
the protein α2-gliadin and is proposed to be the trigger of the
inflammation process in Celiac disease. In vitro experiments have
shown that it presents a supramolecular behavior that could be
related to the development of the disease. In this work we applied
biophysical and computational techniques to evaluate the
interaction between an oligomer of 33-mer peptide and different
biological membranes. Firstly, we obtained the octamer from a
molecular dynamics simulation of ten 33-mer monomers in water.
We saw how eight of them interacted, came close to each other
and formed a primitive oligomer. Afterwards we carried on with the
simulation of the octamer for 50 ns, obtaining a stable structure of
interesting size. The MD simulations were performed with
GROMACS package. The interaction with the membrane was
assessed through calculations of electrostatic energy of interaction,
for different positions and relative orientations, by making use of
the software APBS (Adaptative Poisson-Boltzmann Solver).
Acknowledgments: We want to thank CONICET and UNS.
147
_____________________________________________________
TMB-13] Effects of topology and feedback inhibition on
multistability and robustness of metabolic pathways
1
1
1
2
Cogo, C. , Vallejo, D. F. G. , Melgarejo, A. A. , Lodeiro, A. R.
1
Departamento de Ciencias Básicas, Facultad de Ingeniería,
2
UNLP, IBBM, Facultad de Ciencias Exactas, UNLP-Conicet
Metabolic systems are open systems in steady state out of
equilibrium, which means that the concentrations of metabolites do
not change over time. These systems consist of a large number of
metabolites linked by enzyme-catalyzed reactions, which occur in
linear or cyclic sequences. To analyze how the topology and
regulation of metabolic networks influence its robustness, we
proposed four simple ways in which three metabolites are linked by
reactions catalyzed by enzymes that obey the Michaelis-Menten
kinetics. The regulation of each pathway was modeled as a
feedback inhibition of the latter metabolite on the first reaction.
Furthermore, we assumed that the state of the system is
determined by a vector whose components are the concentrations
of all the metabolites involved. To perturb the system, we randomly
modified the enzyme concentrations of the pathway in a range of
50-fold, generating a multistable steady-state system where each
cell in the population contained a given concentration of each
enzyme, and thus a particular set of metabolite concentrations.
This set of metabolite concentrations defined the state of the
system. By evaluating how these states were dispersed in
response to enzyme variation, we evaluated the robustness of the
system. Our results indicate that cyclic topologies promote
multistable dispersion of system states, while linear topologies
promote robustness. Regulation by feedback inhibition increases
multistability when the topologies contain linear stretches.
148
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TBM-14] Modeling root hydraulic adjustment capacity in
plants submitted to different stress conditions
Vitali, V; Yaneff, A; Amodeo, G.
IBBEA-CONICET, FCEN, UBA, Argentina
Water flow through plants has been described as a passive
diffusion mechanism based on the analogy with Ohm's law1. A
continuum soil-plant-atmosphere is described in order to design a
hydraulic circuit that allows describing the involved resistances
2
through all the pathway . In this model the transpiration demand on
aerial part represents the main driving force to plant water entry by
roots. It is also known that under stress conditions the driving force
is not enough to interpret the circuit as a simple passive diffusion
flow3. The introduction of aquaporins in the scenario creates the
challenge to revisit the root intrinsic properties and its effect on
water movement from soil to xylem. We performed experiments
measuring root hydraulic conductivity (Lpr) as the property that
better reflects the adjustment capacity to modulate water flow. We
focus on discerning the root water pathways in plants submitted to
a salt stress condition, seeking for capable models to explain the
kinetics of water flow, in terms of Lpr and aquaporins (i.e. the
enhancement or not of the transcellular pathway). Our results show
that Lpr changes can highlight the intrinsic root strategy to
overcome the stress situation.
Acknowledgements: Supported by PIP-CONICET, PICT11 2339
and UBACyT14-17 all grants to GA
1. Van der Honert TH. Disc. Faraday Soc. (1948) 146
2. Steudle, E. J Exp Bot (2000) 1531
3. Wegner LH. J Exp Bot (2013) 381
149
_____________________________________________________
TBM-15] Multimethodological study of non-classical
FABPs interactions with biological membrane. Evidence
of a third mechanism.
(a)
(a)
Zamarreño, F . Viso, J.F., Amundarain , M.J., IbáñezShimabukuro M. (b), Córsico B. (b) and Costabel M.D. (a)
a
Grupo de Biofísica, Dpto. de Física, Universidads Nacional del
Sur – IFISUR. bInstituto de Investigaciones Bioquímicas de La
Plata, CONICET-UNLP, Facultad de Ciencias Médicas,
Universidad Nacional de La Plata, Argentina.
Fatty Acid Binding Proteins (FABPs) belongs to a family of
intracellular lipid binding proteins with the general function of lipid
trafficking. A significant amount of In vivo and in silico studies have
shown that different FABPs transfer fatty acids to membranes by
two different mechanisms: collisional or difusional [1,2].
Nevertheless, a small group of FABPs with structural features,
such as As-p18 from parasitic nematode Ascaris suum [3] and
human myelin peripheral membrane protein P2 [4], shows a
different behavior. In this work we show that this group of FABPs
has an ambiguous behavior in its interaction with biological anionic
membranes with a putative third mechanism that seems to be a
merger between collisional and difusional mechanisms. In order to
study the insights of this unusual protein-membrane interaction, we
analyzed the electrostatic involved in the system and developed a
bioinformatics study to determinate plausible structure
conservation.
Acknowledgements to CONICET for grants support.
1. Storch, J. JLR. 2008. S126.
2. Zamarreño, F. BBA-Biomem. 2012. 1691
3. Ibáñez-Shimabukuro, M. Biomol NMR Assing. 2014. 33.
4. Ruskamo S. Acta Crystallogr D Biol Crystallogr. 2014. 165
150
_____________________________________________________
TBM-16] Development of a ligand set enrichment
pipeline for virtual screening using drug and protein
structural similarity searches
Arcon, JP. Radusky, L. Defelipe, LA. Marti, MA. Turjanski, AG.
Departamento de Química Biológica and INQUIMAE-CONICET Facultad de Ciencias Exactas y Naturales - Universidad de Buenos
Aires
Starting from a selected protein target, a key question for insilico structural based drug design is how to select a small set of
potential "drug-like" ligands adequate for performing a virtual
screening procedure. In the present work we show a computational
pipeline that, starting just from the target protein sequence allows
to select a small set of compounds derived from large public
databases such as ZINC or PubChem and gives the opportunity to
download them as complete set of potential 3D conformers ready
for their use in a subsequent docking protocol. The final set
includes different species considering acid-base properties,
common tautomers and stereoisomers (RS and EZ) in their low
energy conformations. The pipeline first determines a potential
protein structure, by either searching in the PDB database or using
comparative modeling, and then finds those ligands that bind the
target protein or other proteins related to it through PFAM or
PubChem BioAssay databases. These compounds are used as
seeds to look for similar ones using chemical fingerprints in
compound databases. Our results show that significant enriched
compound sets are obtained and, when used in combination with
our modified docking protocol, the pipeline yields increasing
amounts of potential new protein-ligands complexes.
Acknowledgements: CONICET – ANPCyT – UBA.
151
_____________________________________________________
TBM-17] SIRAH: a new Coarse-Grained Force Field for
Multiscale Simulations
Pantano S., Machado M.R., Brandner A.F., Gonzalez H.C., Darre
L., Hugo G., Dans P.D., Zeida A., Silva S.
Institut Pasteur de Montevideo, Mataojo 2020, 11400, Montevideo,
Uruguay.
Despite the robustness and reliability achievable by molecular
simulations, a direct comparison with experimental data is often
difficult owing to the large size and long time scales needed for a
proper description of biological systems. This have motivated the
development of coarse-grained (CG) methods aimed to bridge the
gap between experiments and simulations. SIRAH is a CG force
field for molecular dynamics simulations of biomolecular systems,
which includes parameters for water and electrolytes, DNA and
proteins. Interactions are treated using a typical Hamiltonian for
classical molecular dynamics simulations, allowing for a rigorous
treatment of long-range electrostatics via PME and an easy
implementation in commonly used simulation packages.
Furthermore, the CG parameters can be straightforwardly
combined with atomistic force fields to perform dual-resolution
simulations, in which solutes (i.e., proteins or membranes) can be
treated at fully atomistic detail while the bulk water can be
simulated at a supramolecular level; alternatively DNA filaments
can be simulated at CG resolution with regions of interest including
atomistic nucleobases. Interaction parameters, topology files and
tutorials for running simulations in Amber and Gromacs are freely
downloadable from http://www.sirahff.com.
152
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TBM-18] The reaction between hydrogen sulfide and
peroxynitrite: “the yellow nightmare”
Zeida, A, Cuevasanta, E, Carballal, S, Wedmann, R, Morzan, U,
Trujillo, M, Radi, R, Estrín, D, Filipovic, MR, Alvarez, B.
Laboratorio de Enzimología, Facultad de Ciencias, UdelaR,
Montevideo, Uruguay - DQIAQF, INQUIMAE-CONICET, Facultad
de Ciencias Exactas y Naturales, UBA, Argentina - Department of
Chemistry and Pharmacy, University of Erlangen-Nuremberg,
Erlangen, Germany
Hydrogen sulfide and peroxynitrite are endogenously generated
molecules that participate in biologically-relevant pathways. A
revision of the kinetic features of the reaction between peroxynitrite
and hydrogen sulfide revealed a complex process. The rate
constant of peroxynitrite decay, (6.65 ± 0.08) x 103 M-1 s-1 in 0.05 M
sodium phosphate buffer (pH 7.4, 37°C), was affected by the
concentration of buffer. Theoretical modeling suggested that, as in
the case of thiols, the reaction is initiated by the nucleophilic attack
of HS- on the peroxide group of ONOOH by a typical bimolecular
nucleophilic substitution, yielding HSOH and NO2-. In contrast to
thiols, the reaction then proceeds to the formation of distinct
products that absorb near 408 nm. Experiments in the presence of
scavengers and carbon dioxide showed that free radicals are
unlikely to be involved in the formation of the 408 nm products. The
results are consistent with product formation involving the reactive
intermediate HSSH and its fast reaction with a second peroxynitrite
molecule. Mass spectrometry and UV-Vis absorption spectra
predictions suggest that at least one of the products is HSNO2 or
its isomer HSONO.
1. Carballal, S. Free Radic. Biol. Med. 2011. 196.
2. Filipovic, M. R. Biochem. J. 2012. 609.
153
_____________________________________________________
TBM-19] All-atom folding and binding of an Instrinsically
Disordered Protein
1
2
1
Ithuralde, RE ; Roitberg, A ; Turjanski, AG .
Departamentos de Química Biológica y Química Inorgánica,
Analítica y Química Física e INQUIMAE – Facultad de Ciencias
Exactas y Naturales – Universidad de Buenos Aires. 2Department
of Chemistry and Quantum Theory Project – University of Florida
1
Intrinsically Disordered Proteins (IDPs) lack a definite secondary
structure in solution and sample many different configurations.
IDPs may acquire some structure when bound to their partners,
and thus the binding process needs the folding of the protein, that
could follow a conformational selection process, an induced fit one
or a combination of both. c-Myb is a transcription factor involved in
the regulation of hemapoietic cells life cycle. TAD region of c-Myb
is intrinsically disordered (30% of helicity in solution) and folds
upon binding to the KIX domain[1]. The NMR structure of the
complex has been solved[4] Kinetic studies of cMyb-KIX binding
are consistent with a two-state process and theassociation process
has an apparent activation energy of 11 kcal/mol [1,2].. A Φ value
analysis was performed and proposed an ordered transition state,
in contradiction with studies that show that prefolding c-Myb does
not rise kon [2,3]. We carried out an all atom biased MD and GoType coarse grain simulations. Our results show that c-Myb folds
very fast upon binding to KIX, but that pre-folded configurations of
c-Myb can also bind with similar activation barrier. The transition
state has a broad configuration, with helicity going from 30% to
70%. Both all atom and Go-Type simulations show similar results,
indicating folding upon binding follows a native contact mechanism.
To the best of our knowledge this is the first work to provide an
atomistic understanding of the free energy surface of the binding
process of IDPs.
1. Sarah L. Shammas. Journal of Physical Chemystry B.
2013. 13346
2. Stefano Gianni. Biochimecal and Biophysical research
Communications. 2012. 205
3. Rajanish Giri. Proceedings of the National Academy of
Sciences. 2013. 14942.
154
_____________________________________________________
TBM-20] Conformational space analysis of proteins
based on dynamics alignments
Balatti, G E. Fernandez-Alberti, S.
Universidad Nacional de Quilmes.
The comparation of proteins conformers based on its dynamics
can show functionality aspects not revealed in secuential or
estructural alignments. Under this premise, it’s possible to align
protein conformers with dynamics similarities based on the
identification of concerted atomic movements. These movements
can be calculated from the native state 3D structure. Here, we
developed a novel algorithm for dynamics alignment, based on the
Needleman-Wunsch [1] global alignment algorithm, and analice
with them a set of pairs bounded/not bounded proteins, in seek of
new relations beetwen dynamics and function.
1. Needleman, S.B. and Wunsch, C.D. (1970). «A general
method applicable to the search for similarities in the
amino acid sequence of two proteins». Journal of
molecular biology (Elsevier) 48 (3): pp. 443-453.
155
TRANSPORTERS, RECEPTORS AND CHANNELS
_____________________________________________________
TRANSPORTERS,
RECEPTORS AND
CHANNELS
156
_____________________________________________________
TRC-1] The efferent olivocochlear system modulates the
tonotopy of a central auditory nuclei.
1
1
Di Guilmi, MN and Elgoyhen AB .
1
Instituto de Investigaciones en Ingeniería Genética y Biología
Molecular, Dr. Héctor N Torres, Consejo Nacional de
Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
The auditory system in several mammals is immature at birth but
precisely organized in adults. Spontaneous activity in the inner ear
comes into play to guide this process. The rate of this spontaneous
activity is under the control of an olivocochlear efferent pathway
that descends from the brain. Along the auditory pathway neurons
are tonotopically organized, with cells that are grouped according
to their frequency selectivity. The specific aim of this project is to
understand how the tonotopic establishment of auditory circuits is
affected by the early activation of the olivocochlear efferent
system. For this propose, we studied the MNTB tonotopy in a
mouse model with an enhanced efferent functionality (Chrna9L9´T,
KI).
Previous
studies
have
provided
evidence
that
hyperpolarization-activated cyclic nucleotide-gated (HCN) channels
are topographically arranged in the MNTB (Leao et. al., 2006), and
are responsible for hyperpolarization-activated potassium currents
(Ih). Under voltage-clamp mode, Ih currents of wild-type mice were
larger in medial than in lateral MNTB cells. However, this tonotopic
diference disappeared in KI mice, where lateral cells showed the
same amplitudes as medial cells. Current-clamp experiments
supported these observations. A larger voltage sag in response to
hyperpolarizing currents was observed in medial compared to
lateral cells in wild-type mice. This difference was not observed in
the KI. Our current data suggests that the efferent pathway might
be involved in the refinement of the tonotopic map of the auditory
system, ensuring the presence of high frequency coding cells.
[Leao, et al., J Physiol 571 (2006) 563.)
157
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TRC-2] Synaptic gain-of-function effects of mutant CaV2.1
channels in a mouse model of familial hemiplegic
migraine are due to increased basal [Ca2+]i.
1
1
2
Di Guilmi MN ; González Inchauspe C ; Forsythe I ; van den
Maagdenberg A3; Borst JG4; Uchitel OD1.
1
IFIBYNE, UBA- CONICET, Buenos Aires, Arg.; 2MRC Tox. Unit,
U.Leicester, Leicester. UK.; 3 Dep. of Hum Gen & Neurolog and
Dep. of Neurol, Leiden U., Leiden, The Netherlands, 4 Dep. of
Neurosc, Erasmus MC, Rotterdam, The Netherlands.
Specific missense mutations in the CACNA1A gene, which
encodes a subunit of voltage-gated CaV2.1 channels, are
associated with familial hemiplegic migraine type 1 (FHM1), a rare
monogenic subtype of common migraine with aura. We used
transgenic knock-in (KI) mice harboring the human pathogenic
FHM1 mutation S218L to study presynaptic Ca2+ currents and
EPSCs in the MNTB-calyx of Held synapse. Whole-cell patchclamp recordings of presynaptic terminals from S218L KI mice
showed a strong shift of the calcium current I-V curve to more
negative potentials, leading to an increase in basal [Ca2+]i,
increased levels of spontaneous transmitter release, faster
recovery from synaptic depression, and enhanced synaptic
strength despite smaller action-potential-elicited Ca2+ currents. This
synaptic phenotype may explain the misbalance between
excitation and inhibition in neuronal circuits resulting in a persistent
hyperexcitability state and other migraine-relevant mechanisms
such as an increased susceptibility to cortical spreading
depression.
158
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TRC-3] Loop B Serine phosphorylation status
differentially affects pH sensing of PIP aquaporins
a,c
b,c
a
a,c
Yaneff A , Sigaut L , Aliaga Fandiño AC , Alleva K , Pietrasanta
b,c
a,c
LI , Amodeo G
a
IBBEA, CONICET-UBA, Argentina and DBBE, FCEN, UBA,
b
c
Argentina. CMA and DF, FCEN, UBA, Argentina. CONICET,
Argentina.
The crystallization of SoPIP2;1 in an open and close conformation
as well as biophysical evidences have allowed to propose a gating
mechanism in PIP aquaporins. A close state seems to prevail
under different stimuli: cytosolic pH decrease2, intracellular Ca2+
concentration increase and desphosphorylation of specific
serines1. In previous work, we characterize FaPIP1;1 and
FaPIP2;1 in terms of their Pf and pH inhibition response and
demonstrate that heterotetramerization affects pH gating
2
sensitivity . In the light of these findings we decided to explore if
phosphorylation of specific residues is involved in this response.
Several PIP2 mutants where Serine from the loop B was replaced
to alanine showed less activity as water channels when expressed
in Xenopus laevis oocytes3–5 or yeast6,7. However, for several plant
species, loop B serine has shown to be non-phosphorylated in
vivo4,5,8. In this work, we generate loop B mutants (FaPIP2;1S121A
and FaPIP1;1S131A) in order to simulate desphosphorylated state
and characterize their behavior in terms of Pf and pH inhibition
response. Our results show that loop B serine phosphorylation
status affects pH gating sensitivity for FaPIP2;1 but not for
FaPIP1;1. Thus, we propose a crosstalk between different
regulatory
mechanisms
(heterotetramerization,
serine
phosphorylation status and pH sensitivity) that would rule the Pf of
a membrane that express PIPs
1.
2.
3.
4.
5.
6.
7.
8.
Törnroth-Horsefield, S. et al. Nature 439, 688–94 (2006).
Yaneff, A. et al. Proc. Natl. Acad. Sci. U. S. A. 111, 231–6 (2014).
Amezcua-Romero, J. C. et al. J. Biol. Chem. 285, 16739–47 (2010).
Johansson, I. et al. Plant Cell 10, 451–9 (1998).
Van Wilder, V. et al. Plant Cell Physiol. 49, 1364–77 (2008).
Fischer, M. et al. J. Biol. Chem. 283, 33889–92 (2008).
Azad, A. K. et al. Plant Cell Physiol. 49, 1196–208 (2008).
Prak, S. et al. Mol. Cell. Proteomics 7, 1019–30 (2008).
159
_____________________________________________________
TRC-4] Electromagnetic Fields Inhibit Cys-loop Receptor
Function by Inducing a Novel Conformational State
1
2
1
Tolosa M.F. , Cravero W.R. and Bouzat C.
1
INIBIBB, UNS-CONICET, Bahía Blanca.
CONICET, Bahía Blanca.
2
IFISUR,
UNS-
The fact that the population is increasingly exposed to
electromagnetic fields (EMFs) due to the advances in technology
raises concern about their potential health effects. There are many
gaps in knowledge, particularly at the molecular level, still needing
to be filled before better health risk assessments can be made. We
therefore studied the influence of EMFs on two Cys-loop receptors:
muscle nicotinic (AChR) and 5-HT3A receptors. The exposure of
cells expressing these receptors to EMFs (15 Hz-120 kHz)
significantly decreases the peak current as a function of EMF
frequency and increases the rise time of macroscopic currents
elicited by the agonists. The effects on both receptors are
qualitatively similar but more profound for 5-HT3A, indicating
different sensitivity to the EMF within the receptor family. To
understand the molecular basis leading to the macroscopic
changes, we compared single-channel properties before and after
EMFs exposure. Single-channel amplitude, open duration, duration
of activation episodes (clusters) and open probability within
clusters are not affected by the EMF. However, EMF leads to a
profound decrease of the number of clusters as a direct function of
frequency. The analysis reveals that EMFs induce a novel, non
conductive, conformational state that arises from the closed resting
state through a frequency-dependent transition. Thus, the
stabilization of this novel state by EMF sequesters receptors from
the activation pathway. Simulations of macroscopic and singlechannel currents on the basis of a scheme including this new state
well reproduce our experimental data. The identification of a novel
conformational state induced by EMF enhances our understanding
of receptor function, in general, and of the mechanisms by which
EMFs affect neuronal excitability, in particular.
160
_____________________________________________________
TRC-5] Exploring alpha7 positive allosteric modulators
from a single-channel perspective
1
1
1
1
2
Andersen ND , Corradi J , Tolosa MF , Gasparini N , Arias HR ,
1
Bouzat C .
1
UNS/CONICET, Instituto de Investigaciones Bioquimicas de
Bahia Blanca, Bahia Blanca, Argentina. 2Department of Medical
Education, California Northstate University College of Medicine,
Elk Grove, CA,USA.
Through the enhancement of endogenous ACh responses, positive
allosteric modulators (PAMs) of the nicotinic α7 receptor represent
a promising therapeutic approach for the treatment of several
neurological disorders. We combine single-channel and
macroscopic current recordings to explore the molecular basis
underlying potentiation of human α7 by three amide derivatives
compounds named as Compound 2 (3-furan-2-yl-N-p-tolylacrylamide), 3 and 4 (5-100 µM). In contrast to the brief and
isolated openings typical of α7, in the presence of Compounds 2, 3
or 4 opening events show significantly increased durations (~7fold) and appear grouped in bursts of about 10-20 ms. This activity
pattern is similar to that observed in the presence of 5hydroxyindole (5-HI), a type I PAM, which leads to openings of ~2
ms and bursts of ~5 ms. PNU-120596, a type II PAM, produces a
more profound increase in open duration, and opening events
appear in long clusters that last for several seconds. 5-HT3A and
α7-5HT3A receptors as well as an α7 quintuple mutant receptor
that is insensitive to PNU are not potentiated by Compound 2. At
the macroscopic level, Compound 2 and 5-HI increase the net
charge (~5-fold and 8-fold at 50 µM and 2 mM, respectively).
However, such increase is mainly due to the decrease in the decay
rate for Compound 2, which resembles the effect of a type II PAM,
and to the increase of the maximal peak currents for 5-HI. Our
results demonstrate that these novel compounds may bind to the
same intrasubunit transmembrane cavity as PNU, decrease
desensitization as type II PAMs, and modify single-channel activity
similarly as type I PAMs. They also contribute to elucidating the
foundations of α7 modulation.
161
_____________________________________________________
TRC-6] Functional roles of amino acids of nicotinic
receptors are conserved between C. elegans and human
Bergé I., Hernando G., Andreocci E., Roccamo A., Bouzat C.
Instituto de Investigaciones Bioquímicas Bahía Blanca. UNSCONICET
Mutations in ion channels may lead to genetic disorders named as
channelopathies. Congenital myasthenic syndromes (CMS) are
channelopathies produced by mutations in the muscle nicotinic
receptor (AChR). Our goal is to generate models of these human
neuromuscular disorders to be used for drug screening and
development of therapeutic strategies. To this end, we use the
free-living nematode C. elegans, whose muscle AChR (L-AChR) is
essential for neuromuscular transmission and locomotion. We
generated gain-of-function mutant L-AChRs by exchanging
hydrophobic residues at 9' position of M2 segment, which have
been shown to form the gate of the ion channel in vertebrates, by
hydrophilic residues in two essential subunits of the L-AChR.
Single-channel recordings from muscle cells of the mutant
transgenic worms show a dramatic increase (11- to 14-fold) of the
open duration of mutant L-AChR with respect to wild-type.
Macroscopic currents elicited by ACh show a decrease in the
desensitization rate. These functional changes are similar to those
observed in vertebrate AChRs carrying the equivalent mutations,
thus revealing a high degree of conservation of functional roles of
AChR amino acids. Generation and analysis of a mutant strain
carrying in the L-AChR a gain-of-function mutation (T12´M2P) that
in human leads to a severe slow-channel CMS reveals that the
functional changes in the worm mimic those of the patients. These
results open doors for establishing C. elegans models for human
myasthenic syndromes.
162
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TRC-7] Heterotetramerization of plant PIP channels
involves different stoichiometries
1
2
1,3
1,3
Jozefkowicz, C., Sigaut, L., Canessa Fortuna, A., Scochera,
4
2
1
1,3
F., González Flecha, L., Pietrasanta, L.I., Amodeo, G., Alleva,
K.
1
2
DBBE FCEN UBA & IBBEA - CONICET, CMA & DF, FCEN, UBA
3
& CONICET, Dpto. Fisicomatemática, FFyB, UBA, 4IQUIFIB, UBA
– CONICET. Buenos Aires, Argentina.
As other multimeric membrane channels, aquaporins oligomers
composition could be homooligomeric or heterooligomeric.
Although physical and functional interaction among different PIPs
has been proved, it is not elucidated how the different monomers
are organized in a functional oligomer. First, we confirmed the
existence of PIP oligomers as heterotetramers by studying some
structural elements involved in the intermolecular interactions
between PIP monomers (1). Now, we investigate the functional
properties of heterotetramers comprising different PIP1-PIP2
stoichiometry ratios. Our experimental approaches include: i)
performing homo and heterodimeric constructs made of either
PIP1 or PIP2, as well as both subunits; ii) measuring water
transport in control and inhibited (medium acidification) conditions
in Xenopus oocytes; iii) localizing PIPs heterotetramers by confocal
fluorescence microscopy; iv) detecting PIP assemblies by in gel
fluorescence technique. Results indicate that PIP heterotetramers
with stoichiometries PIP1-PIP2 2:2, 3:1 and 1:3 can exist in the
plasma membrane since they are able to assemble by coexpression of PIP1-PIP2 dimers, and by co-expression of dimers
with PIP1 or PIP2 monomers. Moreover, heterotetramer
stoichiometry present biological relevance since it affects water
transport activity and proton-dependent gating.
Acknowledgements: PICT 2010-2042, UBACYT 2013 grants to K.
A. and PICT11-2239, PIP12-14 CONICET grants to G.A.
1. Jozefkowicz, C. et al., Plos One. 2013.
163
_____________________________________________________
TRC-8] Neurotransmitter GABA activates muscle but not
α7 nicotinic receptors
Dionisio, L.; Bergé I.; Bravo, M.; Esandi, C.; Bouzat, C.
Laboratorio de Neurofisiología y Farmacología Molecular –
INIBIBB-CONICET
Cys-loop receptors are neurotransmitter-activated ion channels
involved in synaptic and extrasynaptic transmission in the brain
and are also present in non-neuronal cells. As GABAA and nicotinic
receptors (nAChR) belong to this family, we explored by
macroscopic and single-channel recordings if the inhibitory
neurotransmitter GABA has the ability to activate excitatory
nAChRs. GABA differentially activates nAChR subtypes. It
activates muscle nAChRs, with maximal peak currents of about 10
% of those elicited by ACh and 15-fold higher EC50 with respect to
ACh. At the single-channel level, the weak agonism is revealed by
the requirement of 20-fold higher concentration of GABA for
detectable channel openings, a major population of brief openings,
and absence of clusters of openings when compared to ACh.
Mutations at key residues of the principal binding-site face of
muscle nAChRs (αY190 and αG153) affect GABA-activation
similarly as ACh-activation whereas a mutation at the
complementary face (εG57) shows a selective effect for GABA.
Studies with subunit-lacking receptors show that GABA can
activate muscle nAChRs through the α/δ interface. Interestingly,
single-channel activity elicited by GABA is similar to that elicited by
ACh in gain-of-function nAChR mutants associated to congenital
myasthenic syndromes, which could be important in the
progression of the disorders due to steady exposure to serum
GABA. In contrast, GABA cannot elicit single-channel or
macroscopic currents of α7 or the chimeric α7-5HT3A receptor, a
feature important for preserving an adequate excitatory/inhibitory
balance in the brain as well as for avoiding activation of nonneuronal receptors by serum GABA.
164
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TRC-9] PIP aquaporin N and C terminals involved in the
stability of the tetrameric assembly of the channel
Scochera F., Gómez N., Jozefkowicz C., Amodeo G., Moglioni A.,
Alleva K. and Martini MF.
NANOBIOTEC
CONICET-UBA,
IBBEA
CONICET-UBA,
IQUIMEFA CONICET-UBA, Buenos Aires, Argentina.
PIP is an aquaporin subfamily of tetrameric water channels formed
by different paralogues, PIP1 and PIP2. PIP2 easily reaches the
plasma membrane while PIP1 remains in the cytosol unless is coexpressed with PIP2. This PIP1-PIP2 interaction triggers not only
the formation of heterotetrameric assemblies but also regulates its
biological activity. We recently showed that PIP2 loop A is a key
structural element to control heterotetramerization1 and now we
seek for other structural elements involved in homotetramer, or
even heterotetramer stabilization. To study PIP's quaternary
structure stabilization through N and C inter-monomeric terminals,
we first assayed -by molecular dynamic simulations in an NPT
ensemble- the interaction of these terminal peptides corresponding
to PIP2 or PIP1 but also the hetero-interaction of PIP2 N-ter with
PIP1 C-ter (and viceversa). The interaction energies (as well as
specific interactions) showed differences when discriminating the
type of PIP analyzed. Interestingly, our preliminary results
suggests that the energy interaction between PIP2 N and C intermonomeric terminal is lower than PIP1 one. These results shed
light on N and C terminals as relevant elements in the stability of
PIP assembly.
Acknowledgements: UBACYT 159 2013-2016 to KA, PIP CONICET to
GA. PICT-2011-2606 to MMF.
1. Jozefkowicz, et al., PlosOne 2013, DOI:
10.1371/journal.pone.0057993
165
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TRC-10] pH-dependent blocking action of bupivacaine
on BKCa channel.
1
1
1
2
Piccinini, L ; Moncada, M ; Enrique, N ; González, W ; González,
3
1
1
C ; Martin, P ; Milesi, V .
1
Laboratorio de Canales Iónicos. IIFP. CONICET- UNLP. La Plata.
Argentina. 2Centro de Bioinformática y Simulación Molecular
(CBSM). Universidad de Talca. Chile. 3Centro Interdisciplinario de
Neurociencias de Valparaíso (CINV). Universidad de Valparaíso.
Chile.
Bupivacaine (B) is a local anesthetic that belongs to the amino
amide group (B/BH+ equilibrium, pKa=8.1). Previously, we
demonstrated [1] that bupivacaine inhibits the potassium channel
BKCa, by a very-fast type block and voltage-dependent mechanism
(depolarization increases the blocking ratio). This inhibition occurs
from the intracellular side, by interacting with the channel pore. In
this study we investigated the effect of pH (pH= 7.4 and pH= 7.0)
on bupivacaine blocking action on BKCa channel expressed in
HEK293T cells. In addition, we studied the drug-protein interaction
mechanism (B and BH+), using the computational docking
technique.Using the patch clamp technique in voltage clamp
configuration, G/Gmax curves were built in absence and in the
presence of 300µM bupivacaine at both pH values. The analysis of
the steady-state ionic currents showed that, at pH= 7.0 (BH+/B=
12.6), the addition of bupivacaine generated a 50% decrease in
Gmax. At pH: 7.4 (BH+/B= 5) the decrease in Gmax was 25%.
Moreover, the bupivacaine had no effect on the instantaneous
current, measured on tail currents. We also performed docking
simulations of BH+ and B in the BKCa channel homology model
obtained before by our group. The grid where the docking was
performed contained the pore structure of the BKCa channel. The
lowest energy conformations of the ligands were docked below the
selectivity filter interacting with the residues L377, F380, and V384.
BH+ also interacts with T352 of the selectivity filter. These results
confirm that the blocking effect is greater when the ratio of BH+/B
was higher and that it is mediated by a voltage-dependent
blockade of the channel unitary conductance without affecting the
channel opening probability.
1.
Martin, P. et al. Channels (Austin). 2012. p174-80
166
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TRC-11] Effects of beta amyloid on alpha7 receptor
function.
Matías Lasala, Jeremías Corradi and Cecilia Bouzat
Instituto de Investigaciones Bioquímicas de Bahía Blanca –
Universidad Nacional del Sur.
CONICET – UNS.
Alzheimer’s disease (AD) is the most common form of dementia in
elderly people that produces a severe impairment in mental
function, with profound effects on learning and memory. The beta
amyloid peptide (bA) plays an important role in AD development.
Several studies have shown that bA can affect cholinergic
signaling in the brain but the specific mechanism is still obscure. Of
the nicotinic acetylcholine receptors at risk, the most critical may
be those containing the alpha7 subunit, because they are
widespread, have a high relative permeability to calcium, and
regulate numerous cellular events in the nervous system. Here we
explore the effect of bA1-40 on the human alpha7 nicotinic
receptors at the single-channel level. We observe that nanomolar
concentrations of beta amyloid peptides block human alpha7
receptors. In the presence of 100µM ACh combined with the
positive allosteric modulator PNU, single-channel episodes appear
as openings of ∼200ms grouped in long clusters of ∼3s and high
open probability (>0.9). In the presence of bA there is a clear
reduction in open- and cluster-duration (∼4-fold and 2-fold,
respectively at 500nM of bA). Our results demonstrate that bA
antagonizes the human alpha7 receptor by at least two different
mechanisms, one that produces reduction in the open duration,
probably acting as an open channel blocker, and another that
reduces cluster duration, which is compatible with slow block or
increased desensitization. Understanding the actual mechanism
that leads to the neuronal damage would help to design more
effective and specific treatments for AD patients.
167
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TRC-12] Dual effects of Arachidonic acid on BK channel:
role of β1-subunit
1
1
2
1
Moncada, M ; Piccinini, L ; Castillo, K ; Asuaje, A ; González, C
2
1
1
; Milesi, V ; Martín, P.
1
Laboratorio de Canales Iónicos, IIFP, CONICET-UNLP, La Plata,
2
Argentina.
Centro Interdisciplinario de Neurociencias de
Valparaíso (CINV), Chile.
Arachidonic acid (AA) is a polyunsaturated fatty acid involved in
modulation of several ion channels activity. Previously, we reported
that 10 µM AA activates the high conductance Ca2+ and voltage
dependent K+ channel (BK) in human vascular smooth muscle
cells where the α subunit of BK is expressed together with the
regulatory β1-subunit (β1) [1]. In this work, we studied in depth the
action mechanism of AA using patch clamp technique on BK
channel heterologously expressed with or without β1. 10 µM AA
activated BK only in presence of β1, changing the voltage
dependence of activation (left shift on G-V curve, ΔV1/2= -55,2 mV
± 4,4; n=3; p<0,05), and modifying the voltage sensor movement
(left shift in Q-V curve, ΔV1/2= -17,2 mV ± 8,1; n=5; p<0,05).
Conversely, BK expressed without regulatory subunits, was
inhibited by 10 µM AA (n=3) showing a right shift on G-V curve
(ΔV1/2= 130,3 mV ± 6,4; p<0,05), a decrease on Gmax (Gmax-AA/GmaxCONTROL = 0,66 ± 0,06; p<0,05) and no change on gating currents
(n=5). The inhibitory effect of AA was Ca2+ dependent, since the
inhibition was bigger at 80 µM (first results) than at 8 µM
2+
intracellular Ca (ΔV1/2= 73,0 mV ± 4,0 and Gmax-AA/Gmax-CONTROL =
0,91 ± 0,04; n=3; p<0,05). The AA effect was faster when it was
applied to the intracellular membrane side, suggesting that AA acts
from the intracellular side of the channel. Together, these results
suggest that the effect of 10 µM AA depends on the presence or
absence of β1, being able to activate or inhibit BK, respectively.
1. Martín, P. and Moncada, M. et al Pflugers Arch - Eur J
Physiol. 2014. 1779.
168
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TRC-13] Plant aquaporins PIP2 and TIP2 show different
responses to membrane deformation
1
2
2
2,3
Goldman, R.P. , Jozefkowicz, C. , Sutka, M. , Alleva, K. , Ozu,
1
M .
1
Lab. de Biomembranas, IFIBIO - Houssay, Facultad de Medicina,
UBA-CONICET. 2Instituto de Biodiversidad
y Biología
Experimental y Aplicada, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, CONICET, Argentina.
3
Facultad de Farmacia y Bioquímica, UBA.
In this work we studied the mechanosensitive properties of PIP2
and TIP2, both aquaporins (AQPs) from Beta vulgaris (beetroot).
We expressed the proteins in Xenopus oocyte membranes and
tested their functionality applying different osmotic gradients by
means of the emptied-out oocyte technique [1]. The results
obtained with TIP2 show that the relationship between water flux
(JW) and the osmotic gradient (∆Osm) is similar to that observed
with human AQP1 [2]. However, this seems not to be the case for
PIP2. These results would be reflecting differences between the
sensibility of TIPs and PIPs to changes of membrane tension. In
the case of TIP2, the osmotic permeability coefficient (Pf)
decreases with increasing ∆Osm. This indicates that the closure of
the water pore could be a cooperative phenomenon, occurring in
TIPs in a similar way as in hAQP1 [2]. In conclusion, present
results show that a closure mechanism gated by increasing
membrane tension is present in plant AQPs, as occur in animal
members of the family.
1. Ozu et al, J. Biochem. Biophys. Methods. 63(3). 2005. P
187.
2. Ozu et al, Biophys. J. 104(1). 2013. P 85.
169
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TRC-14] Metal Fluoride Complexes of plasma membrane
calcium pump: Characterization of fluoride-stabilized
phosphoenzyme analogues
De Sautu M., Ferreira-Gomes M.S., Saffioti, N.A., Rossi, J.P.,
Mangialavori, I.C.
IQUIFIB –Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires. [email protected]
The plasma membrane calcium pump (PMCA) belongs to the Ptype ATPase family of active cation pumps. According to the
conventional E1–E2 theory, E1 and E2 are the high-affinity and lowaffinity states for Ca2+, respectively. Gating of the ion pathway is
coupled to the phosphorylation and dephosphorylation of the
ATPase. Phosphoryl transfer from ATP to an Asp residue in the
cytoplasmic domain (i.e., E1 to E1P) closes the cytoplasmic gate,
and the release of ADP triggers a change in the affinity of the Ca2+
binding sites (i.e., E1P to E2P) opening the luminal gate. Hydrolysis
of the aspartylphosphate (E2P to E2) closes the gate. Metal
fluorides like magnesium, beryllium, and aluminum fluorides,
operate as phosphate analogues and inhibit P-type ATPases by
interacting with the phosphorylation site, stabilizing conformations
that are analogues to the specific phosphoenzyme intermediates.
Thus, MgFx, AlFx, and BeFx (MeF) stabilize analogues of the E2P
product state (E2MgF42-), the E2P transition state (E2AlF4-), and the
E2P ground state (E2BeF3 ), respectively.
Recently, we demonstrated that the inhibition of PMCA Ca2+ATPase activity by these complexes have different characteristics
[1]. Our results show that (1) the apparent velocity constant (kobs)
for inhibition of PMCA activity by different fluoride complexes
shows fluoride concentration dependence; (2) its dependence
differs with each MeF; and (3) Ca2+ reverses PMCA inhibition by
these MeF.
With grants of ANPCYT, CONICET and UBACYT
1. Siciliano, N., Ferreira-Gomes, M., Rossi, JPFC. and
Mangialavori, IC. XLII Reunión Anual de la Sociedad
Argentina de Biofísica. ISBN 978-987-27591-2-4. Pag.
TRC 95
170
_____________________________________________________
TRC-15] Effects of metal-fluoride complexes on the
phosphatase activity of the PMCA
Saffioti N.A, Ferreira Gomes, M.S., de Sautu M., Rossi, J.P. and
Mangialavori I.C.
IQUIFIB- Facultad de Farmacia y Bioquímica, UBA
[email protected]
Plasma membrane calcium pump (PMCA) belongs to the P-type
family of active cation pumps. According to the conventional E1–E2
theory, E1 and E2 are the high and low-affinity states for Ca2+,
respectively. When the pump in the E1 state hydrolyses ATP, it
generates an auto-phosphorylated intermediate E1-P which
transform to E2-P. This conformational change allows the calcium
release. Besides of this phosphorylase activity, it have been
described a phosphatase activity where the E2 conformation
hydrolyze p-nitrophenylphosphate in the absence of Ca2+ (1).
Vanadate and metal fluorides like magnesium, beryllium, and
aluminum (MexFy), act as phosphate analogues and inhibit P-type
ATPases, by interacting with the phosphorylation site. In the
sarcoplasmic calcium pump, these metal-fluoride complexes
stabilize analogues of E2P in different conformations (2). In order to
study the effects on PMCA, we characterized the phosphatase
activity and conformational changes of a purified preparation of
PMCA, in the presence of these fluoride-complexes. Our results
show that MexFy inhibit phosphatase activity hyperbolically, with a
similar behavior, except with magnesium fluoride which exhibits a
more complex manner. The apparent inhibition constants were
different from those obtained for the phosphorylase activity in the
same conditions. We also detected conformational changes with
the fluorescent probe TNP-ATP when these complexes bind to
PMCA, showing different structural changes of E2 intermediates.
With grants from UBACYT, ANPCYT y CONICET
1. Mazzitelli I R, Adamo H P. Biochimica et Biophysica Acta.
2007. 1777.
2. Clausen J D. The Journal of Biological Chemistry. 2011.
11792
171
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TRC-16] Human Aquaporin-1: Furosemide Docking and
Protein-Protein Interactions
1
2
1
1
1
Dorr, RA ; Rosi, P ; Ozu, M ; Costa Almar, F ; Toriano, R
1
Lab.
de
Biomembranas-IFIBIO
Houssay-UBA-CONICET;
2
DQIAyQF-FCEN-UBA
In our previous work we showed that the water permeability of
human aquaporin-1 (hAQP1) is specifically inhibited by the
presence of intracellular furosemide and modulated by the tension
of the plasma membrane. These data were obtained in the
Xenopus laevis oocyte membrane expressing water channels. Now
we use different docking protocols to locate sites of inhibition and
modifiers of membrane tension. The first strategy was to perform a
blind docking (with AutoDock) against the monomeric structure of
hAQP1 obtained from electron-crystallographic data (1FQY PDB
code). The results indicate that furosemide binds to both cytosolic
and outside areas of the channel, but never at sites in the
transmembrane region. Coincidentally with the in vitro results, the
cytosolic region was more favored than the extracellular binding
region. To evaluate the influence of protein conformational
changes, we did an unrestricted α-carbon molecular dynamic
simulation (50ns in explicit aqueous solvent, repeating docking
protocol for 10 frames separated by 5ns each). A significant
change in the angle of the α-helices and a pore occlusion
(quantified with PoreWalker server) could be observed. Under
these conditions, the furosemide binding sites are lost quickly. To
simulate hAQP1 in an environment that resembles protein included
in a lipid bilayer, α-carbon fixation was used. In such condition, the
distinction between furosemide binding sites remains. Once again
the intracellular binding site was favored. Based on published data
in which the presence of Na+ channel ENaC modifies the
membrane tension, we also studied the co-expression of hAQP1
and ENaC in Xenopus oocytes. The in vitro data plus the possibility
of formation of a protein-protein interaction (that was found using
the ClusPro server) lead us to hypothesize a physiological and
morphological interaction between the two proteins.
172
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TRC-17] Modulation of Plasma Membrane Ca2+-ATPase
by neutral phospholipids: Effect of the micelle – vesicle
transition and the bilayer
Pignataro MF, Dodes-Traian MM, González-Flecha FL, Sica M,
Mangialavori IC and. Rossi JP.
IQUIFIB- Facultad de Farmacia y Bioquímica, UBA.
[email protected]
The effects of lipid structure on a membrane protein are likely to be
complex and unique for each membrane protein. Here we
employed different detergent-phosphatidylcholine reconstitution
media and study their effects on PMCA. We found that Ca2+ATPase activity largely depends on the length and the unsaturation
degree of the hydrocarbon chain. The biophysical properties of the
detergent-phosphatidylcholine mixtures lead to a biphasic behavior
of PMCA activity regardless the length of the hydrocarbon chain.
Using Fluorescence Correlation Spectroscopy, we monitored the
micelle-vesicle transition in this system and found that the
transition ensues at XPC= 0.3 for DMPC and at 0.6 for DLPC. We
found a decrease in PMCA turnover after the micelle-vesicle
transition, but the time of residence of the phosphorylated
intermediate (EP) increase with the concentration of XPC. Maximal
PMCA activity is obtained in detergent/DMPC micelles in
agreement with functional and structural analysis of the
hydrophobic domain thickness. Results in this paper show that at
optimal protein/phospholipid stoichiometry, ATPase activity is
higher when the hydrophobic thickness of the lipid bilayer is
expected to match the transmembrane domain of the protein,
reducing the thermodynamic cost of exposing either fatty acyl
chains or hydrophobic amino acids to water
With grants from UBACYT, ANPCYT y CONICET
173
_____________________________________________________
TRC-18] The use of Azido Ruthenium photoactivatable
probe to study the Ca2+-binding site of the Plasma
Membrane Calcium Pump.
Ontiveros, M. Q.; Mangialavori, I. C.; Pignataro, M. F.; Martiarena,
J.; Rossi, J. P.; Ferreira-Gomes, M. S.
Instituto de Química y Fisicoquímica Biológicas. ”Prof. Paladini”.
Departamento de Química Biológica, Facultad de Farmacia y
Bioquímica. Universidad de Buenos Aires
[email protected]
The Plasma Membrane Calcium ATPase (PMCA) is a calmodulinregulated P-type ATPase responsible for the maintenance of low
intracellular concentration of Ca2+ in most eukaryotic cells. It
couples the transport of Ca2+ outside the cells with the hydrolysis of
ATP. Since the crystal structure of PMCA is still not resolved, the
2+
information about the Ca -binding site is limited to the comparative
studies with the structure of Sarcoplasmic Reticulum Calcium
ATPase. There are evidences that certain amino acids on the
transmembrane segments M4 and M6 would be linked with the
calcium binding site [1]. The purpose of this work is to identify and
characterize the Ca2+-binding site of PMCA. For this, we
synthesize a Ca2+-like photoactivatable reagent, azido-ruthenium
(AzRu) which binds covalently and specifically to Ca2+-binding
proteins after exposure to ultraviolet irradiation at 290 nm [2]. The
experiments were performed with vesicles and purified PMCA
obtained from human erythrocytes. Results showed that (a) AzRu
inhibited Ca2+-dependent activities with high affinity; (b) AzRu had
no effect on Mg2+-dependent activity and on other ATPases like the
2+
Na-K pump. The ability of AzRu to inhibit Ca -dependent activities
was enhanced when PMCA-AzRu was exposed to irradiation,
suggesting that photoactivation leads to an irreversible binding of
the probe to the enzyme. These results provide a new approach to
localize the Ca2+-binding site of PMCA in combination with MALDITOF spectroscopy.
With grants of ANPCYT, CONICET and UBACYT.
1. Rinaldi D., Adamo H. Biochimica et Biophysica Acta. 2009.
2404.
2. Israelson A., Zilberberg N. & Shoshan-Barmatz, V. Nature
Protocols. 2006. 111.
174
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TRC-19] Transport of divalent cations mediated by the
Plasma Membrane Calcium Pump
Ontiveros, M. Q.; Mangialavori, I. C.; Martiarena, J.; Verstraeten,
S.; Rossi, J. P.; Ferreira-Gomes, M. S.
Instituto de Química y Fisicoquímica Biológicas.”Prof. Paladini”.
Departamento de Química Biológica, Facultad de Farmacia y
Bioquímica. Universidad de Buenos Aires.
[email protected]
The plasma membrane calcium (PMCA) pump maintains the
homeostasis in eukaryotic cells by transporting Ca2+ in a process
coupled to the ATP hydrolysis. PMCA is activated by a Ca2+calmodulin complex, by controlled proteolysis or by acidic
phospholipids [1, 2]. The aim of this work was to investigate the
effect and regulation of different divalent metal ions on PMCA
activity. The experiments were performed with purified PMCA and
inside-out vesicles from membranes of human erythrocytes. We
evaluated the transport of Be2+, Sr2+, Ba2+, from the alkaline earth
metals and Co2+, Cd2+ and Pb2+ from the fourth, fifth, and sixth
periods, respectively. Results show that: (a) PMCA is able to
transport Ca2+, Sr2+, Ba2+, Co2+ and Pb2+ with different Vmax and
affinities, while Be2+ and Cd2+ are not transported; (b) activated
PMCA showed an increasing to the apparent affinity for Ca2+, Sr2+
and Ba2+, however for Co2+ and Pb2+ the apparent affinity was not
modified; (c) the closer the cation is to the Ca2+ radius, the
transport is more efficient, at exception of Cd2+, which inhibits
PMCA reacting with Cys residues. These results suggest that
PMCA may contribute to the mechanism of toxicity/detoxification
process under the eventual access of other divalent cations into
the cell.
With grants of ANPCYT, CONICET and UBACYT.
1. Mangialavori et al. J. Biol. Chem. 2009. 4823
2. Filomatori et al. J. Biol. Chem. 2003. 22265
175
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TRC-20] A comparison of the E2P-like states induced by
metal-fluorides complexes in the Na,K-ATPase and H,KATPase
Centeno, M.; Ferreira-Gomes, M.S.; Monti J.L.; Rossi, R. C. and
Montes, M. R.
Instituto de Química y Fisicoquímica Biológicas, Dr. Alejandro C.
Paladini - Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires. [email protected]
Na,K-ATPase and H,K-ATPase are membrane-bound ion pumps
that generate electrochemical gradients of cations across the
plasma membranes of animal cells fueled by the hydrolysis of ATP.
They undergo phosphorylation and dephosphorylation reactions
and oscillate between two major conformations, E1 and E2.
Fluorinated complexes of Mg2+, Al3+ and Be2+ inhibit the activity of
several ATPases because they imitate the phosphoryl group and
stabilize the intermediate ground (BeFx), transition (AlFx) and
product (MgFx) states in the dephosphorylation sequence: E2-P
(ground state)→ E2·P (transition state)→ E2·Pi (product state)→
E2 + Pi. We have studied the kinetics of the E1→E2P
conformational change, assessed by eosin fluorescence
measurements, produced by metal-fluorides complexes in the
Na,K-ATPase and H,K-ATPase at 25 °C in media with 25 mM
imidazole-HCl, pH 7.4. Our results show that BeFx induces the E2P ground state with a similar rate in both enzymes, while MgFx
induces the E2·Pi product state with a much slower rate in the H,KATPase than in the Na,K-ATPase. These results are in agreement
with the difficulties reported to isolate the state of the H,K-ATPase
containing occluded K+.
With grants of ANPCYT, CONICET and UBACYT
1- Morth JP. Nature. 2011. 3031.
2- Mónica R. Montes. BBA. 2011. 316.
176
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TRC-21] Mechanism of activation of the 5-HT3A receptor
by partial agonists.
Jeremías Corradi and Cecilia Bouzat
Instituto de Investigaciones Bioquímicas de Bahía Blanca –
Universidad Nacional del Sur.
CONICET – UNS.
Partial agonists have emerged as attractive therapeutic molecules.
2-Me-5HT and tryptamine have been defined as partial agonists of
5-HT3 receptors on the basis of macroscopic measurements.
Because several mechanisms may limit maximal responses we
took advantage of the high-conductance form of the mouse 5-HT3A
receptor to understand their molecular actions. Individual 5-HTbound receptors activate in long episodes of high open probability,
consisting of groups of openings in quick succession. The
activation pattern is similar for 2-Me-5HT only at very low
concentrations since profound channel blockade takes place within
the activating concentration range. In contrast, activation episodes
are significantly briefer in the presence of tryptamine. Generation
of a full activation scheme reveals that the fully-occupied receptor
overcomes transitions to closed pre-open states (primed states)
before opening. Reduced priming explains the partial agonism of
tryptamine. In contrast, 2-Me-5HT is not a genuine partial agonist
since priming is not dramatically affected and its low apparent
efficacy is mainly due to channel blockade. The analysis also
shows that the first priming step is the rate-limiting step and partial
agonists require an increased number of priming steps for
activation. Molecular docking suggests that interactions are similar
for 5-HT and 2-Me-5HT but slightly different for tryptamine. Our
study contributes to understanding 5-HT3A receptor activation,
extends the novel concept of partial agonism within the Cys-loop
family, reveals novel aspects of partial agonism, and unmasks
molecular actions of classically-defined partial agonists. Unraveling
mechanisms underlying partial responses has implications in the
design of therapeutic compounds.
177
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TRC-22] Study of the effect of hypotonic stimuli on the
mouse epithelial sodium channel (ENaC) activity
expressed in Xenopus laevis oocytes: role of the
intracellular sodium concentration.
Galizia L, Marino GI, Palma A, Kotsias BA.
Laboratorio de Canales Iónicos, Instituto de Investigaciones
Médicas Alfredo Lanari, Universidad de Buenos Aires, IDIMCONICET, Buenos Aires, Argentina.
The regulation of the epithelial Na+ channel (ENaC) during cell
volume increase is relevant in cellular processes involving osmotic
challenges. ENaC function is affected by changes of the
intracellular sodium concentration. Its sensitivity to hypotonicinduced swelling was investigated in the Xenopus oocyte
expression system with the injection of the three subunits of the
mouse ENaC (mENaC) [1]. We used the voltage clamp technique
to measure the amiloride-sensitive Na+ currents (INa(amil)) in order
to study the role of intracellular sodium on the ENaC regulation
mediated by hyposmotic challenges. ENaC-injected oocytes under
low intracellular sodium conditions, showed no significative
reduction of INa(amil) inward currents measured at -100 mV (Ina
(amil)ISO : -5.15 ± 0.84 µA vs INa(amil)HIPO:-4.20 ± 0.48 µA, NS, n
= 6 ). Moreover, the high intracellular sodium condition elicited an
inhibitory response of INa(amil). Oocytes expressing a DEG
mutant of the β-ENaC subunit (β-S518K), which produce an open
probability equal to 1 showed a reduced INa(amil) hypotonic
mediated inhibitory response in both conditions of intracellular
sodium concentration. Based on these results, we suggest that
hypotonicity-dependent ENaC inhibition, due to open probability
changes is mediated by an intracellular sodium dependent
mechanism.
1- Galizia et al J Membr Biol. 2013. 246 P 949
178
INDICE DE AUTORES
_____________________________________________________
APELLIDO
POSTER
PÁGINA
Alarcón, L.M.
LBM 12
72
Albano, J.
TMB 3
138
Alonso, S. del V.
LBM 16
76
Amundarain, M.J.
TMB 12
147
Andersen, N.D.
TRC 5
161
Andujar, S.
TMB 9
144
Angelani, C.R .
PNA 7
101
Angiolini, J.
TMB 6
141
Arcon, J.P.
TMB 16
151
Avila, C.L.
LBM 21
81
Balatti, G.E.
TMB 20
155
Bergé, I.
TRC 6
162
Bianchi, M.
NTA 4
88
Boron, I.
PNA 6
100
Bucci, H.A.
PNA 31
125
Burgos, M.I.
NTA 7
91
Cababie, L.A.
EZ 1
58
Cámara, C. I.
LBM 7
67
Cámara, C. I.
LBM 19
79
Cámara, C. I.
LBM 22
82
Caruso, B
PNA 13
107
Castro, I.
PNA 14
108
Chavez, Silvina.
PNA 23
117
Cogo, C.
TMB 13
148
Colmano, G.N.
LBM 4
64
Corradi, J.
TRC 21
177
Curto, L.M
PNA 21
115
179
INDICE DE AUTORES
_____________________________________________________
APELLIDO
POSTER
PÁGINA
Curto, L.M.
PNA 22
116
Curto, L.M.
PNA 8
102
Cutró, A.
LBM 11
71
Daza Millone, M.A.
NTA 3
87
De Rossi M.C.
SID 2
130
De Sautu, M.
TRC 14
170
Di Guilmi, M.N.
TRC 1
157
Di Guilmi, M.N.
TRC 2
158
Diaz, C.
NTA 6
90
Dionisio, L.
TRC 8
164
Dorr, R.A.
TRC 16
172
Faraj, S.E.
PNA 19
113
Ferreira-Gomes, M.S.
TRC 20
176
Franchini,G.R.
PNA 26
120
Gabriel, M.
NTA 8
92
Galizia, L.
TRC 22
178
Garay, A.S.
LBM 5
65
Gauto, D.F.
PNA 17
111
Gennaro, A.M.
LBM 2
62
Gianotti A. R.
PNA 10
104
Giudice, F.
LBM 3
63
Gómez, G.E.
NTA 1
85
González Lizárraga, M.F.
PNA 16
110
Grasso, E.J.
LBM 1
61
Grille, L.
PNA 18
112
Guauque-Torres M. P.
PNA 29
123
Herlax, V.
PNA 15
109
180
INDICE DE AUTORES
_____________________________________________________
APELLIDO
POSTER
PÁGINA
Herrera, M.
PNA 12
106
Herrera, M.G.
PNA 27
121
Hollmann, A.
LBM 6
66
Hoyos Obando, A.
LBM 17
77
Iglesias, D.E.
BET 1
54
Ithuralde, R.E.
TMB 19
154
Jozefkowicz, C.
TRC 7
163
Klinke, S.
PNA 24
118
Labanda, M.S.
NTA 5
89
Lasala, M.
TRC 11
167
Lavaisse, L.M.
LBM 13
73
Ledesma, A. E.
PNA 9
103
Lichtig, P.
TMB 5
140
Lopez, L.
SID 6
134
Manssour-Triedo, F.
PNA 28
122
Martínez, D.
TMB 11
146
Martínez-Rodríguez, S.
PNA 25
119
Marzari, G.
LBM 8
68
Maté, S.
LBM 15
75
Maturana, P.
LBM 18
78
Menéndez C.A.
TMB 7
142
Moncada, M.
TRC 12
168
Montes de Oca, J.M.
EZ 2
59
Morini, M.
LBM 10
70
Ontiveros, M. Q.
TRC 18
174
Ontiveros, M. Q.
TRC 19
175
Ozu, M.
TRC 13
169
181
INDICE DE AUTORES
_____________________________________________________
APELLIDO
POSTER
PÁGINA
Pallavicini, C.
NTA 9
93
Pantano, S.
TMB 17
152
Perrotta, R.
LBM 9
69
Piccinini, L.
TRC 10
166
Piegari, E.
SID 3
131
Pignataro, M.F.
TRC 17
173
Placenti, M.A.
PNA 33
127
Rios, A.S.
PNA 1
95
Rodrigues, D.E.
LBM 20
80
Romero J. M.
PNA 3
97
Rosa, A.S.
LBM 23
83
Rossi Fernández, A.C.,
NTA 2
86
Saffioti, N.A.
TRC 15
171
Sain, P.
LBM 14
74
Samus, S. I
PNA 20
114
Scochera, F.
TRC 9
165
Sferco, S.J.
TMB 8
143
Sigaut, L.
SID 4
132
Siri, M.
PNA 4
98
Toledo, P,
PNA 2
96
Tolosa M.F.
TRC 4
160
Torchio, G.
PNA 30
124
Valdez, L.B.
BET 2
55
Valsecchi, W.M.
PNA 5
99
Vazquez D.S
PNA 32
126
Vettorazzi, M.
TMB 4
139
Veuthey, T.
PNA 11
105
182
INDICE DE AUTORES
_____________________________________________________
APELLIDO
POSTER
PÁGINA
Villarruel, C.
SID 5
133
Viso, J.F.
TMB 10
145
Vitali, V.
TMB 14
149
von Bilderling C.
SID 1
129
Wood, I.
TMB 1
136
Wood, I.
TMB 2
137
Yaneff, A.
TRC 3
159
Zamarreño, F.
TMB 15
150
Zaobornyj, T.
BET 3
56
Zeida, A.
TMB 18
153
183
INDICE DE AUTORES
_____________________________________________________
184

SOCIEDAD ARGENTINA DE BIOFÍSICA XLIII Reunión Anual

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