Beruflich Dokumente
Kultur Dokumente
-
decarboxylation switch that arrests the sensor domain of BlaR1 in an activated state required for
signal transduction,
2,3
we have characterized BlaI binding to its operator region, and we have shown
that the in vivo concentrations account for the basal level transcription of the resistance genes.
4
We
have also presented evidence that support the hypothesis that BlaR1 fragmentation is a means for
turnover,
5
a process required for recovery from induction of resistance in S. aureus in the absence
of the antibiotic challenge, and that BlaR1 is indeed a metallo-protease that degrades the gene
repressor BlaI.
6
Regarding the DD-transpeptidase PBP2a, we have recently reported the
identification of an allosteric binding site that regulates the opening of the active site to permit
substrate entry, through a multiresidue conformational change.
7
In my group, we are currently
working on the elucidation of the topology and structure of the sensor proteins BlaR1 and MecR1.
Acknowledgements: The PEW Charitable Trusts, NIH, ANPCyT, CONICET
1 Llarrull LI, Fisher JF, Mobashery S. Antimicrob. Agents Chemother.2009. 4051.
2 Borbulevych O, Kumarasiri M, Wilson B, Llarrull LI, et al. J. Biol. Chem. 2011. 31466
3 Kumarasiri M, Llarrull LI, et al. Journal of Biological Chemistry. 2012. 8232.
4 Llarrull LI, Prorok M, Mobashery S. Biochemistry. 2010. 7975
5 Llarrull LI, Toth M, Champion MM, Mobashery S. Journal of Biological Chemistry. 2011. 38148
6 Llarrull LI, Mobashery S. Biochemistry, 2012. 4642.
7 Otero LH, Rojas-Altuve A, Llarrull LI, et al. Proc. Natl. Acad. Sci. USA. 2013. 16808.
Conferencias (lectures) SAB2013
15
Mini-conferencia II / Short conference II
One enzyme two pathways: Single molecules studies on Nitrite Reductase
Tabares LC
Service de BionergtiqueBiologieStructuraleetMcanismes, CEA-Saclay, France.
Leiden Institute of Physics, University of Leiden, The Netherlands.
Single enzymes measurements have changed the way we look to these molecular machines mode
of operation, providing new, previously unobtainable, information. However, most of these
measurements were restricted to enzymes which undergo conformational changes, have
fluorescent cofactors or use/produce fluorescent compounds. We have developed a new method
for monitoring the redox state of a metalloprotein based on fluorescence resonance energy transfer
(FRET) between a fluorescent label and the protein metal center. The method was applied to the
study of copper-containing nitrite reductase from Alcaligenesxylosoxidans. Nitrite reductase is a key
enzyme in bacterial denitrification and plays an important role in the global nitrogen cycle. In our
single molecule studies we found new evidence showing a heterogenic distribution in two
populations of molecules which correspond to two distinct catalytic pathways [1]. In order to obtain
the kinetics parameter we apply a microfluidic trapping device to allow, for the first time,
measurement of single enzymes in solution [2]. Our results reconcile a long-standing dispute about
the mode of action of this enzyme bringing together the previously proposed binding-first,
reduction-first and random mechanisms.
References
1 Tabares LC, Kostrz D, Elmalk A, Andreoni A, Dennison C, Aartsma TJ, Canters GW. Chem. Eur.
J. (2011) 17:12015-9.
2Goldsmith RH, Tabares LC, Kostrz D, Dennison C, Aartsma TJ, Canters GW*, Moerner WE*.
Proc. Natl. Acad. Sci. (2011) 108:17269-74.
SAB2013
16
S2: Estructura y funcin de protenas (protein structure and function)
17
SIMPOSIOS / SYMPOSIA
S1: Biofsica de biomembranas e interaccin lpido-protena / lipid-protein
interaction and membrane biophysics
S1.1_ Novel lipid binding proteins from helminth parasites. Structural and functional
analysis.
Marina Ibez Shimabukuro*, Florencia Rey*, Gisela R. Franchini*, Malcolm W. Kennedy
#
, Alan
Cooper
#
, Brian O. Smith
#
and Betina Crsico*
* Instituto de Investigaciones Bioqumicas de La Plata (CONICET-UNLP), Facultad de de Ciencias
Mdicas, UNLP. Argentina
# Institute of Biomedical & Life Sciences, University of Glasgow. UK
Parasitic helminths express lipid-binding proteins (LBPs) that are structurally distinct from host
LBPs. These proteins bind a wide range of lipid classes such as fatty acids, retinoids, eicosanoids,
triglycerides, phospholipids and cholesterol. Due to helminths limited lipid metabolism, LBPs have
been proposed to participate in parasites development and in the interaction with the host. To
understand the mechanisms involved, we have selected three important types of LBPs from highly
pathogenic helminth parasites: a) a novel class of fatty acid and retinol binding proteins with a
structure that has no known counterpart, b) relatives of the fatty acid binding protein family,
including members that are structurally modified in ways that are unique to nematodes, and c)
nematode polyprotein allergens. The atomic structures are under analysis employing NMR
spectroscopy, for which we already have obtained high quality data and full structure determination
is in progress. Protein's interactions with ligands employing NMR spectra show the changes
registered during the binding process when stripped and reloaded samples are compared. We are
also analyzing their ligand-binding parameters employing fluorescence-based systems. The studies
confirm these LBPs bind natural ligands and fluorescent analogues in the sub-micromolar range.
Structural and functional studies will enhance our understanding of the unique features of helminth
LBPs that may be related to the survival of the organisms and could be used as potential drug
targets.
S1.2_The power of being at the interface: mechanism of DesK thermosensing
Cybulski L
Instituto de Biologa Molecular y Celular de Rosario (IBR)- CONICET and Departamento de
Microbiologa, Facultad de Ciencias Bioqumicas y Farmacuticas, UNR, Rosario, Argentina.
The thermosensor DesK is a five-pass transmembrane (TM) histidine kinase that senses
and signals temperature changes in Bacillus. Temperature sensing involves a built-in instability
caused by two motifs of hydrophilic residues located at both, the N-terminus and C-terminus of the
TM domain. The N-terminus has two hydrophilic amino acids (K10 and N12) below the lipid/water
interface, and the C-terminus has a hydrophilic motif composed of three serines located on one side
of the helix. These interfacial hydrophilic motifs render the protein sensitive to membrane thickness
and to the extent of interfacial hydration, which would in turn depend upon temperature changes. A
conformational changein the linker connecting the TM sensing domain with the cytoplasmic catalytic
domain is triggered by the interplay of these interfacial motifs to control DesK activity.
S2: Estructura y funcin de protenas (protein structure and function)
18
S1.3_ Conformation of peripherally bound membrane proteins: the influence of the
lipid phase state
Mara Beln Decca
Departamento de Qumica Biolgica-CIQUIBIC, Facultad de Ciencias Qumicas, Universidad
Nacional de Crdoba. Haya de la Torre y Medina Allende,5000, Crdoba, Argentina.
The transfer of soluble proteins into the interface between the lipid membrane and the aqueous
phase is recognized as a key step for several cellular processes. This translocation represents a
major change in the protein environment that can stabilize different protein conformations with
possible consequences on its biological activity. Using as a model the peripherally bound protein L-
BABP we found that conformation can be modulated by the phase state of the lipid membrane.
When L-BABP was bound to lipids in the gel phase, the secondary structure was similar to the
native structure in solution, membrane transition to the liquid-crystalline phase produced the partial
unfolding of the protein. This was observed with anionic phospholipids with different polar
headgroup and different melting transition temperature, and it was sensitive to the ionic strength.
We explored changes in surface potential as possible triggers of protein unfolding at the interface.
We measured membrane electrokinetic potential at different temperatures and we found a
correlation with protein conformation: membrane-bound, native-like protein occurred under
conditions in which lipid vesicles have low surface potential and unfolded state was observed in
membranes with higher values of surface potential. Therefore, changes in protein conformation
coupled to lipid phase transitions can result as a consequence of the modification of electrostatic
surface potential during lipid melting. We demonstrate the linkage between lipid organization,
protein conformation, strength of binding, and membrane electrostatic surface potential.
S1.4_Phospholipid modulation of membrane protein thermal stability
Santiago Martnez, Diego I. Cattoni
1,2
, Jos M Argello
3
and F. Luis Gonzlez Flecha
1
1
Laboratorio de Biofisica Molecular. IQUIFIB , Universidad de Buenos Aires-CONICET, Argentina
2
Centre de Biochimie Structurale, INSERM, Universit de Montpellier, France.
3
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, USA
Despite recent progress in understanding membrane protein folding, little is known about the
mechanisms stabilizing these proteins. Here we characterize the effects of phospholipids on the
kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu
+
-ATPase from Archaeoglobus
fulgidus. The enzyme was purified and reconstituted in mixed micelles composed by detergent
(DDM) and different phospholipids. In all the conditions CopA retained its thermophilic
characteristics with maximum activity at 75 C. Incubation of CopA in the absence of substrates at
temperatures in the 66-85 C range led to an irreversible exponential decrease in enzyme activity
suggesting a two-state process involving fully-active and inactive molecules. The lowest thermal
stability was obtained for CopA reconstituted in detergent micelles, and the highest for the enzyme
located in E coli membranes. Remarkably, the activation energy was similar for all the reconstitution
systems assayed. Transition state theory analysis of the kinetic data allowed to evaluate the
enthalpic and entropic contributions. S
#
values were similar in membranes and mixed micelles, but
higher than those obtained for CopA reconstituted in detergent micelles, whereas H
#
followed an
inverse order respect to that observed for the kinetic coefficients. These results suggest that
phospholipids promotes charge and H-bonds distributions between the native and the transition
state and increased the degrees of freedom of the protein solvent system in the transition state.
With grants from UBACyT and ANPCyT
S2: Estructura y funcin de protenas (protein structure and function)
19
S2: Estructura y funcin de protenas / protein structure and function
S2.1_Structural disorder and induced folding in the nucleoproteins and
phosphoproteins of paramyxoviruses
Longhi, S
1
, Habchi, J
1
, Blocquel, D
1
, Beltrandi, M
1
, Erales, J
1
, Dosnon, M
1
, Papageorgiou, N
1
,
Blangy, S
1
, Communi, G
2,3
, Ringkjobing-Jensen M
2
, Blackledge, M
2
, Ruigrok, RWH
3
1
AFMB, UMR 7257, CNRS and Aix-Marseille University, Marseille, France,
2
Institut de Biologie
Structurale, Grenoble, France,
3
UVHCI, Univ Grenoble Alpes-EMBL-CNRS, Grenoble, France
In the last decade there has been an increasing amount of experimental and computational
evidence pointing out that the proteome of eukaryotes and viruses is enriched in intrinsically
disordered proteins (IDPs) and/or intrinsically disordered regions (IDRs). IDPs/IDRs are ubiquitous
functional proteins that lack stable II and III structures under physiological conditions in the absence
of a partner and that rather exist as highly dynamic conformational ensembles. IDPs are often
involved in biological processes implying manifold protein-protein interactions, such as cellular
regulation, transcription and signal transduction.
In the course of the structural and functional characterization of the measles virus replicative
complex, we discovered that the nucleoprotein (N) and the phosphoprotein (P) contain long (up to
230 residues) disordered regions possessing sequence and biochemical features that typify IDPs.
More recently, by combining computational and experimental approaches, we extended these
results to the N and P proteins from the newly emerged Nipah and Hendra viruses. My talk will
focus on (i) the identification and characterization of disordered regions of the N and P proteins of
these paramyxoviruses, (ii) the assessment of their structural state in the context of the full-length N
and P proteins, (iii) the investigation of the molecular mechanisms underlying the induced folding
events triggered by binding partners. Finally, the functional implications of disorder within the
replicative complex of these viruses will be discussed.
S2.2_ Elucidating the mechanisms of action of BCL2 family proteins in apoptosis
using in vitro reconstituted systems
Landeta, O, Landajuela A, Garcia-Valero J, Bustillo, I, Flores-Romero H, Terrones, O, Basaez, G
Unidad de Biofsica, Consejo Superior de Investigaciones Cientficas - Universidad del Pas
Vasco/Euskal Herriko Unibertsitatea (CSIC-UPV/EHU), Barrio Sarriena s/n, Leioa, 48940, Spain,
During apoptosis, mitochondrial membranes undergo dramatic changes in permeability and
morphology. The principal components involved in these processes are the BCL2 family proteins,
with the assistance of an increasing number of mitochondrial protein/lipid effectors. Despite the
remarkable progress made in uncovering the molecular underpinnings of apoptotic cell death in the
last decade, the precise mechanisms by which BCL2 family proteins regulate the structure and
functioning of mitochondrial membranes remains a key and controversial issue in the field of cell
death. Given the inherent complexity of the cellular apoptotic network, we use in vitro reconstituted
systems bearing physiological relevance to try elucidating the mode of action of specific members
of the BCL2 family and/or their effectors at the membrane level, using a multidisciplinary approach
based on biophysical, biochemical, and molecular biology techniques. Here, I will explain our recent
progress in the role of apoptosis-related mitochondrial lipids on BCL2 family protein function. I will
also discuss the mechanism by which BAX and BAK form the lethal mitocondrial apoptotic pore.
S2: Estructura y funcin de protenas (protein structure and function)
20
S2.3_Equilibrium Unfolding of the PDZ Domain of b2-Syntrophin
Torchio, G
1,2
; Burgos, I
3,2
; Fidelio, G
3,2
; Arn, M
4,2
; Gallo, M
4,2
; Ermcora, M
1,2
; Sica, M
5,2
.
1
.Laboratorio de Plegado y Expresin de Protenas, Univ. Nac. de Quilmes-IMBICECIC, Argentina.
2
.CONICET, Argentina.
3
.Centro de Investigaciones de Qumica Biolgica,Univ. Nac. de Crdoba,
Argentina.
4.
Fundacin Instituto Leloir, Argentina.
5
.Laboratorio de Bioenergas. IEDS. Centro
Atmico Bariloche, Argentina.
2-syntrophin, a dystrophin-associated protein, plays a pivotal role in the insulin secretion by
pancreatic -cells. It contains a PDZ domain (2S-PDZ) that, in a complex with protein-tyrosine
phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation
state of 2-syntrophin allosterically regulates the affinity of 2S-PDZ for ICA512, and the disruption
of the complex triggers the mobilization of the insulin granule stores. We have investigated the
thermal unfolding of 2S-PDZ at different conditions and applying various techniques. Our results
indicate that, unlike other PDZ domains, 2S-PDZ is marginally stable. Thermal denaturation
experiments show broad transitions and cold denaturation, and a two-state model fit reveals a
significant unfolded fraction under physiological condition. Furthermore, Tm and Tmax denaturant-
dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest
that two-state and three-state models fail to explain properly the equilibrium data and are in better
agreement with downhill scenario. DSC and NMR experiments are also consistent with this view.
Additionally, a higher stability at pH above 9, and molecular dynamics simulations indicate that this
behavior of 2S-PDZ might be related to its charge distribution. All together, our results suggest a
link between the conformational plasticity of the native ensemble this PDZ domain and the
regulation of the insulin secretion.
S2.4_ A large scale search for protein sequence- structure -function relationship.
Bisch, PM.
Instituto de Biofsica Carlos Chagas Filho Universidade Federal do Rio de Janeiro, Brazil.
In the last years high throughput sequencing of DNA has produced an increasing amount of
biological data. Analyzing gene sequence similarities and putative homology, an inference about
biological function can be assigned to some of the new sequences. Although, it still has a lot of
sequences classified as hypothetical or unknown function. Further, it is well accepted that structures
of proteins are close related to the genes function, unfortunately experimental determination of
protein structure still be a very difficult task. So, now days, for a millions of know protein sequences
only thousands of structures are in the structure data bank. It seems that to overcome this question
a theoretical and computational effort are in order. The emerging System Biology field should
provide a means to overcame this difficult task by exploiting the concept of sequence-structure-
function relationship in a new computational high throughput approach. Fortunately, computational
means and new information technologies have also a big increase in last years. Combined with new
concepts we should deal with a large number of data and increase the knowledge about genes and
their interaction relationship. We show a few examples where we have combine the use
bioinformatics analysis of a large number of unknown sequences, construction of structural models
and molecular dynamics simulations to revel their biological functions.
Acknowledgements; CNPq, CAPES, FAPERJ, FINEP.
1 Campos ,RA et al.: Genet Mol Res. 2012. 2122.
2 Maranho, AG et al.: Infect Genet Evol.. 2012. 1397.
3 Lery, LMS et al.: BMC Genomics. 2010. S7.
S3: Transportadores y canales de membrana (Transporters and channels in membranes)
21
S3: Transportadores y canales de membrana / Transporters and channels in
membranes
S3.1_Small and Large conductance potassium channels: Where is the difference?
Diaz-Franulic, I (1)., Navarro, N.(1), Gonzlez-Nilo, F.(2), Seplveda, R.(2), and Naranjo, D:(1).
(1) Centro Interdisciplinario de Neurociencia, Universidad de Valparaso, Valparaso, Chile, (2)
Center for Bioinformatics and Integrative Biology (CBIB), Universidad Andrs Bello, Santiago, Chile.
Potassium channels are membrane proteins that allow the passage of K+ ions across the
hydrophobic core of the membrane. They display an extremely conserved signature sequence
capable of eliciting high ion transport rates with exquisite K+ selectivity among ions with similar
radii. Despite of this conservation, closely related potassium channels display differences of up to
100-fold in their single channel conductance, suggesting that the ion transport rate limiting step is
somewhere else in the pore. Because the Pro475Asp substitution -near the internal entrance
dramatically increases Shaker K+ transport rate by 7-8 fold, we suggested that such anrise could
result from higher pore occupancy. Then, to test this hypothesis, we introduced charged residues
along the pore of Shaker to fill the permeation pathway and compared their maximal single channel
conductance to that of BK channels (600pS). Fully occupied Shaker variants (as tested with
Molecular Dynamic simulations) were still far below of BK single channel conductance. A possible
explanation for this finding could be that the inner entrance dimensions limit the maximal ion
transport rate. To test this idea we estimated the radius of capture Shaker variants by measuring
the diffusion limited currents in solutions containing additional 2M of sucrose to increase viscosity.
Our result shows that Kv channels have a smaller inner entrance than large conductance K-
channels which imposes an upper limit for the maximal transport rate of K-channels.
This work was supported by FONDECYT 1120818 (DN), 1131003 (FGN), and CINV (Millenium
Initiative, 09-022-F). RS and IDF are CONICYT and MECESUP doctoral fellows, respectively.
S3.2_Achieving maximal speed of solution exchange for patch clamp experiments
in purinergic receptors
Auzmendi, JA; Moffatt L.
INQUIMAE, FCEN, UBA CONICET
Purinergic receptors are cationic channels comprised by three subunits; they form a pore with only
six transmembrane domains. The crystal structure of zP2X4 has been recently determined both in
the closed and the open state
1
and efforts are being made to study the molecular dynamics of the
coupling mechanism of binding and gating. In this context, the ability to obtain kinetic information of
high quality would be inestimable to experimentally ground the possible molecular mechanisms. As
this coupling occurs in the tens to hundreds of microseconds, we focus our latest efforts in the
development of the experimental ability to expose the patch clamp preparation to the agonist during
shorter and shorter periods of time. We developed the ability of applying pulses of 25 microseconds
measured at the open tip of the patch pipette
2
. In this way, the brief intermediate states that occur
between the binding of the agonist and the opening of the pore would be accessible to experimental
study not only for purinergic but also for fast open channels like AMPA and Ach receptors.
This study has been funded by the ANPCyT (PICT 06 1902) and UBACyT (20020100100636)
1 Hattoriet al.Nature 2012: 207
2 Auzmendi et al. PLoS ONE 2012: e42275
S3: Transportadores y canales de membrana (Transporters and channels in membranes)
22
S3.3_Cationic Amino Acid Transporters: insights from a non-transportable
enantiomer
Peluffo, R. D.
Universidad de la Repblica, Regional Norte, Salto, Uruguay.
Cationic amino acid transporters are highly selective for L-enantiomers such as L-arginine (L-Arg).
Because of this stereoselectivity, little is known about the interaction of these transporters with D-
isomers. To study whether these compounds provide information on the molecular mechanism of
transport, inward currents activated by L-Arg with low apparent affinity were measured in whole-cell
voltage-clamped cardiomyocytes as a function of extracellular L-Arg and D-Arg concentrations. D-
Arg inhibited L-Arg currents in a membrane potential (V
M
)-dependent competitive manner, indicating
the presence of D-Arg binding sites in the carrier. Accordingly, D-Arg-dependent charge movements
were also detected in these cells. Analysis of steady-state currents showed that L- and D-Arg
binding reactions dissipate a similar small fraction of the membrane electric field. Since D-Arg is not
transported, these results suggest that enantiomer recognition occurs at conformational transitions
that prepare amino acid translocation. Simulations of the V
M
dependence of maximal current levels
with a four-state alternating model suggest that inward currents arise from the outward movement of
a negative charge in the unliganded transporter. Translocation of the L-Arg-bound complex, on the
other hand, appears to be an electroneutral process. To our knowledge, this study provides first
quantitative data on electrogenic reactions that accompany low-affinity L-Arg transport.
This work was supported by Award Number R01HL076392 from the National Heart, Lung, and
Blood Institute (R.D.P.).
S3.4_ Flexibility in the ion transport pathway of P-type ATPases?
Berlin, J.R.
Dept. of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark,
New Jersey, USA
P-type ATPases are a large family of enzymes responsible for the active transport of ions and
phospholipids across cell membranes. In this family of enzymes, biochemical, mutagenesis and
structural data for the sacroplasmic/endoplasmic reticulum Ca
2+
-ATPase (SERCA) have led to
detailed mechanistic proposals for how biochemical reactions, ATP binding, enzyme
phosphorylation, and P
i
hydrolysis drive vectorial transport of Ca
2+
across the membrane domain of
this enzyme. There seems little doubt that these biochemical reactions are highly conserved
across the P-type ATPase family. However, less data exist as to whether the mechanism of
vectorial transport is also highly conserved. In order to address this question, we have begun to
study a plant P-type H
+
-ATPase from Arabidopsis thaliana, AHA2. The question that we are
investigating is whether, during H
+
transport, conformational changes of the alpha helices located in
the membrane domain of AHA2 could be consistent with those postulated to occur during Ca
2+
transport by SERCA. Cysteine scanning mutagenesis experiments were performed with AHA2
expressed in Saccharomyces cerevisiae. Accessibility of amino acids substituted with cysteine to
extracellular thiol-reactive reagents was tested and H
+
transport rate was measured. Accessibility
of residues in the first transmembrane alpha helix did change with different AHA2 turnover rates;
however, all together, the data suggested that vectorial H
+
transport by AHA2 could follow a
different ion transport pathway than has been postulated for Ca
2+
in SERCA or for Na
+
in the Na,K-
ATPase. These results lead us to postulate an alternative transport pathway for H
+
through the
membrane domain of AHA2.
S4: Modelado molecular (Biomolecular modeling)
23
S4: Modelado molecular / Biomolecular modeling
S4.1_Strategies for the de novo design of protein-protein interactions
Nir London
a,b,
Xavier Ambroggio
a
a
Rosetta Design Group LLC, Burlington, Vermont, USA
b
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA
The great chemical diversity of amino acid side-chain functional groups coupled to the flexibility of
protein backbones makes the computational design of proteins for specific functions a challenging
feat. Early successes in computational design tackled the problem of designing amino acid
sequences that would adopt a specified target conformation, also known as the inverse protein
folding problem, by employing algorithms to sample side-chain conformations and model, with
sufficient accuracy, the physiochemical forces of a folded protein. These successes laid the
groundwork for general computational design algorithms and methodology, however, in order to de
novo design in complex functions, such as protein-protein interactions or protein-ligand interactions,
new strategies have emerged. The strategy of using well-defined models for the desired binding
interaction modes and the development of new methodologies for incorporating those models into
the context of nave scaffolds has led to many recent successes in the de novo design of protein-
protein interactions. We present here an overview of these exciting studies and the strategies they
employed along with some of our experiences in the design of protein-protein interactions and
supramolecular assemblies.
S4.2_ Botulinum neurotoxins and SNARE complexes: A new structural view from
modeling and simulations.
Sergio Pantano
Institut Pasteur de Montevideo, Uruguay. spantano@pasteur.edu.uy
The very high affinity and specificity of Botulinum neurotoxins for SNARE proteins lead to the
paralysis of neuromuscular junctions; making these toxins attractive for therapeutics, cosmetics and
even bioterrorism. However, the molecular details of the neurotransmitter release apparatus remain
still elusive.
Comparison of the mode of actions between different Botulin serotypes suggests that multiple
SNARE complexes associate on a radial super complex. Modelling and simulations are used to
derive 3D information of this nanomachine and identify protein-protein contacts within this
quaternary arrangement. The role in neuroexocytosis of amino acids at the putative inter SNARE
surface is confirmed by electrophysiology measurements on neuromuscular junctions of transgenic
flies, providing support for a radial arrangement of SNARE complexes and furnishing novel insights
on the self-assembly and regulation of biomolecular nanomachines.
- Pantano S and Montecucco C. The Blockade of the Neurotransmitter Release Apparatus by
Botulinum Neurotoxins. Cell. Mol. Life Sci. 2013, DOI:10.1007/s00018-013-1380-7.
- Megighian A, et al. Evidence for a radial SNARE super-complex mediating neurotransmitter
release at the Drosophila neuromuscular junction. J. Cell. Sci., 2013, 136: 3134.
- Megighian A, et al. Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila
melanogaster neuromuscular junction. J Cell Sci. 2010, 123:3276.
- Montecucco C, Schiavo G, Pantano S. SNARE Complexes and Neuroexocytosis: How Many, How
Close? Trends Biochem. Sci. 2005, 30:367.
S4: Modelado molecular (Biomolecular modeling)
24
S4.3_Predictive Biomolecular Modeling Applied to Protein Engineering and
Proteomics
Isabelle F. T. Viana
1,2
, Ranieri V. Carvalho
3
and Roberto D. Lins
1
1
Department of Fundamental Chemistry, Federal University of Pernambuco, Recife, PE, 50740-560,
Brazil;
2
Department of Infectious Diseases and Microbiology, University of Pittsburg, 9022 BST3,
Pittsburgh, PA, 15261, USA;
3
Center of Informatics, Federal University of Pernambuco, Recife, PE,
50740-560, Brazil
Atomic-scale biomolecular modeling is predominantly directed towards finding properties of a
specific system. This presentation will focus on the use of computational biophysics techniques to
address issues in a global and predictive manner. Such approach will be showcased by studies
capable of i. predicting the primary sequence of peptides based on calculated ion mobility mass
spectrometry data; and, ii. de novo design of gp41-based conformation-specific HIV-1 epitopes
grafted onto highly-stable scaffolds aimed to point-of-care diagnostic kits and vaccines.
Keywords: molecular dynamics, protein engineering, ion mobility spectrometry
This work is supported by FACEPE, CNPq, NanoBiotec-BR/CAPES and nBioNet and STINT.
Computer allocation was provided by the Environmental Molecular Sciences Laboratory located at
the Pacific Northwest National Laboratory and Argonne National Laboratory.
S4.4_Study of Frataxin folding
Romn E.A.
IQUIFIB-Buenos Aires, Argentina.
Frataxin is globular protein that appears in all the three biological kingdoms and is related to the
iron intracellular homeostasis. In humans, the lack of this protein yields Friedreich's Ataxia. This
pathology is associated to the cellular redox equillibrium and ATP synthesis. The absence of
functional frataxin results in an increase in the free radical content and also problems in iron
delivery to other protein targets.
In our laboratory, we are interested in the study of the mechanisms of frataxin iron binding, and in
the relation between the stability and functionality of this protein. In this sense, we faced ligand
binding studies, and the stability and folding mechanism analysis and study of this variant.
Previous experimental results suggest that the carboxi-terminal region of human frataxin could be
participating as a limitant step in the folding process of the human variant. Moreover, other
laboratory studies showed that this region is closely related to the global stability of this protein.
In this talk, we will introduce the computational simulation results where we studied by coarse
grained techniques the folding process of human frataxin. From these experiments we obtained
information on the global stability of this variant which could be related to its structural topology.
Also, we inferred the presence of, at least, one folding intermediate that could be related to
structural and energetically to destabilized variants that appear in patients with Friedreich's Ataxia.
The analysis of these results and experimental folding kinetics determinations would make us able
to perform a detailed characterization of this folding process.
S5: Difraccin de rayos X y SAXS en bioestructuras (X-Ray difraction and SAXS to study biostructures)
25
S5: Difraccin de rayos X y SAXS en bioestructuras / X-Ray difraction and SAXS to
study biostructures
S5.1_Structural studies on a two-component system activated by blue light in
Brucella abortus
Klinke S., Rinaldi J. J., Sycz G., Paris G. & Goldbaum F. A.
Fundacin Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435 (C1405BWE), Ciudad de
Buenos Aires, Argentina. E-mail: sklinke@leloir.org.ar
Brucella abortus is an intracellular pathogen that causes a worldwide zoonosis called brucellosis,
an endemic disease that causes abortion and infertility in cattle with consequent huge economic
losses. One of the projects in our lab focuses on the study of a particular two-component signal
transduction system (TCS) in Brucella that is activated by blue light and was shown to be a key
virulence factor [1]. This TCS is composed by (i) the sensor histidine kinase LOV-PAS-HK, which is
a three-domain protein able to sense blue light through a bound FMN molecule, and (ii) two cognate
response regulators called PhyR and CheY.
In this talk, we will present our latest results regarding the structural description of this system using
protein X-ray crystallography. Explicitly, we were able to solve the crystal structure of the isolated
LOV [2] and HK domains in the sensor histidine kinase, as well as the structure of the response
regulator PhyR.We will also describe our present strategies for the resolution of multi-domain
constructs and complex structures. To finish, we will show the technical aspects of synchrotron
radiation application for fast diffraction data collection and automated structure solving of the
proteins described here, according to our experience at the SOLEIL synchrotron in France.
Overall, the structural information on this TCS, complemented with biochemical studies that are
being performed in our lab, correspond to an excellent starting point for the understanding of the
signal transduction effect between the different domainsin LOV-PAS-HK and the general activation
of histidine kinases.
Acknowledgements: CONICET and MINCyT (funding). SOLEIL and Institut Pasteur Montevideo
(access to X-ray data collection) [1] Swartz, T.E. et al. (2007) Science317, 1090-1093. [2] Rinaldi,
J.J. et al. (2012) J. Mol. Biol.420, 112-127.
S5.2_Small Angle X ray Scattering to study liposomes for gene therapy
Balbino, T.
1
, Gasperini, A.
2
, Oliveira, C.
3
, Azzoni, A.
3
, Cavalcanti, L.
2
, de La Torre, L.
1
1
Univ. Campinas,
2
Brazilian Synchrotron Light Lab (LNLS),
3
Univ. So Paulo, BRAZIL
In this talk we will present a characterization study of complexes formed by cationic liposomes (CL)
and pDNA with main application in gene delivery systems. We found that conventional physico-
chemical properties were nearly unaffected at the studied ranges of molar charge ratio between
pDNA and CL, for which the results from in vitro transfection showed significant differences. We
then used small angle x-ray scattering (SAXS) to determine the lipoplex structural modifications
trying to comprehend the transfection properties. The SAXS results revealed that pDNA/CL
complexes can be described as being composed of single bilayers, double bilayers and multiple
bilayers, depending on the charge balance between pDNA and CL
1
. These results were used to
explain the observed transfection differences and allowed proper correlation of the physico-
chemical and structural properties of pDNA/CL complexes with the in vitro transfection, contributing
to a better understanding of the gene delivery process.
Acknowledgements: The SAXS experiments were made at LNLS. The authors TB and AG are
granted by FAPESP agency. 1 Balbino et al, Langmuir (2012) 28:11535
S5: Difraccin de rayos X y SAXS en bioestructuras (X-Ray difraction and SAXS to study biostructures)
26
S5.3_Structure and function of ICA2, a receptor involved in insulin secretion
Ermcora Mario R.
Structural Biology and Biotechnology Group, Imbice, UNQ-Conicet
ICA512 is a type 1 membrane protein located in pancreaticcell, insulinsecretory granules and in
electrodense granules of other neuroendocrine cells. One of its intracellular domains is a tyrosine
phosphatase (PTP) and the protein as a whole is a receptor PTP (RPTP).
ICA512 was discovered in the '90, as an autoantigen associated to autoimmune diabetes, and it
has since been utilized for the precocious diagnosis of the disease. Latter, it became notorious
because of the discovery of its involvement and multiple roles in the process of insulin secretion:
ICA512 modulates the mobility of secretory granules, as well as the expression of insulin and other
genes related to the granular integrity and cell proliferation.
Beside its role in the endocrine pancreas, ICA512 participates in the secretion of pituitary hormones
and its deficiency causes infertility. Today, the receptor and interacting proteins are considered
promising targets for the development of new medicines and as attractive subjects of basic and
medical investigations.
The mature ectodomain of ICA512 (MPEICA512) may be involved in oligomerization process and
cell signalling. Our laboratory solved the structure of MPEICA512 by Xray crystallography under
different conditions relevant for the granulogenesis process. The structural information, along with
evidence obtained in experiments in vivo established the basis for the preparation of 3D model of
the entire receptor. In the presentation, the progress in the preparation of such model will be
discussed.
S5.4_ Synchrotron radiation experiments on the biomineralization of ferritin
Ceoln, M.
Instituto de Investigaciones fsico-Qumicas Tericas y Aplicadas (INIFTA, UNLP-CONICET).
Diagonal 113 y 64 (1900) La Plata
Transmission Electron Microscopy (TEM), X-ray Absorption Near Edge Spectroscopy (XANES),
Electron Energy-Loss Spectroscopy (EELS), Small-Angle X-ray Scattering (SAXS), and SQUID
magnetic studies were performed in a batch of horse spleen ferritins from which iron had been
gradually removed, yielding samples containing 2200, 1200, 500, and 200 iron atoms. Taken
together, findings obtained demonstrate that the ferritin iron core consists of a polyphasic structure
(ferrihydrite, magnetite, hematite) and that the proportion of phases is modified by iron removal.
Thus, the relative amount of magnetite in ferritin containing 2200 to 200 iron atoms rose steadily
from 20% to 70% whereas the percentage of ferrihydrite fell from 60% to 20%. These results
indicate a ferrihydrite-magnetite core-shell structure. It was also found that the magnetite in the
ferritin iron core is not a source of free toxic ferrous iron, as previously believed. Therefore, the
presence of magnetite in the ferritin cores of patients with Alzheimers disease is not a cause of
their increased brain iron(II) concentration.
The author is in debt to CONICET (Argentina) and LNLS (Brazil). The participation of several co-
authors as part of the project (Dr. J:M.Dominguez-Vera and his crew) is also deeply acknowledged.
1 N.Galvez, B.Fernandez, P.Sanchez, R.Cuesta, M.Ceolin, M.C.Leon, S.Trasobares, M.Lopez-
Haro, J.Calvino, O.Stephan and J.M.Dominguez-Vera. J.American Chemical Society (2008) 130
8062.
SAB 2013
Resmenes de posters/Poster abstracts
Biofsica de lpidos y membranas / Biophysics of lipid and membranes (BLM)
Biofsica de protenas y cidos nuclicos / Biophysics of proteins and nucleic acids (BPA)
Enzimologa/ Enzymology (ENZ)
Bioenergtica y transferencia electrnica / Bioenergetics and transfer (BTE)
Teora y modelado de sistemas biolgicos / Theory and modeling of biological systems (TMSB)
Transportadores, receptores y canales / Transporters, receptors and channels (TRC)
Sealizacin y Dinmica Intracelular / Signaling and Intracellular dynamics (SDI)
Nuevas tcnicas en biofsica / New techniques in biophysics (NTB)
Biofsica: Aplicaciones biotecnolgicas / Biophysics: Biotechnological applications (BBA)
SAB 2013
SAB 2013 Biofsica de lpidos y membranas (Biophysics of lipid and membranes)
29
BLM1_Mechanical properties of membranes from spheroplast using optical tweezers.
Colque, A., Gonzalez Montoro, M.A., Valdez-Taubas, J. y Wilke, N.
Instituto de Qumica Biolgica de Crdoba (CIQUIBIC-CONICET), Departamento de Qumica Biolgica,
Facultad de Ciencias Qumicas, Universidad nacional de Crdoba.
Laser tweezers and optical microscopy have been used for the determination of the rheological properties
of model biomembranes
1
and of membranes of mammal cells
2
. Here, we used the technique to explore
the deformability of membranes of spheroplast of yeast Saccharomyces cerevisiae. It was reported that
the shear viscosity of this membrane is very different from mammal cells but the out-of-plane
deformability has not been studied yet. The wall of Saccharomyces cerevisiae cells was digested with
zymoliase and 200 L of a suspension of spheroplasts were placed on a glass cover-slide previously
treated with piranha solution for 2 h and poly-lysine in bufferborate overnight. A 10L-drop of an aqueous
solution of micro-spheres (anionic, 3 m diameter or cationic, 1 m diameter) was added to the
spheroplasts. After an hour, some beads were attached to the spheroplast surface, permitting to catch the
bead with the optical trap and move it apart from the spheroplast. This movement generated a membrane
nanotube between the cell and the bead, which allowed to test the membrane deformability, by tracking
the bead position relative to the spheroplast in time after the trap was turned off. The nanotube relaxation
process suggested that the spheroplast membrane was more elastic than the membrane of mammal
cells.
Acknowledgements: Sistema Nacional de Microscopa, SeCyT-UNC, FONCyT, CONICET.
1- Sorre Betal.PNAS. 2012. 173-178. 2- Pascoal P.et al. Lab Chip. 2010. 2235-2241.3- Valdez-Taubas J.
et al. Curr. Biol2003 1636-1640.
BLM2_Distribution of Salmon Calcitonin in Phospholipid Membranes. A Thermodynamic
study with models of Langmuir-Blodgett (LB-films) and Atomic Force Microscopy (AFM)
Meneses K.
,1
, Giordani C.
,,2
, Giraldo M.
,3
Grupo de Investigacin Biofsica (GIBF), Instituto de Fsica, Universidad de Antioquia, Medelln,
Antioquia, Colombia.
Departimento de Tecnologie e Salute, Istituto Superiore di Sanit, Viale Regina Elena 299, 00161 Roma,
Italy.
1
kayumesi@fisica.udea.edu.co,
2
cristiano@fisica.udea.edu.co,
3
m.a.giraldo@fisica.udea.edu.co
Nowadays over 30 diseases for which no cure is available (e.g. Alzheimer`s and Parkinson`s diseases)
are associated with amyloid-forming proteins. Up-to-date, research activities suggest that amyloid
oligomers may be the toxic species. Although the target has not been yet identified, the oligomer
properties suggest that neurons may be annihilated by unregulated permeabilization of the membrane,
possibly through the formation of a pore amyloid. The studies of Schubert and coworkers showed that
Calcitonin, in analogy with other amyloid proteins, show an aggregative behavior that is toxic to cultured
cells. These observations as well as the slow aggregation rate of the calcitonin, suggest the usage of
salmon calcitonin, sCT, as a "probe" to study the formation of amyloid neurotoxicity. This work aims to
study the molecular mechanism of the interaction of sCT with lipid membrane models which mimic the so-
called lipid rafts through the use of thermodynamic techniques (via the Langmuir-Blodgett trough or also
known as LB-trough) and atomic force microscopy (AFM).
BLM SAB 2013
30
BLM3_Studies of the Interaction of a Galleria mellonella Peptide with Molecular Models of
Gram-Negative Bacteria Membranes
Villanueva G.
1
, Correa W.
1
, Oate J.
1
, Patio E
1
., Espejo. F.
2
, Brandenburg K.
3
Manrique-Moreno M
1
.
1
Instituto de Qumica, Universidad de Antioquia, Medelln, Colombia.
e-mail: mmanrique@exactas.udea.edu.co, mmanriqm@hotmail.com
2
Escuela de Qumica, Facultad de Ciencias, Universidad Nacional, Medelln, Colombia
3
Forschungszentrum Borstel, LG Biophysik, parkalle 10, D-23845 Borstel Germany
Antimicrobial peptides are important components of the immune system of several living organisms, and
they are considered promising alternatives to conventional antibiotics [1]. The aim of this study was to
evaluate the interactions of a G. mellonella peptide with bacterial membranes. Previously we studied the
antimicrobial activity of a synthetic G. mellonella peptide against S. typhimurium and E. coli. Based on
these results we carried out peptide-membrane studies using liposomes of the main lipid components of
Gram-negative bacteria membranes (lipopolysaccharides, dimyristoylphosphatidylglycerol and
dimyristoylphosphatidylethanolamine) [2]. Experiments were performed by Fourier-transform infrared
spectroscopy and Fster resonance energy transfer spectroscopy. Results showed that the peptide
affected the liposome stabilities and, therefore the bacterial membranes. Our lab is presently focused on
the design and synthesis of potential antimicrobial peptides based on the G. mellonella amino acid
sequence in order to improve their biological activity.
[1] M. Pushpanathan, P. Gunasekaran, J. Rajendhran. International Journal of Peptides, 2013, 1.
[2] J.M. Sanderson. Organic and Biomolecular Chemistry, 2005, 201.
BLM4_Effect of the presence of domains on the stiffness of lipid monolayers.
Wilke N., Caruso B., Mangiarotti A.
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC), Departamento de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Pabelln Argentina, Ciudad
Universitaria. X5000HUA Crdoba.
The compressibility of monolayers composed of two lipids with low lateral miscibility was determined with
the aim of investigating the effect of the mechanical properties of each phase on the stiffness of the
composite. In nine different systems, the stiffness of each phase and the texture at the plane of the
monolayer were studied and the results are discussed in the light of the following two hypotheses: a. The
stiffness of the composite is a weighted sum of the stiffness of each phase, regardless of the distribution
of the phases in the plane of the monolayer; b. The stiffness of the composite is dominated by the
mechanical properties of the continuous phase. Our results were better explained by this latter proposal,
as in all the analyzed mixtures it was found that the mechanical properties of the percolating phase were
the determining factors
1
.
On the other hand, the effect of the presence of domains in the dilute regime did not follow a common
trend. This different behavior did not correlate with different domaindomain interactions or differences in
the shape/size of the domains between the systems. We hypothesize that the differences are a
consequence of the cooperativity of the percolation process in each monolayer.
Acknowledgements: SeCyT-UNC, FONCyT, CONICET.
1- Caruso B., Mangiarotti A., Wilke N. Langmuir 2013, 29, 10807-10816.
SAB 2013 BLM
31
BLM5_Interaction of magnetic nanoparticles with phospholipids in Langmuir monolayers
Menzaque, A.
a
, Lanterna, A.
a
, Maggio, B
b
., Granados, A.
a
, Krause, R.
c
, Vico, R.
a
a
INFIQC-CONICET, Departamento de Qumica Orgnica,
b
CIQUIBIC-CONICET, Departamento de
Qumica Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba.
c
Medicinal
Organic and Nanomaterials Chemistry, Department of Chemistry, Rhodes University.
A growing number of innovations have emerged in the fields of nanobiotechnology/nanomedicine and
new engineered nanomaterials are posing new challenges to understand the full spectrum of interactions
at the nanobio interface. Among them, magnetic nanoparticles (MNP) are greatly promising because of
their potential application in fields such us drug delivery, magnetic resonance imaging (MRI), heat source
in magnetic fluid hyperthermia.
1
We are studying the interaction of MNPs with phospholipids in Langmuir monolayers with the aim of
identify the influence of nanoparticle surface functionalization in their biomolecular properties. Here we
present the results of the interaction of MNPs covered with Oleic Acid (MNP@OA) and Stearic Acid
(MNP@SA) with dipalmitoylphosphatidylcholine (DPPC). The presence of either MNP cause phase
condensation of DPPC with respect to the pure phospholipid and significantly modify the surface
topography of DPPC (observed by BAM) along the whole isotherm, even if the MNP constitute only a
small mole fraction of the mixture.
1
Mout, R., Moyano, D., Rana, S., Rotello, V. Chem. Soc. Rev. 2012, 2539.
BLM6_Phase coexistence in films composed of DLPC and DPPC: a comparison between
different model membrane systems.
Mangiarotti A., Caruso B., Wilke N.
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC), Departamento de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Pabelln Argentina, Ciudad
Universitaria. X5000HUA Crdoba.
For the biophysical studies of membranes, a variety of model systems are used to measure different
parameters and to extract general principles of processes that occur in cellular membranes. However,
there are very few reports in which the results obtained with the different models are compared. In this
work, we quantitatively compared the phase coexistence in Langmuir monolayers, freestanding bilayers
and supported films composed of a lipid mixture of DLPC and DPPC. Two-phase segregation was
observed in most of the systems for a wide range of lipid proportions using fluorescent microscopy. The
lipid composition of the coexisting phases was determined and the distribution coefficient of the
fluorescent probe in each phase was quantified, in order to explore their thermodynamic properties. The
comparison between systems was carried out at 30 mN/m since it is accepted that at this or higher lateral
pressures, the mean molecular area in bilayers is equivalent to that observed in monolayers.
Our study showed that Langmuir monolayers and GUVs have a similar phase behavior. On the other
hand, supported films showed a different composition of the phases and the distribution coefficient of the
fluorescent probe was close to one. Our results suggest that in supported membranes, the presence of
the rigid substrate could be leading to a stiffening of the liquid-expanded phase due to a loss in the
degrees of freedom of the lipids when interact with the supporting material and this impacts on the
properties of the coexisting phases.
Acknowledgements: SeCyT-UNC, FONCyT, CONICET.
BLM SAB 2013
32
BLM7_Study of hemorheological properties of hypercholesterolemic rats treated with
proantocianidine extract of Ligaria cuneifolia (Lc)
Garca G
1
, Crosetti D
1
, Dominighini A
1
, Urli L
1
, Galliano S
1
, Gonzalvez J
1
, Monti J
2
, Ronco MT
2
, Wagner
M
3
, Carnovale CE
2
, Luquita A
1
1
Biofsica, Fac. Cs. Mdicas, UNR,
2
Fisiologa, Fac. Cs. Bioq. y Farm.-IFISE-UNR-CONICET,
3
Farmacobotnica, Fac. Farm. y Bioq.-UBA
Lc or creole mistletoe is an Argentine semiparasitic plant. We have previously demonstrated that crude
Lc extract decreases plasma Cholesterol (Cho), increases blood viscosity and reduces erythrocyte
deformability. Objective: to analyze the effect of treatment with an enriched fraction in proanthocyanidine
(PLc) on Cho plasma concentration and erythrocyte rheological properties in hypercholesterolemic rats.
Adult male Wistar rats (70 days), care according International Rules, were fed 28 days with Standard diet
plus Cho (97% purity) 0.8g/100g of diet (28% corn oil wt/wt diet). Control group (C) n=12 received ip
saline. Experimental group (E) n=12 received ip PLc 3mg/100g BWt every 24h, during 10 days. On day
11 the rats were anesthetized (Na-pentobarbital 50 mg/kg BWt) to obtain blood samples by cardiac
puncture. Results: (mean SE). Plasma Cho (enzymatic method)(mg%): C: 119.741.26, E: 68.501.86*,
HDLCho: C: 28.800.62, E:23.170.86*; LDLCho: C.25.300.84, E:18.160.59*. Blood: Morphological
Index (light microscopy): C:-2.350.48, E:-1.98 0.29*; SIII(%): C:40.1214.23, E:27.2611.74**; Rigidity
Index (nucleopore membrane filter): C:8.010.96; E:6.300.92**; Membrane erytrocyte Cho (mg%):
C:1.050.15, E: 0.770.23*.(**p<0.001 and *p<0.05 vs. C). Conclusion: PLc treatment diminishes plasma
Cho, HDLCho and LDLCho. Decrease of Cho from erythrocyte membrane leads to a morphological
change from discocyte to stomatocyte III. In agreement with other reports the morphological change
could point out to an interaction between PLc and the inner layer of the erythrocyte lipid bilayer
membrane altering its curvature; this could explain the erythrocyte deformability increasement i.e.,
Rigidity Index decreases in hypercholesterolemic rats.
BLM8_Molecular amphipathicity impacts on peptide surface behaviour
Ernesto Ambroggio and Gerardo Fidelio
CIQUIBIC, CONICET, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Ciudad
Universitaria, X5000HUA-Crdoba, Argentina.)
Protein primary structure has all the information for a specific protein/peptide folding. This sequence can
define specific amphiphilic regions in the molecules important for the interaction with interfaces. In order
to shed light how amphipathicity has a role in the surface properties of proteins, we designed three pairs
of peptides where all six type of molecules have the same hydrophobic residues but different hydrophilic
amino acids: positively, negatively and non-charged. The sequence of the peptides is inverted in
comparison to their pair. This inversion provokes that one kind of peptide has the N-terminal region
hydrophilic and the C-terminus hydrophobic and the other kind of peptide, with the same type of amino
acids, the opposite distribution. We evaluated how amphiphilicity has a role in peptide lateral stability at
the air-water interface by using the Langmuir monolayer technique. We also performed peptide/peptide
mixtures to understand whether there is a coupling within opposed charges and if amphipathicity may
contribute to the global lateral stability of the peptide film. In addition we measured membrane
destabilization (leakage of a fluorescent solution trapped inside liposomes) and membrane association of
these molecules. Finally we analyzed the peptide secondary structure by ATR-FTIR. Our results show a
distinctive surface behaviour for each kind of molecule whereas all presented the same secondary
structure.
SAB 2013 BLM
33
BLM9_Role of phase coexistence and composition of ternary lipid dispersion containing
cholesterol and ceramide on spontaneous curvature and structural stability.
Giudice F., Maggio B., Fanani M.L.,
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC), Departamento de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba, Haya de la Torre y Medina
Allende, Ciudad Universitaria, X5000HUA, Crdoba, Argentina.
Lipid dispersion of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Cholesterol (Chol) and
Palmitoyl ceramide (P-Cer) in a wide range of relative composition were prepared by the traditional
method of extrusion and compared with lipid dispersions formed by the Rapid Solvent Exchange
method (RSE) [1], which allows lipid vesicles to adopt its spontaneous curvature, without external
influences such as filtration or sonication. Thus, spontaneous curvatures of the ternary dispersions were
evaluated and related to the different phase states characterized in these mixtures [2]. The data shows
that, as we move from a fluid phase to the fluid and gel phases coexistence regions, the vesicles showed
a higher polydispersity in diameter, which appears to depend on the composition of the fluid phase that, in
turn, depends on the relative cholesterol/ceramide ratio.
This work was supported by: FONCyT, CONICET and SECyT-UNC
1- J. T.Buboltz, G. W.Feigenson. Biochi.Biophys. Acta 1417 (1999) 232.
2- B. M. Castro, L. C. Silva, A. Fedorov, R.F.M. de Almeida, and M. Prieto. J.Biol.Chem. Vol 284,NO.34
pp.22978.
BLM10_A simple model to predict the number of unilamellar liposomes in a suspension
Montanari, J, and Alonso, S. del V.
Laboratorio de Biomembranas, GBEyB (IMBICE-CONICET), Universidad Nacional de Quilmes, Roque
Saenz Pea 352, (1876) Bernal, Buenos Aires, Argentina. E-mail: jmontanari@unq.edu.ar
In particulate systems such as liposomes, concentration units are not enough to describe the drug
distribution in suspensions, as they are not homogeneous. In certain in vitro assays, exposure to different
number of particles (containing the same amounts of active in the same global volume) introduces an
extra variable regarding to contact phenomena. Volume and area of liposomes decrease upon extrusion,
while their quantity grows, as new vesicles are formed. Aiming to a rapid estimation of the number of
unilamellar liposomes present in a suspension, a mathematical method was developed, based on the
area and molecular weight of lipids and the mean size of the liposomes. Unilamellar liposomes loaded
with a probe were prepared by extrusion and counted by fluorescence microscopy and Tunable Resistive
Pulse Sensing (Q-Nano). Size was determined by Dynamic Light Scattering. There was about a 90%
coincidence between the theoretical results and the number of unilamellar liposomes counted for two
liposomes populations of different mean size. This model could be a useful complement for interpretation
of in vitro experiments in which results could depend on the distribution of actives into different quantities
of liposomes.
BLM SAB 2013
34
BLM11_A new method for the accurate determination of refractive index on Langmuir
Monolayers by Reflectometry
Pusterla, J.M. and Oliveira, R.G
CIQUIBIC (CONICET) and Departamento de Qumica Biolgica, Facultad de Ciencias Qumicas,
Universidad Nacional de Crdoba, Haya de la Torre y Medina Allende, X5000HUA- Crdoba, Argentina.
Monolayers at the air/water interface have been widely used as biomembrane models under controlled
intermolecular organization (lateral molecular area). Surface pressure, surface potential, reflectivity (R)
and other magnitudes can be precisely determined on these planar monomolecular films. However, some
physical parameters such as the refractive index (n
m
) still remain elusive. This is important because its
precise quantification allows for the determination of the thickness of the film by R, for instance on a
Brewster Angle Microscope (BAM)
1
. The uncertainties of n
m
determine important errors in the calculation
of monolayers thickness that propagates non-linearly.
Here we present an analytical method for the experimental determination of n
m
in monolayers based on
the principle of refractive index matching as used in bulk suspensions of liposomes
2
. By using a BAM
setup and monolayers spread over subphases with different and known refractive index (n
s
), a minimum
in R is search as a function of n
s
. In these conditions, the n
m
is equal to the known n
s
. The results shown
correspond to monolayers of isolated bovine myelin and two lipids with n
m
previously reported, DMPC and
cerebrosides. The n
m
values remain approximately constant, slightly increasing in a range of 0.005 to
0.01 upon compression. The obtained values allows for determination of thickness.
Acknowledgements: We thank funding from CONICET, SECyT-UNC and FONCYT.
1- Hnon, Sylvie. Review of Scientific Instruments. 1991. 936.
2- Ardhammar, Malin. PNAS. 2002. 15313.
BLM12_Interfacial behavior of a novel non-ionic azobenzene amphiphile. Towards a new
membrane photoswitch
Benedini, L
a
; Sequeira; M. A.
a
; Fanani;L.
b
, Maggio;B
b
; Dodero, V.I
a
a. INQUISUR-CONICET, Baha Blanca, Buenos Aires.b. CIQUIBIC-CONICET, Crdoba, Crdoba.
The comprehension of the stimulus-response behavior in biological is a great challenge [1]. In this
context, azobenzenes amphiphiles are versatile compounds which could be used as molecular switches
triggered by light [2]. We have obtained a new non-ionic amphiphile with photomodulated capacities that
allow using it as a membrane photoswitch. The film properties at different temperatures of the pure
derivative in both conformations were evaluated in Langmuir films at the air-water interface. The
capability of interaction and modification of a membrane model was evaluated by polarized optical
microscopy, electron microscopy and differential scanning calorimetry. In Gibbs monolayers, penetration
experiments showed that both isomers were able to interact differentially with the lipid monolayer. The (Z)
isomers showed the highest surface pressure cut off probably related to the increased the polarity of the
molecule.
Acknowledgements: Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1. Alberts, B. ; Bray, D. ; Lewis, J. ; Raff, M. ; Roberts, K. ; Watson, J.D. Molecular Biology of the cell,
5rd ed, Garland Publishing: New York, 2008.
2. Sebai, S. C.; Cribier, S.; Karimi, A.; Massotte, D.; Tribet, C. Langmuir. 2010. 14135
SAB 2013 BLM
35
BLM13_Surface behaviour of sphingomyelines with very long chains (C28-C32) PUFAs
and their interaction with premixed or enzymatically generated ceramides
Pealva DA
1
, Wilke N
2
, Maggio B
2
, Aveldao MI
1
, Fanani ML
2
1
INIBIBB, CONICET-UNS, Baha Blanca,
2
CIQUIBIC, UNC-CONICET, Crdoba, Argentina.
E-mail: lfanani@fcq.unc.edu.ar
Molecular species of sphingomyelin (SM) with nonhydroxy (n) and 2-hydroxy (h) very long-chain
polyunsaturated fatty acids (n- and h- 28:4, 30:5 and 32:5) abound in rat spermatogenic cells and
spermatozoa. These SMs are exclusively located on the sperm head, where they are converted into the
corresponding Cer by sphingomyelinase after completion of the acrosomal reaction. The aim of this study
was to gain some insight into the surface properties of this unique type of sphingolipids and how such
properties change by the SM Cer conversion. SM and Cer species were isolated by HPLC [1] and
organized in Langmuir films, alone and in SM/Cer mixtures. Compression isotherms for all six SMs under
study were compatible with a liquid-expanded state and showed large mean molecular areas. Only the
longest SMs (n- and h-32:5 SM) underwent phase transition upon cooling. h-28:4 SM show typical
general properties whereas h-28:4 Cer exhibited an easily compressible liquid condensed phase [2]
which may results from its higher conformational freedom in such phase. In premixed and enzymatically
generated h-28:4 SM / h-28:4 Cer films, Cer-rich domains with a high incorporation of SM were formed. In
conclusion, while the SMs from sperm behave in a regular way, the corresponding Cers show atypical
properties that may be relevant for the structural rearrangement occurring in the acrosome-reacted sperm
membrane.
This work was supported by FONCyT, CONICET, SECyT-UNC and SECyT-UNS.
1- D.A. Pealva, et al, J. Lipid Res. (2013) 54:2225-35.
2- D.A. Pealva, et al, Biochim Biophys Acta (2013), accepted.
BLM14_Study of the effect of steric interaction in lamellar phases
Barbara B. Gerbelli, Rafael L. Rubim, Emerson R. Silva, Frdric Nallet, Laurence Navailles, Cristiano L.
P. Oliveira, Elisabeth A. de Oliveira.
Some biological process such as membrane fusion and organization of membranes in stacks are
governed by interactions between membranes, which are formed by a mixture of lipids organized in
bilayers. Different structures like sponge phase, vesicles, or stacks can emerge, depending on the lipid
fraction with respect to the solvent, and on the packing of lipids in the bilayers. In this work we investigate
the behavior of multilamellar phases composed of lecithin and a commercial co-surfactant (Simusol),
which is a mixture of ethoxylated fatty acids. Composition of membrane was varied from 100% of lecithin
to 100% of Simulsol. Using X ray scattering and a new procedure to fit the data, relevant parameters
characterizing the lamellar structure were determined as a function of membrane composition. Scattering
data illustrating the swelling of the lamellae for different amounts of co-surfactant are presented with the
respective behavior of the Caill parameter. The incorporation of co-surfactant to lecithin membrane
shifted the equilibrium bilayer separation
Acknowledgements: To CNPQ, FAPESP, CAPES, INCT /CNPQ, Egide. Soleil synchrotron kindly
allocated beam time on the SWING station, proposal number 20101084.
BLM SAB 2013
36
BLM15_Surface properties of liposomes mixtures of phosphatidylcholines and
phosphatidylethanolamines.
Sierra,M.B
1
., Pedroni,V
1
., Morini,M
1
., Disalvo,E.A
2
., Alarcon,L
1
., Appignanesi,G
1
.
1- Laboratorio de Fisicoqumica Dpto. de Qumica. INQUISUR Universidad Nacional del Sur
2 -Laboratorio de Biointerfases. Centro de Investigacin y Transferencia, de Santiago del Estero
Phase properties studies of mixtures of DMPC/DPPC and DMPC/DMPE performed by means of
differential calorimetry and fluorescence microscopy, determined the existence of phase segregation as a
function of temperature and composition. It is inferred from these studies that the phases in contact
produce areas of contact, which affect surface properties of the membranes in contact with the aqueous
medium. However, there are few systematic studies about the surface potential that arise from the
presence of segregated phases. Thus, in this paper the zeta potential of multilamellar liposomes of
DMPC/DPPC and DMPC/DMPE mixtures was determined at 50C and 60C respectively, which are the
phase transition of the lipids, in a 1mM KCl medium. For the DMPC/DPPC zeta potential discontinuities
are observed as mole fraction of DPPC increases. Two mole fractions show particularities, which coincide
with what was reported for these mixtures in gel state [1] and with bigger sized liposomes. Since these
lipids are miscible in the whole range of molar fractions, the results could be explained as a consequence
of the organization of polar headgroups at the lipidic interphase. This conclusion is also supported by the
behaviour of unilamellar and multilamellar mixtures of DMPC/DMPE where the polar headgroup plays an
important role in the stabilization due to steric factors being this influence more noticeable than the one
related to the change of length of the hydrocarbon chain, as shown by the DMPC/DPPC mixtures.
Results are in agreement with Makino [2] proposal for the placement of PC headgroups on the liposome
interphase according to its phase state.
Acknowledgements: With funds from CONICET and UNS.
1- M. del C. Luzardo,G. Peltzer, and E. A. Disalvo. Langmuir. 1998. 5858.
2- K. Makino, T. Yamada, M Kimura. T Oka, H. Ohshima and T. Kondo. Biophys.Chem.1991. 175.
BLM16_Interaction of amphiphilic cyclodextrins with phospholipids in Langmuir
monolayers
Pinzn Barrantes, J.
a
,Hoyos de Rossi, R.
a
, Maggio, B
b
., Vico, R.
a
a
INFIQC-CONICET, Departamento de Qumica Orgnica,
b
CIQUIBIC-CONICET, Departamento de
Qumica Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba.
Cyclodextrins (CDs) are water-soluble molecular cages with a hydrophobic interior that allows the
inclusion and hence solubilization of various molecular species. These biocompatible cyclic
oligosaccharides, with low toxicities and no immune response are currently used as hydrophobic drug
carriers. CD-containing organized supramolecular structures can be obtained through assembly of
amphiphilic CDs
1
or by their insertion into integrated systems such as lipid membranes.
2
In previous work
we study the capability of amphiphilic CD to form Langmuir monolayer as well as the properties of these
films.
1,2
Here we present the properties of mixed films formed by an amphiphilic CD with palmitoyl oleyl
phosphatidyl choline (POPC) at different mole fractions. The miscibility and interactions of the
components have been characterized by A isotherms and the excess of free energy of mixing (G
exc
).
The results indicate non ideal mixing. Additionally, the more stable composition is dependent, al low
the more stable film is enriched in POPC, while at high there are two minima in G
exc
at X
POPC
=0.20 and
X
POPC
=0.8.
1
Vico, R., de Rossi, R.H., Maggio, B. Langmuir 2010. 8407.
2
Roux, M., Sternin, E., Bonnet, V., Fajolles,C., Djedani-Pilard, F. Langmuir 2013. 3677.
SAB 2013 BLM
37
BLM17_Folic acid: pro-oxidant action and characterization of a system based in SPC
liposomes
Marsanasco M.
a
, Piotrkowski B.
b
, Calabr V.
b
, Chiaramoni N.S.
a.
and Alonso S. del V
a
a Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
b Fisicoqumica, Facultad de Farmacia y Bioqumica, UBA e Instituto de Bioqumica y Medicina
Molecular-CONICET
The aim of this work was to study the antioxidant action of folic acid (FA) and characterize liposomes
encapsulating FA, before and after pasteurization. Soy phosphatidylcholine (SPC) liposomes were
obtained by the Bangham method with FA and/or in combination with stearic acid (SA) or calcium
stearate (CAS), with vitamin C (VC) and in combination with vitamin E (VE). Oxidative stability was
studied with TBA and ORAC methods. Characterization was performed by means of surface charge (Z
potential), membrane packing (probes MC540 and laurdan), size distribution by dynamic light scattering
(DLS) and rheological behavior.
The pro-oxidant action of FA was observed via TBA method, even after pasteurization. The VE resists the
pro-oxidant effect of the FA. With the ORAC method, no effect was observed with FA alone or in
combination with VE. The three formulations with FA had similar size distributions and Z potential values.
FA favors Merocyanine 540 (MC540) interaction with SPC and SPC:SA and increased laurdan GP when
combined with VE, in all of the formulations studied. Although the heat treatment decreased the viscosity
of SPC and SPC:SA and increased the viscosity of SPC:CaS; all systems showed Newtonian behavior,
which made these systems attractive for industrial applications.
Acknowledgements Universidad Nacional de Quilmes, Universidad de Buenos Aires and CONICET.
BLM18_Effect of hormone thyroids on ripple gel phase membranes
Marcelo C. Sosa Morales
a
, Ana C. Juarez
b
, Doly M. Chemes
b
, Rosa Maria S. lvarez
a
a
INSIBIO (CONICET-UNT), Instituto Superior de Investigaciones Biolgicas, Chacabuco 461, San M. de
Tucumn, Tucumn, T4000ILI, Argentina
b
Instituto de Qumica Fsica, Universidad Nacional de Tucumn, San Lorenzo 456, San. M. de Tucumn,
Tucumn, T4000ILI, Argentina
Since some time ago, our interest was focused on the study, from a microscopic point of view, of the
action mechanisms of certain biomolecules on cell membranes. The elucidation of the structural
characteristics of the molecules involved in these processes is carried out primarily by Raman
spectroscopy. This technique is a powerful tool to understand the interactions between phospholipid
membranes and biologically important molecules such as thyroid hormones (TH) (1). As yet, the
experimental evidence indicated that the membrane in gel phase hinders access of HT to the
hydrophobic region of the bilayer, in such a way that a significant portion of the hormones remains
anchored in the polar region. This fact might lead to interactions between PO2 and CO lipid groups with
the alanine moiety of the HT. Comparison between the DMPC/HT and DMPG/HT systems allow us to
better understanding of the nature of the interactions that take place in the hydrophilic region of the
membrane, while helping us to predict the degree of insertion of hormones in the hydrophobic region
according to the structural characteristics of the bilayers.
1- A.A.Petruk, M.C.Sosa Morales, R.M.S.lvarez, Spectrochim Acta Part A, (2013) 112, 403
BLM SAB 2013
38
BLM19_Interaction of nisin with anionic membranes. Purification and Raman study
Marcelo C. Sosa Morales
a
, Augusto Bellomio
a
, Ana C. Juarez
b
, Rosa Maria S. lvarez
a
a
INSIBIO (CONICET-UNT), Instituto Superior de Investigaciones Biolgicas, Chacabuco 461, San M. de
Tucumn, Tucumn, T4000ILI, Argentina
b
Instituto de Qumica Fsica, Universidad Nacional de Tucumn, San Lorenzo 456, San. M. de Tucumn,
Tucumn, T4000ILI, Argentina
Nisin is an antibiotic peptide ribosomally sintetized by Lactococcus lactis that inhibits the growth of a wide
range of gram-positive bacteria. It has a net positive charge (+5) and its weight is approximately 3kDa.
Commercially, it is available as a lyophilized powder containing 2.5% (w/w) nisin.
Purification was carried out using chromatographic methods (1). The bactericidal activity of fractions
obtained was visualized on agar plates. In addition, Tricine SDS-Page and Mass Spectrometry were
performed to confirm the purity of nisin.
Raman Spectroscopy was used to study the interaction of this small peptide with anionic models
membranes. We evaluated and interpretated signal spectral changes considered specific markers of the
lipid packaging, the number of conformers gauche in the hydrocarbon chains and the degree of hydration
of the polar heads. For this purpose, dipalmitoylphosphatidylglycerol (DPPG) and
dilauroylphosphatidylglycerol (DLPG) were utilized. Measurements were made at room temperature.
Analysis of DPPG/Nisin and DLPG/Nisin systems will allow us to know if lipid phase state impact on the
interaction and hence to have a better understanding about the action mechanism of nisin.
1- Abts, Andr, International Journal of Peptides, (2011), Volume 2011, Article ID 175145
BLM20_Interactions and Elastic Properties Of Lipid Membranes: The Role of A
Cosurfactant
Rubim, R L
1
; Bougis, K
2
; Gerbelli, B B
1
; Silva, E R
1
; Oliveira, C L P
1
; Navailles, L
2
; Nallet, F
2
; Oliveira, E
A
1
.
1
Complex Fluids Group, Experimental Physics Department, Institute of Physics, University of So Paulo,
So Paulo, Brazil
2
Research Centre Paul Pascal, Pessac, France
In this work we investigate interactions between lipid membranes and their elastic properties, with the
incorporation of ethoxylated fatty acids, commercially known as Simulsol. Small Angle X-ray Scattering
(SAXS) experiments were carried out in lamellar phases in order to obtain structural information and
using a model to the scattered radiation, we can obtain the thickness of the membrane and the Caill
parameter, which is related to the flexibility of the membrane. Applying an osmotic pressure to the
membranes we can access information about the interactions between the bilayers and evaluate the
effect of changing their flexibility and the interface. Combining the information obtained from both
methods it is possible to characterize the elastic parameters of the membrane. We observe that the
insertion of the cosurfactant increases the flexibility of the membranes, as well as the diluted and
repulsive interactions. This study brings a new method to evaluate interactions between membranes and
characterize its elastic properties.
Acknowledgements: This work was supported by FAPESP, process number 2011/16149-8, and INCT-
FCx.
1- DOI: 10.1021/la402962c
2- C. Oliveira et al. J. Appl. Cryst.. 2012. 1278.
SAB 2013 BLM
39
BLM21_Insertion of Glyphosate into a lipid bilayer: Study by molecular dynamics
Frigini, E.; Martin, O. A. and Porasso, R. D.
IMASL-CONICET, Departamento de Fsica, Universidad Nacional de San Luis, Italia 1556, 5700-San
Luis, Argentina
Herbicides are very useful for agriculture, allowing the elimination of weeds for the optimum development
of crops. In the last decades, glyphosate has been the most widely used herbicide in the world. Currently,
it is used without an accurate knowledge of its adverse effects on human health. Independent studies
have shown that the glyphosate present some toxicity in laboratory animals
(1-3)
. In order to understand
how the glyphosate interacts with a cell, in a first approximation, we model the process of insertion of this
herbicide molecule into a lipid bilayer, using the molecular dynamics technique. The main goal is to
determine the precise location of the glyphosate within the membrane, determining the free energy
profiles of this process
(4,5)
.
Acknowledgements This work was supported by PIP-112-2011-0100030 from CONICET and P-328402
from the UNSL, Argentina
1- E. A. Smith and F. W. Oehme. Vet Hum Toxicol. 1992. 531
2- C. Cox. Glyphosate. J. Pesticides Reform, 1995. 14.
3- A. Paganelli et al, Cem. Res. Toxicol, 2010,1586
4- G.M. Torrie and J.P. Valleau. J. of Computational Physics. 1977. 187.
5- Porasso, R, et al. J. Phys. Chem. B. 2009. 9988
BLM22_To Be Or Not To Be In Membrane Domains: Transbilayer Asymmetry And
Sphingomyelin-Dependent Preferential Partitioning Of The Acetylcholine Receptor
Perillo V.L
a
; Pealva, D.A.
a
; Vitale, A.J.
b
, Aveldao, M.I.
a
; Barrantes F.J
c
; Antollini S.S.
a
a
Instituto de Investigaciones Bioqumicas de Baha Blanca / Universidad Nacional del Sur, Argentina.
b
Instituto Argentino de Oceanografa / Universidad Nacional del Sur, Argentina.
c
Lab. Molec. Neurobiol.
Biomed. Res. Institute, UCA-CONICET, Argentina.
The preferential partitioning of the nicotinic acetylcholine receptor (AChR) in liquid-ordered (Lo) domains,
heterogeneous membrane domains commonly known as rafts,is thought to be a part of its clustering
mechanism. Previous studies from our group have shown that AChR lacks preference for Lo domains
when reconstituted in sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) model systems. Here we
study the effect on the possible Lo-preferential partitioning of purified AChR reconstituted in two model
systems (POPC:Chol, 1:1 and POPC:Chol:SM, 1:1:1) under: a) induced transbilayer asymmetry, by
addition of brain sphingomyelin (bSM) to the external hemilayer; and b) the presence of different pure SM
species in the model membrane (bSM, 16:0-SM, 18:0-SM or 24:1-SM). AChR distribution was evaluated
by fluorescence resonance energy transfer efficiency between the AChR intrinsic fluorescence and
Laurdan or dehydroergosterol fluorescence, and also by determining the presence of AChR in detergent-
resistant and detergent-soluble domains (1% Triton X-100, 4C). Both studies show that the induction of
transbilayer asymmetry or the presence of 16:0-SM or 18:0-SM, as opposed to bSM or 24:1-SM, leads to
a preferential partitioning of AChR in Lo domains. Thus, the localization of AChR in Lo domains strongly
depends on the characteristics of the host lipid membrane.
BLM SAB 2013
40
BLM23_Changes in biophysical properties of membranes containing sphingomyelin with
very long chain PUFA induced by its hydrolysis
Pealva DA
1
, Fanani ML
2
, Maggio B
2
, Aveldao MI
1
, Antollini SS
1
1
INIBIBB, CONICET-UNS, Baha Blanca, and
2
CIQUIBIC, UNC-CONICET, Crdoba, Argentina.
E-mail: dpenalva@criba.edu.ar
Very long-chain (C24 to C36) polyunsaturated fatty acids (VLCPUFA) are important acyl groups of
sphingomyelin (SM) and ceramide (Cer) of mammalian spermatozoa. In rat sperm, SM species containing
PUFA with 28-32 carbon atoms are exclusively located on the heads. After inducing the acrosomal
reaction almost complete hydrolysis such SMs occurs, leading to gametes considerably enriched in the
corresponding Cer species. The aim of this study was to evaluate the effects of the sphingomyelinase-
induced conversion VLCPUFA-SM VLCPUFA-Cer on the biophysical properties of a binary model
system (POPC/SM). The VLCPUFA-containing molecular species of SM were isolated from rat testes by
a combination of chromatographic techniques. Egg-SM, whose properties are widely known, was used for
comparison. Unilamellar liposomes (LUVs) were prepared and Dynamic Light Scattering was used to
evaluate their structures/sizes before and after enzymatic hydrolysis. The potential increase in the
interaction between different populations of liposomes (fission/fussion) after the SM Cer hydrolysis was
evaluated by FRET assays using NBD-PE as donor and Rh-PE as acceptor, and the lateral segregation
of phases after Cer generation was followed by anisotropy of different fluorescence probes (laurdan, DPH
and NBD-PE). In all biophysical properties measured, the SM species containing VLCPUFA contrasted
with those of the egg-derived saturated SM. The longer and bulkier acyl chains of VLCPUFA-Cer may
play a role in favouring the fusion/fission events that occur in the head of spermatozoa during the
acrosomal reaction, a process that requires topological lipid intermediates with negative curvature.
BLM24_Surface activity of L- ascorbic acid derivatives
Mottola, M.
1, 2
, Villanueva, M.
2
, Fanani, M.L.
1
, Vico, R
2
.
1
CIQUIBIC CONICET, UNC.
2
INFIQC - CONICET, Departamento de Qumica Orgnica, UNC.
E-mail: mili_m26@hotmail.com
L-ascorbic acid derivatives are molecules of potential pharmacological interest (as well as alimentary and
cosmetic) due to its antioxidant properties and amphiphilic nature.
These vitamin C derivatives (ASC
n
n= 10, 12, 14) were synthesized according to a modified method of
the main procedure already reported in literature [1] and were compared with the commercial derivative
ASC
16
. These molecules were characterized through
1
H
13
C NMR and IR, showing stability during three
weeks.
We found that, contrary to ASC
10
and ASC
12
, ASC
n
with n= 14 and 16 form stable Langmuir monolayers
at room temperature. ASC
16
films show phase transition from a liquid-expanded (LE) to a liquid-
condensed (LC) or crystalline phase, whereas the other derivatives presents only a LE phase.
All these compounds have a complex surface behavior and display a favorable penetration into
phospholipid monolayers with strong interaction among them.
This work was supported by: SECyT-UNC, FONCyT and CONICET. MLF and RV are Carrer Researchers
of CONICET, MM and MV are undergraduate students of UNC.
1- Capuzzi, G.; Nostro, P. Lo; Kulkarni, K.; Fernandez, J. E. Langmuir 11 (1996) pp. 3957
2- M. Mottola, N. Wilke, L. Benedini, R.G. Oliveira, M.L. Fanani. Biochimica et Biophysica Acta 1828
(2013) pp. 2496.
SAB 2013 BLM
41
BLM25_Characterization of the interaction of polyphenols with model membranes.
Effects upon viscosity and protection against oxidative agents.
de Athayde Moncorvo Collado A, Morero RD, Minahk CJ
Instituto Superior de Investigaciones Biolgicas (INSIBIO) CONICET-UNT. Chacabuco 461, San Miguel
de Tucumn.
Polyphenols are secondary metabolites commonly found in a wide variety of plants and vegetable-origin
aliments, characterized by the presence of two or more phenolic groups. These substances are subject of
great interest in the last years, due to their potential multiple bio-active features, including their well-
known antioxidant capacity. Importantly, a widely accepted characteristic about polyphenols is their ability
to interact with biological membranes.
For the present study, four polyphenols from different chemical families (resveratrol, naringenin,
epigallocatechin gallate and enterodiol) were tested for their effect on membrane viscosity by
fluorescence techniques. For that purpose, liposomes of dipalmitoyl phosphatidylcholine (DPPC) alone or
in combination with 40% cholesterol were prepared by extrusion and labeled with either laurdan, DPH or
TMA-DPH. Anisotropy was measured at different temperatures. On the other hand, protection against
oxidative agents such as hydrogen peroxide and Cu II was spectrophotometrically studied following the
increase of conjugated dienes. Fluorescence studies showed that the presence of polyphenols induced a
marked increase in the viscosity with a concomitant alteration in the order of the phospholipids, which
was polyphenol-dependent. On the other hand, all compounds tested were able to decreased the
oxidation levels induced by the Fenton reaction. However, little correlation was found with the membrane
interaction inferred from anisotropy measurements.
Results presented in this work indicate that polyphenols are able to closely interact with biological
membranes. Therefore, further studies in this area would result in a better understanding of the potential
uses beyond their anti-oxidant properties.
BLM26_Effect of pressure in assembly and disassembly of surfactant aggregates
Espinosa Y.R
1
, Grigera J.R
1,2
1
Centro de Qumica Inorgnica, CEQUINOR (UNLP-CONICET).
2
Departamento de Ciencias Biolgicas,
Facultad de Ciencias Exactas, Universidad Nacional de La Plata. Argentina.
Hydrophobic interaction manifests itself in numerous phenomena, including the processes of self-
assembling, formation of micelles and protein folding
[1]. We studied the effects of high pressure,
temperature and solvent on hydrophobic interactions, responsible for micellization of amphiphilic
surfactants, using Molecular Dynamic simulations in a system of sodium dodecyl sulphate (SDS) in water,
heavy water and a pure non polar solute (Lennard-Jones) Preliminary results indicate that (i) At
pressures around 1-1,5 kbar causes disassembly of the formed micelles. (ii) In the range of the pressure
higher than 1,5 kbar, the pressure effects is completely reversed, the number aggregation increases
causing a redistribution of surfactant molecules changing the geometry of the aggregates, in agreement
with experimental observation. (iii) In Lennard-Jones solvent reverse micelles are formed, due to the lack
of hydrophobic interaction. (iv) Thermal analysis indicated than at low temperature the SDS molecules
are aligned in parallel, similar to lamellar phase diminishing the solvent accessible surface area; when
temperature is increased; the structural transition change producing predominantly micellar configurations
with a high aggregation number and high solvent accessible surface area.
1- Meyer, E.E.; Rosenberg, K.J.; Israelachvili, J. PNAS 2006, 15739.
BLM SAB 2013
42
BLM27_Characterization of Vesicle-Micelle transitions by Fluorescence Correlation
Spectroscopy (FCS) and Isothermal Titration Calorimetry (ITC)
Dodes Traian MM
1,2
, Pignataro MF
1
, Levi V
2
, Gonzlez Flecha FL
1
1
IQUIFIB-CONICET, Dpto. de Qumica Biolgica, FFyB, Universidad de Buenos Aires
2
IQUIBICEN-CONICET, Dpto. de Qumica Biolgica, FCEyN, Universidad de Buenos Aires
Structural and kinetic studies of membrane proteins require their reconstitution in amphiphilic systems to
ensure their proper folding and stability in aqueous media. The structure of these systems ranges from
micelles to vesicles depending on the relative concentrations of phospholipids and detergent. To
characterize the behaviour of these systems and the transitions among them we used Fluorescence
Correlation Spectroscopy (FCS) and Isothermal Titration Calorimetry (ITC). We used FCS to measure the
diffusion coefficient of the particles and thus this technique allowed us to gather information about the
heterogeneity of the studied systems. ITC allows detecting membrane solubilisation by measuring the
heat exchanged upon the addition of detergents. Working with a system similar to that used in membrane
activity protocols we could observe the abrupt change in size characteristic of a micelle-vesicle transition
at a phospholipid / detergent mole fraction around 0.75 and by ITC around 0.8 mole fraction the heat
signature changes from endothermic to exothermic. These experiments contribute to our understanding
of the correlation between the physical properties of the amphipilic systems and the function of
membrane proteins inserted in them.
With grants from ANPCyT and UBA.
BLM28_Biophysical and Biochemical characterization of Proteoliposomes harboring
Annexin V and Alkaline Phosphatase
M. Bolean
1
, A.M.S. Simo
1
, Kiffer-Moreira, T.
2
, M.F. Hoylaerts
3
, J.L. Milln
2
and P. Ciancaglini
1
1
Departamento de Qumica, FFCLRP-USP, Ribeiro Preto, SP, Brazil,
2
Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA,
USA,
3
Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
In this work, we standardized the proteoliposomes harboring Annexin V (AnxA5) and alkaline
phosphatase (TNAP) in order to obtain the best lipid composition to optimize the incorporation and
function of proteins. The kinetic parameters for the hydrolysis of the substrates by TNAP, in the absence
and presence of AnxA5, were determined at physiological pH. The proteins were incorporated into
liposomes constituted by DPPC and DPPC:DPPS (5, 10 and 15%). The best catalytic efficiency was
obtained for the system containing DPPS 10%. DSC studies show that with the DPPS concentration
increased in the DPPC liposomes is observed a progressive broadening of the transition peaks. The
presence of AnxA5 causes a decrease in H values and when TNAP is present in the proteoliposomes,
that effect is even greater. AnxA5 was able to mediate Ca
2+
influx into the DPPC and DPPC:DPPS 10%-
vesicles. The presence of TNAP in the proteoliposomes did not affect AnxA5-mediated Ca
+2
uptake.
However, the presence of AnxA5 affected significantly the hydrolysis of the substrates by TNAP. Studies
with Giant Unilamellar Vesicles (GUVs) confirmed the functional reconstitution of AnxA5 in the mimetic
system. Acknowledgements: CNPq, CAPES, FAPESP.
SAB 2013 BLM
43
BLM29_Structural properties of CHAPS micelles, studied by molecular dynamics
simulations.
Herrera, Fernando E.
a
; Garay, A. Sergio
a
; Rodrigues, Daniel E.
a,b
a
Departamento de Fsica, Facultad de Bioqumica y Ciencias Biolgicas, Universidad Nacional del Litoral
(UNL), Ciudad Universitaria, 3000 Santa Fe, Argentina.
b
INTEC (CONICET-UNL), Gemes 3450, 3000
Santa Fe, Argentina.
Detergents are essential tools to study biological membranes. They are frequently used to solubilize lipids
and integral membrane proteins, and to evaluate the strength of interaction of the membrane
components. The nondenaturing zwiterionic detergent usually named CHAPS was designed for
membrane biochemistry and integrates the effects of sulfobetaine-type detergents and bile salts. Despite
of the available experimental data little is know about the molecular structure of its micelles.
We performed Molecular Dynamics simulations to study the aggregation in micelles of several numbers of
CHAPS (1-16) starting from an homogeneous water dilution. We developed and validated the forcefield
parameters to describe the interactions of the molecule. After 50ns of simulation all the systems result in
the formation of stable micelles. We characterize the molecular shape (gyrate radii, volume, surface) of
the micelles. We analyzed the molecular structure (RDF, salt bridges, H-bonds, SAS) and found that the
main interactions that lead to the stability of the micelles are the electrostatic ones among the polar
groups of the tails and the OH's from the rings moiety. At variant with micelles of other compounds,
CHAPS- show a grain-like heterogeneity with hydrophobic micro-pockets. Our results agree with avalaible
information from NMR and X-ray. The rotational diffusion of the micelles is also characterized to compare
with EPR spectroscopy results.
BLM30_Design, Synthesis and Characterization of a novel azoniosome.
Sequeira; M. A.; Mari, I.; Benedini, L.; Dodero, V. I
Depto de Qumica- INQUISUR, Universidad Nacional del Sur-CONICET, Baha Blanca (AR).
Niosomes are promising new systems for Drug Delivery. They possess a vesicular lamellar structure
similar to liposomes; however they are constituted by mixtures of water insoluble ionic surfactants and
cholesterol [1]. Various groups have attempted to use this event to design vesicular systems based on
azoamphiphiles using either ionic or non-ionic head. However, the selection of the polar group head and
its relationship with the tail of the amphiphile was not adequate to achieve the formation of vesicular
aggregates and/or the photomodulation in water [2, 3].
Through the design and chemical synthesis, we obtained a new non-ionic azoamphiphile whose relative
head / linker/ tail is optimal for the formation of photoswitchable vesicles. This system has potential use
as a phototriggered drug delivery system or azoniosome.
Acknowledgements: Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1. Gupta S. et.al. Polymer 2012, 53, 3053.
2. Kordel C.; Popeney C.; Haag R. Chem. Commun. 2011, 47, 6584.
3. Orihara Y. et. al. Langmuir 2001, 17, 6072.
BLM SAB 2013
44
BLM31_Compressibility modulus and states of water in lipid monolayers.
Fras, M.A.,Salcedo, C.L.,Cutro, A.C. and Disalvo, E.A..
Laboratorio de Biointerfases y Sistemas Centro de Investigacin y Transferencia, de Santiago del Estero
(CITSE)
The lipid monolayer properties have been studied by compressing lipids spread in a large surface up to
its collapse. In this methodology, the lipids spread at huge areas are considered as being in a gas phase
and are compressed at a temperature below the critical one (the transition temperature) until
condensation. The thermodynamic treatment is compared with a real gas in which the condensation is
analyzed as a consequence of the manifestation of the intermolecular forces between the lipids. In other
words, lipid interacts between them as gas particles in vaccum. A more realistic approach to account on
monolayer behavior is that lipids, even at large areas, are in contact with the water phase. Upon
compression, the energy input is not merely used to make work of compression but to overcome the
friction of hydrated lipid molecules with the water (stationary) phase. Thus, the lipids drag water during its
compression, force its reorganization and/or a displacement work can occur. In order to clarify the
hydration role in the compression/expansion isotherms we have compared and evaluated the
compresibility of monolayers compressed from the very diluted state (gas phase) with that obtained after
the stabilization of the lipids in a force-free state. This study allows to comprehend the role of water
confined at the interphase processes on compressibility.
Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
1- K. Ariga, Y. Okahata, Langmuir 10 (7) (1994) 2272
BLM32_Enhanced antiradical activity of gallic acid included in lipid interphases.
Cutr, A.C.
1
, Salcedo, C.L.
1,2
, Fras, M.A., Nazareno, M.A.
2
, Disalvo, E.A.
1
Laboratorio de Biointerfases y Sistemas Biomimticos
1
y Laboratorio de Antioxidantes y Procesos
Oxidativos
2
, Centro de Investigacin y Transferencia, de Santiago del Estero (CITSE)
Polyphenols are well known by its effects as antioxidant agents and by its effects on the hydration layers
of lipid interphases. In this regard, it is known that gallic acid and its family are able to decrease the dipole
potential and to act in water as a strong antioxidant. In this work we have studied both effects on lipid
interphases in monolayers and bilayers of phosphatidylcholines. The results show that the antiradical
activity of gallic acid adsorbed to DMPC membranes increases five-fold with respect to that found in
water. In parallel, the gallic acid increases the negative surface charges of LUVs and decreases the
dipole potential. As a result, the positively charged radical species such as ABTS
+
are able to penetrate
more easily the membrane, enhancing the antiradical activity. The role of interfacial water in the
antiradical activity and the possible association of gallic acid with the phospholipid molecules were
studied by means of surface pressure/area curves, dipole potential measures and FTIR spectroscopy.
These results allow discussing the effect of interfacial water on the antioxidant reaction catalysed by
lipids.
Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
1- Huh NW, Porter NA, McIntosh TJ, Simon SA. Biophys J, 1996. 71, 3261.
2- Salcedo C.L., Lpez de Mishima B.A., Nazareno M.A. Food Res. Int., 2010. 43, 1187.
SAB 2013 BLM
45
BLM33_FTIR interaction between glycine and DPPC in hydrated state.
Norma M. Ale
1
, Aida Ben Altabef
1,2
, Andrea Gmez Zavaglia
3
and Sonia B. Daz
1
1
Instituto de Qumica Fsica. Facultad de Bioqumica, Qumica y Farmacia. U.N.T. San Lorenzo 456.
T4000CAN Tucumn, Argentina. e-mail:sobedi63@yahoo.com.ar.
2
Instituto de Qumica del Noroeste (INQUINOA)-CONICET-Tucumn, Argentina.
3
(CIDCA)-CONICET- Universidad de La Plata, Argentina.
This glycine`s family (betaine, carnitine, dimethylglycine or glycine) takes part in a variety of biochemical
reactions. The main function of a protector is to regulate loss and exchange of water in the biological
structures. The protective capacity of these compounds on the membranes has been studied on
numerous occasions. The existing information is fundamentally empirical, and do not know the
mechanisms of interaction of these compounds with the membrane at molecular level.
The aim of this work is analyze the interaction of the main functional groups of dipalmitoylphosphatidyl
choline (DPPC) with glycine (gly) by infrared spectroscopy Fourier Transform (FTIR) in hydrated state.
Our results revealed intermolecular interactions between the headgroups of DPPC and gly. These
interactions would indicate that the phosphate group of the lipid membrane forms hydrogen bonds with
gly in replacement of structured water.
1. Gmez Zavaglia, A.; Tymczyszyn, E.; De Antoni, G.; A. Disalvo J. Appl. Microbiol., 95(6): 1315-1320
(2003).
2. Daz, S. B.; Biondi de Lpez, A. C. and Disalvo, E. A. Chemistry and Physics of Lipids 122:153-157
(2003).
3 Crowe, L.M.; Mouradian, R.; Crowe, J.H.; Jackson, S.A.; Womersley, C.; Biochim. Biophys. Acta,
769:141150 (1984).
BLM34_Raman and FTIR interaction between l-cysteine methyl ester and DPPC in
anhydrous state
J. M. Arias
2
, M. E. Tuttolomondo
1
, S. B. Daz
1
and A. Ben Altabef
1,2
1
Instituto de Qumica Fsica. Facultad de Bioqumica, Qumica y Farmacia. Universidad Nacional de
Tucumn San Lorenzo 456. T4000CAN Tucumn.
2
INQUINOA-CONICET.
This work is the contribution of a number of studies about the cysteine family (L-cysteine, L-cysteine ethyl
ester and L-cysteine methyl ester) to understand lipid membrane interaction. The cysteine family has a
thiol group that takes part in a variety of biochemical reactions. The possible formation of weak hydrogen
bonds at receptor sites is of considerable interest, as it might contribute to a biological response.
The objective this work is understand and analyze the interaction of the complex of L-cysteine methyl
ester.HCl (CME) with lyophilized liposomes of dipalmitoylphosphatidylcholine (DPPC) by Raman and
infrared (FTIR) spectroscopies.
Our results revealed intermolecular interactions between the headgroups of DPPC and CME. These
interactions would indicate that the phosphate group of the lipid membrane forms hydrogen bonds
probably with S-H groups of CME in replacement of structured water.
The Raman analysis shows that the CME has little interaction with the hydrophobic region of DPPC. The
intensity ratio analyses show that the density of lateral packing of the acyl chain (I
2881
/I
2846
) and the
intensity ratio relevant to Gauche-trans rotamers (I
1096
/I
1062
) do not have significant changes. The I
2933
/I
2846
intensity ratios show an increase in movement and rotational disorder without an increase of the number
of Gauche-trans rotamers.
1- J.L.R. Arrondo, F.M. Goi, Chem. Phys. Lipids 96 (1998) 5368.
2- C. B. Fox, R. H. Uibel, J. M. Harris, J. Phys Chem. 2007, 111, 1142811436.
BLM SAB 2013
46
BLM35_Surface topography of lipid-peptide mixtures at the air-water interface using
BAM: role of physical state of the lipid.
Caruso, B, Ambroggio EE, Wilke, N, Fidelio GD.
CIQUIBIC,Depto.QumicaBiolgica, Fac. Cs.Qumicas, CONICET, UNC.
Previous studies in our laboratory showed that the interaction of peptides with membrane lipids depend
on the secondary structure of the former and the physical state of the latter (1, 2). Here, we contribute to
this subject by imaging of Langmuir monolayers using the Brewster Angle Microscopy technique (BAM).
Monolayer isotherms of the well-known Melittin did not present compressional hysteresis, in keeping with
a prevalence of -helix structure. Melittin form homogeneous monolayers at BAM, but when mixed with a
lipid that forms Liquid Expanded (LE) monolayers (T
c,dmPC
21C) it induced domain formation (phase
segregation) at lateral pressures that dependent on film composition. Although pH affects the isotherm of
pure Melittin, this did not affect the phase diagram of Melittin/DMPC. On the contrary, when mixed with
the more liquid POPC (T
c,poPC
-2C), the domains were not observed. On the other hand, the -sheet
A1-42 peptide exhibits a different behaviour when mixed with either DMPC or POPC. In A1-42/DMPC
mixtures, although phase segregation did not occur at a defined lateral pressure, a fibrillar domain pattern
became apparent upon compression. In A1-42/POPC mixtures no domains were observed. We
conclude that the physical state of lipid phase is not only important for lipid-peptide miscibility but also for
lateral topography.
Acknowledgements: Supported by grants from SeCYT-UNC, CONICET and FONCYT (Argentina).
1- Fidelio et al. BBA.1986. 49.
2- Ambroggio et al. BiophysicalJournal. 2005. 2706.
BLM36_Lipid bilayer fluid phases are not always fluid: a simulation experiment.
Pinto, O.A (1), Fras, M.A.(2), Disalvo, E. A.(2)
(1) Laboratorio de Sistemas Nanoestructurados y Electroqumica., (2) Laboratorio de Biointerfases y
Sistemas Biomimticos. Centro de Investigacin y Transferencia, de Santiago del Estero (CITSE).
FTIR measurements showed that the macroscopic disordered state of the lipid phase has, at the
molecular level, different arrangements of the methylene groups according to the lipid composition. Thus,
fluid membranes are not always fluid because membrane has, according to the acyl chain composition,
different ratios of connected and isolated methylene populations. In this work, we reproduced the typical
curves of changes in the asymmetric stretching modes of CH
2
of phosphatidylcholines with temperature
by modelizing the membrane system as a Lattice Gas in which each element (the CH
2
residues of the
acyl chains) is coordinated with six neighbors. The free energy change of each element state is given by
modulated by a cooperativity parameter accounting for the interaction with the first neighbors. The
Montecarlo simulation was run using the dynamic of Glauber or single spin flip and the algorithm of
Metropolis. The results show that the introduction of water blocks the membrane elements frustrating
some of the elements to contribute to the transition. In consequence, the freezing/frustration of some
elements changes the number of isolated elements as found by FTIR spectroscopy. It is concluded that
the state of the cooperative units in the so-called fluid state are not similar due to the chemical
composition of the acyl change and the water amount buried in it.
1- E.A.Disalvo, A.M. Bouchet, M.A. Fras, Biochim. Biophys. Acta 2013, 1828, 1683.
2- Thermal Biophysics of Membranes, Thomas Heimburg, Published Online: 20 SEP 2007 Print ISBN:
9783527404711, Online ISBN: 9783527611591 DOI: 10.1002/9783527611591
SAB 2013 BLM
47
BLM37_Liposomes and L-cysteine in the treatment of respiratory diseases
Perrota, R., Fernandez Ruocco, M.J., Siri, M., Chiaramoni, N.S., Alonso, S. del V.
Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
Respiratory diseases are very common and affect a large proportion of the population worldwide. For
therapeutic agents to reach the target cells is essential to bypass the barriers of the lung architecture.
With the main goal to overpass these barriers and destabilize them, we developed lipid transporters that
can encapsulate L-cysteine This amino acid, with sulfur in its structure may break the disulfide bonds of
the epithelial barrier and disrupt mucus network [1].
These lipid transporters were formulated with polymerizable lipid DC8,9PC mixed with DMPC . When
irradiated with UV light DC8, 9PC has the ability to form conjugated double bonds increasing stability [2,
3]. Transporters were formulated with two amino acid concentrations: 10 and 50 mol % (relative to lipid).
In order to characterize these systems we studied the polymerization efficiency and the hydrophobic
defects in the bilayer surface. Interactions were analyzed by FTIR. We found that increasing amino acid
concentration, polymerization efficiency decreases presumably due to increased hydrophobic surface
defects. FTIR data obtained confirmed that the L-cysteine interacts strongly with the liposome surface.
The design formulation should be considered as a strong candidate to overcome the mucus barrier. To
confirm this efficiency in vivo testing is going to be carried out in the Laboratory of Dr. Marcelo Morales
(UFRJ, Brazil).
1- Poelma, F.G.J., et al., Int. J. Pharm. 1990, 64: 161-169.
2- Chiaramoni, N.S., et al., J. Liposome Res. 2010, 20(3): 191-201.
3- Temprana, C.F., et al., Chem. Phys. Lipids. 2012, 165(5): 589-600.
BLM38_Molecular properties involved in the effects of steroids on membrane biophysical
state
Wenz J.J.
Instituto de Investigaciones Bioqumicas de Baha Blanca, Universidad Nacional del Sur, Camino La
Carrindanga Km 7, B8000FWB Baha Blanca, Argentina. Tel +54(291)4861201. jwenz@criba.edu.ar.
In order to learn about the factors that govern the effects of steroids on the physical state of membranes,
a library of 82 steroids was studied regarding their ordering, rigidifying, condensing and/or raft promoting
activity on membranes, concurrently with several molecular properties.
Steroids were classified according to their reported activity on membranes into one of two categories by
defining a dichotomous variable (0s and 1s). Each molecular structure was subjected to geometry
optimization by means of a semi-empirical procedure (AM1) and several molecular descriptors were next
computed for the optimized low energy conformations. The obtained data were analyzed by means of
logistic regression and mean contrasting.
Comparing steroids displaying ordering, rigidifying, condensing and/or raft promoting activity with their
counterparts having the opposite effect, substantial differences were found in the partition coefficients
(octanol-water), surface area, volume, number of rotatable bonds (mostly present in the carbon alkyl side-
chain), molar refractivity and molecular weight. Despite the intrinsic correlation between the molecular
properties, the results collectively point toward a positive correlation between steroids lipophylicity and
their ordering, rigidifying, condensing and/or raft promoting effects on bilayers.
BLM SAB 2013
48
BLM39_Surface and hysteresis properties of lipid interphases composed by head group
substituted phosphatidylethanolamines.
Salcedo, C.L.
1,2
, Bouchet, A.M.
1
, Nazareno, M.A.
2
, Disalvo, E.A.
2
, Frias, M.A.
1
Laboratorio de Biointerfases y Sistemas Biomimticos
1
y Laboratorio de Antioxidantes y Procesos
Oxidativos
2
, Centro de Investigacin y Transferencia de Santiago del Estero (CITSE, UNSE-CONICET)
This work analyzes the surface properties of PE-containing membranes modified at the head group
region by the addition of methyl and ethyl residues at or near the amine group. These residues alter the
lipid-lipid and lipid-water interactions by changes in the hydrogen bonding capability and the charge
density of the amine group thus affecting the electrostatic interaction. The results obtained by measuring
the dipole potential, the zeta potential, the area per lipid and the compressibility properties allow to
conclude that the H-bonding capability prevails in the lipid-lipid interaction. The non-polar groups attached
to the C
2
-carbon of the ethanolamine chain introduce a steric hindrance against compression and
increases the dipole potential. The analysis of areas suggests that lipids with methylated head groups
have a much larger compressibility at expense of the elimination of hydration water, which is congruent
with the broader extent of the hysteresis loop.
Acknowledgements: With funds from CONICET, UNSE and ANPCyT.
1- Salcedo, C.L., Bouchet, A.M., Nazareno, M.A., Disalvo, E.A.Frias, M.A. Colloid & Surfaces, B
Biointerfaces, 2014. 113, 243.
2- BouchetA.M., FrasM.A., AlmaleckH., LairionF., MartiniF., DisalvoE.A., GordilloG., Biochim.
Biophys. Acta, 2009. 1788,918.
BLM40_Vitamin C: antioxidant and pro-oxidant action in SPC liposomes
Marsanasco M.
a
, Piotrkowski B.
b
, Calabr V.
b
, Alonso S. del V
a
. and Chiaramoni N.S.
a
a Laboratorio de Biomembranas (LBM), GBEyB, UNQ-IMBICE-CONICET, Quilmes, Buenos Aires
b Fisicoqumica, Facultad de Farmacia y Bioqumica, UBA e Instituto de Bioqumica y Medicina
Molecular-CONICET.
The aim of this work was to study and characterize liposomes with vitamin C (VC) before and after
pasteurization. Liposomes were obtained according to Bangham method with soy phosphatidylcholine
(SPC) and SPC in combination with stearic acid (SA) or calcium stearate (CAS). Then VC or VC
combined with Vitamin E (VE) was added. Oxidative stability was studied with TBA and ORAC methods.
They were characterized by surface charge (Z potential) and membrane packing (MC540 and Laurdan),
size distribution (DLS) and also its rheological behavior. Results showed that the TBA method has an
influence per se on the antioxidant action of VC that maintains its activity even after pasteurization,
probably due to the protective effect of liposomes on the thermolbil VC [1]. VC changes Z potential from
negative to positive, shift that induces liposomal aggregation. The VC favors the entry of merocyanine
540 in SPC and SPC:SA liposomes and increament of laurdan GP in SPC:CaS. Similar trends were
obtained when combining both vitamins: VC and VE. Rheometric studies showed Newtonian behavior
and only SPC had increased viscosity post pasteurization. In summary, these results demonstrates that
SPC based liposomes have a potential application in food industry encapsulating both vitamins (VC and
VE).
Acknowledgements Universidad Nacional de Quilmes, Universidad de Buenos Aires, CONICET .
1- Marsanasco, M., Marquez, A.L., Wagner, J., Alonso, S. del V and Chiaramoni, N.S. (2011). Food Res
Int 44: 30393046.
SAB 2013 BLM
49
BLM41_Discussing the use of light scattering in the characterization of polidisperse
colloidal systems
Nomura, D. A. , Enoki, T. A., Silveira, N. P. and Lamy, M. T.
Grupo de Biofsica, Instituto de Fsica, Universidade de So Paulo, SP, Brazil
Dynamic (DLS) and Static (SLS) light scattering are widely used techniques in the study of colloidal
aqueous systems, like polymers, proteins or lipid aggregates dispersions. In SLS, the intensity of the
scattered light is measured, and the particle radius of gyration calculated. DLS measures time fluctuation
of the scattered light intensity, what allows the calculation of the particle diffusion coefficient, and, with the
use of the Stokes-Einstein equation, an effective particle radius. With aqueous dispersions of polystyrene
nanospheres of discrete sizes ((21.0 1.5) nm, (46.0 2.0) nm and (92.0 3.7) nm) we use DLS and
SLS techniques, at different scattering angles, and discuss the sensitivity of each technique to
characterize monodisperse systems, and the dimensions of particles in a multicomponent dispersion. For
monodisperse systems, SLS was not sensitive to particles of 21 nm, being more limited than DLS in the
characterization of small particles. In multicomponent dispersions, we show that it is very important to
have in mind that both SLS and DLS techniques give Z-average diameter values, that are weighted by
(radius)6, what means that larger particles contribute much more to the final result. Moreover, with DLS,
the size distribution analysis through double exponential or Contin methodologies can be rather
misleading. Also found unreliable is a polidispersity coefficient obtained from the method of Cummulants.
Discussion presented here is fundamental to the use of SLS or DSL in the characterization of unknown
colloidal dispersions.
Financial Support: USP, FAPESP and CNPq.
BLM42_Gold Nanoparticles Functionalized with Cell Penetrating Peptides -Synthesis,
Characterization and Interaction with Biomembranes-
Berberin M.V.
1,2
, Mayorga L.S.
2
, Ciocco Aloia F.
1
and Del Popolo M.G
1
Instituto de Ciencias Bsicas (ICB)
1
e Instituto de Histologa y Embriologa Mendoza (IHEM)
2
,
Universidad Nacional de Cuyo, Mendoza, Argentina
Over the past decades nanoparticles (NP) have prompted a large amount of research in pharmaceutical
sciences, as they offer novel alternatives in the design of drug delivery systems. It is noticeable, however,
that despite the large body of bibliography in this area there is no consensus yet on the way NP are
internalized and distributed within living cells. Size, shape and chemical composition of the surface are
among the factors that control internalization rates. Experiments show that NP intake can occur through
diffusion across the cell membrane, either mediated by the formation of a pore or the invagination of the
lipid bilayer, or follow an endocytic pathway.
In the present work we investigate the interaction of NP with the plasma membrane of living cells, and
explore a possible non-endocytic internalization mechanism. We use human sperms as model cells. Gold
NPs (10100 nm) are synthesized by the reduction of HAuCl
4
in sodium citrate, and the particle surface is
subsequently covered with two different cell-penetrating peptides (CPP). The interaction of the
functionalized NP with the sperms membranes is monitored by electron microscopy. Our results indicate
that, depending on the particle size, concentration, and incubation time, the NP show large affinity for the
cell surface and are able to penetrate the plasma membrane, reaching the acrosome and the cell
nucleus. In order to rationalize the interaction between the CPP-covered nanoparticles and the lipids of
the cell surface, we have investigated by Molecular Dynamics simulations the adsorption and penetration
of a standard CPP molecule (Arg
9
) into a model lipid bilayer (DPPC). Simulations predict a strong a
binding energy to the lipid surface, while the penetration of the CPP into the membrane core is
accompanied by the formation of a water channel.
BLM SAB 2013
50
BLM43_Monitoring CHAPS self-assembly by EPR spectroscopy and Factor Analysis
Rodi, Pablo
1
; Bocco Gianello, M.D.
1
; Gennaro, A.M.
1,2
1
Departamento de Fsica, Facultad de Bioqumica y Ciencias Biolgicas, UNL, Santa Fe
2
IFIS Litoral (CONICET-UNL), Santa Fe
CHAPS is a zwitterionic synthetic detergent derivative from bile salts. It is a facial detergent, and due to
its peculiar characteristics, CHAPS self assembly has been subject of several works in the last years, and
there are open discussions regarding the characteristic of the resulting micelles. In this work we obtain
EPR spectra of the spin label 12-SASL in CHAPS solutions of a wide range of concentrations. The
spectra show strong changes with concentration. Using Principal Factor Analysis we demonstrate that all
the spectra can be reproduced as linear combinations of only three fundamental spectra, and obtained
their relative weights at each detergent concentration. One of them corresponds to 12-SASL free in water,
and the other two correspond to the label in motionally restricted environments. One of the motionally
restricted components appears at a concentration coincident with the experimentally reported cmc of
CHAPS, and it was assigned to 12-SASL in a Type 1 micelle. The relative weight of this component
gradually decreases at increasing concentrations, with the rise of the component assigned to a Type 2
micelle. Rotational correlation times of the spin labels for each of the micelles were obtained by fitting the
spectra with slow motion conditions, and compared with the micelle rotational diffusion times calculated
by MD (poster Herrera et al.). This comparison allows us to make estimations about the aggregation
number.
BLM44_Oncoproteins induce multilamellar structure formation in DMPC vesicles
Borioli G, Gaggiotti MC and Del Boca M
CIQUIBICCONICET, Dep. de Qumica Biolgica, Fac. de Ciencias Qumicas, UN Crdoba.
gborioli@mail.fcq.unc.edu.ar
The Immediate Early oncoproteins c-Fos, c-Jun and Fra-1 are intrinsically unstructured amphitropic
proteins that interact with lipid membranes. In this work, we explored the effects of c-Fos, c-Jun and Fra-1
on membrane remodeling. We have used two complementary techniques, Small Angle X Ray Scattering
(SAXS) and Transmission Electron Microscopy (TEM), to evaluate a system of pure dimiristoyl
phosphatidyl choline (DMPC) or binary (DMPC/PIP
2
, phosphatidyl inositol diphosphate) 100 nm
liposomes , incubated with one of the oncoproteins. SAXS reveals that the three proteins induce the
formation of highly correlated multilamellar structures over the phospholipid phase transition [1], while
TEM shows two coexisting populations of membranes, one of aggregated vesicles and the other of
multilamellar structures, similar to the so-called myelin figures [2]. The spacing differs between SAXS and
TEM measurements, which could be accounted for by the difference in the hydration degree of the
samples. On the other hand, the spacing also differs between the c-Fos and Fra-1 induced multilamellae.
This is a very interesting contribution which may be important to the biological role of oncoproteins.
Acknowledgements: Brazilian SynchrotronLight Laboratory (LNLS),SECyT-UNC, CONICET, FONCyT.
1- Interactions of the transcription factors c-Fos and c-Jun with phospholipid vesicles. Torriani. I.; Borioli
G.A.; del Boca M. Activity Report, Brazilian Synchrotron Light Laboratory 2006
2- Gaggiotti, M.C. Tesis doctoral 2011. Fac. de Ciencias Qumicas, Univ. Nac. de Crdoba
SAB 2013 BLM
51
BLM45_Thermotropic and structural study of sphingolipid mixtures
Dupuy, F; Oliveira, R; Maggio, B.
Centro de Investigaciones en Qumica Biolgica de Crdoba CIQUIBIC-CONICET/UNC, Haya de la Torre
S/N, Ciudad Universitaria, Crdoba, Argentina. Email: fdupuy@fbqf.unt.edu.ar
The hydrocarbon chain of the lipids is a major determinant of the phase behavior of these molecules: long
and saturated acyl chains induce gel/solid ordered phases with a high degree of intermolecular
association. However, lipid phase states can be dramatically altered in multicomponent systems by non-
ideal mixing. In Langmuir monolayers, we demonstrated the formation of an azeotropic mixture between
the short chain ceramide C10:0 Cer and the sphingomyelin N-acylated with palmitic acid C16:0 SM, in
which a condensed phase is obtained at surface pressure values where both components are in
expanded state. In this work, we studied the thermotropism of the sphingolipids C10:0 Cer, C16:0 SM and
their binary mixtures by high sensitivity differential scanning calorimetry. Also, structural information of the
different lipid dispersions was obtained from small angle synchrotron X-ray diffraction. The results indicate
lipid mixing in the gel state but phase separation upon melting. Also, bilayer mesomorphism was
stabilized in the mixed state, although pure C10:0 Cer formed an unusually ordered inverted phase.
Acknowledgements: We thank to the Brazilian Synchrotron Light Laboratory LNS (CNPEM/MCT) for X-
ray beamtime at SAXS2 beamline under project D11A SAXS1 10716.
BLM46_Interfacial stabilization of the antitumoral drug Paclitaxel in monolayers of
ganglioside GM1
Heredia,V.
a
, Dupuy, F.
b
, Maggio, B.
c
, Beltramo, D.
a
a
Unidad de Biotecnologa,CEPROCOR- Min.CyT Pcia. de Crdoba;
b
INSIBIO-CONICET/UNT;
c
CIQUIBIC-CONICET/UNC, Depto. Qca. Biolgica, Facultad de Ciencias Qumicas, UNC
Paclitaxel (Ptx) is an antitumoral drug that inhibits microtubule depolymerization. However, its low water
solubility (less than 1 g.ml
1
) is a drawback in treatment of cancer patients. Recently, we demonstrated
that paclitaxel (Ptx) can be loaded into GM1 ganglioside micelles, to render a stable water soluble
formulation that increases the drug solubility up to 6.3 mg.ml
-1
. In order to get insights on the membrane
interaction of Ptx, we studied the interfacial behavior of the drug in the absence and in the presence of
GM1 by means of the monolayer technique at the air-water interface. Gibbs adsorption experiments and
compression isotherms were carried out in a Langmuir balance, equipped with surface potential detection
and amenable to Brewster angle microscopy. This allows to evaluate the affinity of Ptx towards GM1, the
lateral packing of the mixtures and the surface topography of the molecules in the film. The results
indicate that Ptx becomes stabilized at the interface in the presence of the ganglioside, leading to an
enhanced stability of the mixed GM1/Ptx, film at higher surface pressures compared to pure PTx.
Biofsica de protenas y cidos nuclicos (Biophysics of proteins and nucleic acids ) SAB 2013
52
BPA1_Protein-DNA interactions in extremophiles.
Ariel Aptekmann, Alejandro Nadra
IQUIBICEN-UBA
Protein-DNA interactions are central to cell activity regulation including transcription initiation. The TATA
box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP
is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable
and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs),
from below 0 C to more than 100 C. Thus, the archaeal TBP family is ideally suited to study the
evolutionary adaptation of proteins to an extremely wide range of temperatures (1).
We expect TBP and TATA box to coevolve responding to a number of factors, adaptation to temperature,
pressure, salinity, and other extreme physico chemical conditions.
We characterized the TATA box for different archeal phylum in terms of length, base composition,
information content, and analized its relation with OGT, which is predicted to have an effect in those traits
by Schneider's molecular information theory (2).
1 - Kopitz A, Soppa, J Spectrochimica Acta Part A : Molecular and Biomolecular Spectroscopy, 2009 ,
page 799
2 - Schneider. J. Mol. Biol. 1986, page 415
BPA2_Biochemical Characterization of Human Receptor-Type Protein Tyrosine
Phosphatase ICA512 Ectodomain expressed in Pichia pastoris.
Samus, I.
1
, Noguera, M. E.
1
, Ferreyra, R. G.
1,2
, Ermcora, M. R.
1,2
1
Departamento de Ciencia y Tecnologa, Universidad Nacional de Quilmes.
2
IMBICE-CONICET.
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 processes
1
. 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. Previously, we solved the structure of the mature ectodomain (ME ICA512, residues
449-575) and identified potential dimerization interfaces involved in the regulation of the secretion
2
.
Furthermore, 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 express ME ICA512 in Pichia pastoris. The protein secreted to the culture
medium was purified and characterized by mass spectrometry and size-exclusion chromatography. The
implications of these findings for biological activity will be discussed.
Acknowledgements: CONICET, UNQ and ANPCyT.
1- Hermel et al. Eur J Neurosci. 1999. 8:2609.
2- Primo et al. J Biol Chem. 2008. 283:4674.
SAB 2013 BPA
53
BPA3_Defining the role of the Sco proteins in the assembly of the Cu
A
site
Morgada MN
a
, Abriata LA
b
, Vila AJ
a
a
Instituto de Biologa Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioqumicas y
Farmacuticas, Universidad Nacional de Rosario-Conicet, Rosario, Argentina
b
Swiss Federal Institute of Technology, cole polytechnique fdrale de Lausanne (EPFL), Lausanne,
Switzerland
The dinuclear copper center Cu
A
is the electron entry point of the cytochrome c oxidase (COX). It funnels
the electrons from reduced cytochrome c to the Cu
B
center at subunit I of COX where O
2
is reduced to
water molecules. The correct assembly of this metal center is essential for the function of the complex
and thus for the survival of the cell.
Metallochaperones are proteins capable of binding copper ions and transfer them to their target proteins
that use them as a cofactor, and at the same time reduce the toxic effect. In the case of the assembly of
the Cu
A
site in humans, two proteins of the Sco family have been found as essential for its formation:
Sco1 and Sco2.
1
Both proteins binds copper ions through a CxxxCx
n
H motif, being able not only to transfer copper but also
able to transfer the electrons to maintain the Cys from the Cu
A
protein in its reduced state for the binding
of the copper ions.
2
Using a model of the eukaryotic oxidase, we followed by NMR the interactions between these two
putative metallochaperones finding that Sco1 is able to transfer to copper while Sco2 is important to
maintain the Cys ligands in the reduced state.
1- Robinson, N.J. and D.R. Winge, Annu Rev Biochem, 2010. p. 537-62.
2- Abriata, L.A., et al., Nat Chem Biol, 2008. p. 599.
BPA4_Rheological properties of regular insulin and aspart insulin Langmuir monolayers
at the air/water interface: condensing effect of Zn
2+
in the subphase.
Grasso, E.J., Oliveria, R.G., Maggio B.
Centro de Investigaciones en Qumica Biolgica de Crdoba (CIQUIBIC-CONICET), Dpto. de Qumica
Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba. Pabelln Argentina, Ciudad
Universitaria, X5000HUA, Crdoba, Argentina.
The interfacial behavior of regular insulin (Reg-insulin) and aspart insulin (Asp-insulin) was critically
affected by the presence of Zn
2+
in the subphase. This cation induced a condensed-like behavior in the
compression isotherms, especially apparent for Reg-insulin films when observed by Brewster angle
microscopy. Immediately after spreading, Reg-insulin, but not Asp-insulin, showed bright patches that
moved in a gaseous-like state. Moreover, Zn
2+
caused marked variations of the surface electrostatics of
both insulin monolayers and considerable hysteresis of their molecular organization. By oscillatory
compression-expansion cycles, we observed in all cases the development of a dilatational response to
the surface perturbation, and both monolayers exhibited well-defined shear moduli in the presence of
Zn
2+
, which was higher for Reg-insulin. Development of a shear modulus indicates behavior resembling a
nominal solid, more apparent for Reg-insulin than for Asp-insulin, suggesting the presence of viscoelastic
networks at the surface.
This work was supported by: CONICET, FONCyT and SECyT-UNC.
BPA SAB 2013
54
BPA5_Topological study of the -lactam-sensor proteins from Methicillin resistant
Staphylococcus aureus (MRSA).
Peris,D.; Belluzo,B.S.; Llarrull, L.I.
Instituto de Biologa Molecular y Celular de Rosario (IBR), Ocampo y Esmeralda, Predio CONICET -
(2000) Rosario
The membrane proteins BlaR1 and MecR1 play key roles in resistance to -lactam antibiotics in MRSA
strains. It is proposed that they sense and transduce the signal to unleash the transcription of the
resistance genes.
1
However, the topology of these proteins and the molecular events that are involved in
the transmission of the signal remain controversial. Here we report a topology study of the proteins
expressed in E. coli (where we have previously shown that BlaR1 is active) based in a strategy that
involves the construction of C-terminal fusions to EGFP.
2
This reporter does not fold correctly when
translocated to the periplasmic space through the Sec System and, therefore, it does not fluoresce when
fused to a periplasmic loop. In order to determine the number of membrane-spanning domains and the
localization of the different domains, we characterized the different fusion proteins by fluorescence
spectroscopy in whole cells and by fluorescence microscopy, and we normalized the expression of the
different constructions in E.coli by Western blot analysis.
Acknowledgements
The PEW Charitable Trusts, ANPCyT, CONICET
1- Llarrull, L.I., Fisher, J.F., Mobashery, S. Antimicrob. Agents and Chemother. 2009. 4051.
2- McKenzie, N.L., Nodwell, J.R. Microbiology. 2009. 1812.
BPA6_The structure of plant pri-miR172 and its complex with HYL1.
Suarez, IP
1
; Hbarnter, C
2
; Rasia, RM
1
1 Instutute of Molecular and Cellular Biology of Rosario (CONICET-UNR). Ocampo y Esmeralda, predio
CONICET, 2000, Rosario, Argentina.
2 Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Gttingen, Germany.
MicroRNAs are essential gene regulators in multicellular organisms. Plant miRNA precursors (pri-
miRNAs) are processed in the nucleus by a protein complex formed by DICER-LIKE1 (DCL1), HYL1 and
SERRATE. HYL1 is a double stranded RNA binding protein, with no catalytic activity, that ensures the
accuracy of the processing reaction. The mechanism by which itguides the RNAse DCL1 to the correct
location within the precursor is unknown. pri-miRNAs display highly variable secondary structure features
that could be essential to define the location of the miRNAs within their sequences. In this work we
characterized the secondary structure of Arabidopsis thaliana pri-miR172 and a variant that hinders
processing in vivo by means of enzymatic probing. The mutant shows an alteration in the secondary
structure distribution of the upper stem. We mapped the location of HYL1 within the precursor by RNAse
and DMS protection assays. The protein binds to the lower stem of the precursor, and the size of the
protected region suggests that both RNA binding domains bind in line on the precursor. Binding of HYL1
induces conformational changes on the upper stem region. Remarkably, the first nucleotide of the miRNA
sequence becomes particularly sensitive to DMS modification in the complex with HYL1. Altogether our
results show that HYL1 recognizes specifically the pri-miRNA structure and induces conformational
changes on distant parts of the precursor that contribute to the specificity of the processing reaction.
Acknowledgements: ANPCyT, CONICET, DAAD
SAB 2013 BPA
55
BPA7_Overexpression of the BlaR1 membrane protein implicated in resistance to -
lactams in Staphylococcus aureus
Belluzo, BS, Llarrull, LI
Instituto de Biologa Molecular y Celular de Rosario (IBR), Ocampo y Esmeralda, Predio CONICET -
(2000) Rosario
BlaR1 is a membrane receptor implicated in -lactam antibiotic resistance in Staphylococcus aureus. This
protein is composed of various transmembrana helixes, followed by a cytoplasmic domain and an
extracellular sensor domain capable of detecting de presence of antibiotic (1). The predicted cytoplasmic
domain contains Zn(II)-dependent protease activity and it has been proposed that it is activated in
response to binding of the antibiotic to the sensor domain (2). Activated BlaR1 degrades the gene
repressor BlaI, which triggers the expression of the BlaZ-lactamase. We are interested in the
spectroscopic study of the signal transduction mediated by BlaR1. In order to overexpress this protein, we
usedactive natural variants lacking the first three transmembrane helixes. We evaluated the expression
as fusions to different soluble tags that were shown to help in the overexpression of several membrane
proteins, and we are now developing a protocol of purification from inclusion bodies, denaturation and
refolding.
Acknowledgements
The PEW Charitable Trusts, ANPCyT, CONICET
1- H. Z. Zhang, C. J. Hackbarth, K. M. Chansky, H. F. Chambers, Science 291, 1962 (2001).
2- L. I. Llarrull, S. Mobashery, Biochemistry 51, 4642 (2012).
BPA8_Expression and purification of a novel flavohemoblobin from Mycobaterium
tuberculosis.
Piccinni, F.E
1,3
. Bonamore A.
3
, Marti M. A.
1,5
, Nadra A.D.
1,2
, Boffi, A.
3
and Estrin, Daro A.
4,5
1
Depto. Qumica Biolgica Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aire,
2
IQUIBICEN-CONICET,
3
Laboratory for advanced technology in the production of pharmaceuticals,
Department of Biochemical Sciences, Sapienza University, Roma, Italia.
4
Depto Qumica Inorgnica,
Analtica y Qumica Fsica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Argentina
5
INQUIMAE-CONICET
Flavohemoglobins, widely found in bacteria and yeast, have both a globin and a ferrodoxin reductase
domain (FAD-binding domain). The flavohemoglobin from E. coli has been thoroughly characterized as
an important feature against nitrosative stress. A novel flavohemoglobin studied in M. tuberculosis shows
some structural similarity to the latter one from E. coli. However, it is thought to have a different role in
protection against oxidative damage that does not include NO scavenging. The cell membrane is
protected by its cycling between the ferrous and ferric state due to a D-lactate dehydrogenase activity.
The globin domain can be easily obtained in BL21 DE3 Star E. coli strain. Since it is a rather soluble
protein, its purification consists of a two-steps column protocol and results in a high purity product.
Therefore, several characteristics can be studied simply by UV-visible spectrophotometry. The globin
domain shows a high affinity for oxygen and appears to be able to bind fatty acids in the ferric and ferrous
states. It can be oxidized with dithionite or ferricianide, which will allow further studies on the nature of the
binding (i.e.: by gas mass chromatography). The understanding of this protein might help unravel the
pathogenic pathways of M. tuberculosis.
1- Alessandra Bonamore and Alberto Boffi. IUBMB Life. 2008. p. 19.
2- Kanak L. Dikshit and col. The Journal of Biological Chemistry. 2012. p. 16435.
BPA SAB 2013
56
BPA9_Coagulation of casein micelles induced by bacterial enzymes: effect of cations
Lombardi, J
1,4
; Folmer, AP
2
, Brandelli, A
2
; Risso, P
1,3,4
; Boeris, V
1,4
1
UNR.
2
Universidade Federal do Rio Grande do Sul.
3
IFIR.
4
CONICET.
The aim of this study was to determine the effect of Ca
2+
, Na
+
, Zn
2+
and Mg
2+
on the milk coagulation
process induced by the enzymatic pool P7 (EPP7), secreted by Bacillus sp. P7. The significance of the
concentration of each cation on the time of coagulation (t
c
) and fractal dimension of clots obtained (D
f
)
was analized. The turbidity () of casein micelle (CM) suspensions in the range of 450-650 nm were
recorded. parameter, which is related to the particle size, was obtained from the slopes of the linear
graphs of log vs. log time (t). D
f
, which estimate the degree of compactness of the aggregates formed, is
the maximum value achieved and t
c
was determined as the time at which stabilizes after the sharply
rising. The effect of these four cations on the EPP7 enzymatic activity and on the initial size (initial ) of
the particles were also studied. The presence of Ca
2+
, Na
+
or Zn
2+
increased initial values, which
corresponds to the formation of larger CM. This makes sense since the shielding of the negative casein
charges facilitate their aggregation. The variables t
c
and D
f
proved to be also dependent of Ca
2+
, Na
+
and
Zn
2+
. On the other hand, Mg
2+
has not a significant effect on these variables (p>0.05). As the
concentration of Ca
2+
increased (up to 15 mM) the D
f
decreased; however, the increase in the
concentration of Zn
2+
(up to 0.25 mM) the D
f
increased. The concentration of Na
+
(up to 100 mM)
changes the effect of Zn
2+
on the D
f
: at higher NaCl concentration, less is the effect of Zn
2+
(the ionic
strength is probably the responsible of this change, not the Na
+
specifically). These three cations caused
a decrease of t
c
. This finding confirms that the t
c
is not directly related to the enzymatic activity of EPP7,
since Ca
2+
and Mg
2+
had a stimulatory effect on EPP7 activity, while Na
+
and Zn
2+
caused a decrease on
its activity. In conclusion, the cations evaluated have a differential effect on the CM aggregation process.
BPA10_Auto catalytic ROS dependent proteolysis of ICA512
Toledo P., Ros A., LLovera R., Primo E., Ermcora M.
Laboratorio de Expresin y Plegado de Protenas, Univ. Nac. de Quilmes
In previous works of our group several structural features of the membrane proximal ectodomain of
ICA512 (MPE ICA512; residues 469-557) were clarified and two main different association modes were
described; namely 4-4 and 2-2. These two possible interfaces of dimerization have similar
crystallographic properties but differ in critical aspects. While 4-4 comprises a region with high degree
of sequence conservation within proteins of the same family, 2-2 involves the surface responsible for
the dimers found in solution. To gain further insight in this topic an extended version of MPE ICA512
including residues 469 to 576 was prepared and characterized. The added residues link the ectodomain
to the transmembrane domain (residues 576-600). The new construction allowed to test hypothesis
regarding the topology or the ectodomain and its orientation on the membrane as well as to assess the
mechanism and participation of the added residues in the autocatalytic, ROS dependent, proteolysis of
the receptor reported in previous works by our group.
1- Primo ME, Klinke S, Sica MP, Goldbaum FA, Jakoncic J, Poskus E, Ermcora MR. J Biol Chem.
2008. 283:4674.
2- Primo ME, Jakoncic J, Noguera ME, Risso VA, Sosa L, Sica MP, Solimena M, Poskus E, Ermcora
MR. PLoS One. 2011. 6:e24191.
SAB 2013 BPA
57
BPA11_DSC and RMN approach to study the complex unfolding mechanism of a pdz
domain
Torchio, G
1,2
; Burgos, I
3,2
; Fidelio, G
3,2
; Arn, M
4,2
; Gallo, M
4,2
; Ermcora, M
1,2
; Sica, M
5,2
.
1.Laboratorio de Plegado y Expresin de Protenas, Univ. Nac. de Quilmes-IMBICE-CIC, Argentina
2.CONICET, Argentina. 3.Centro de Investigaciones de Qumica Biolgica,Univ. Nac. de Crdoba,
Argentina. 4.Fundacin Instituto Leloir, Argentina. 5.Centro Atmico Bariloche, Argentina
Many PDZ domains have been extensively studied from a biophysical perspective since they are
important and ubiquitous modules of protein-protein interactions, with particular characteristics of folding
and binding. Our model of study is the PDZ domain of the 2-syntrophin protein (2S-PDZ), which is
directly involved in the regulation of insulin secretion. We have shown that thermal unfolding curves of
2S-PDZ are not as expected for a protein that follows a two state nor a three state model of folding (1),
as reported for other PDZ domains. Moreover, the unfolding curves of 2S-PDZ are more reminiscent of
a downhill regime, or multiple state mechanism of folding (2-3).
Here we present additional evidence that supports the hypothesis of a complex folding mechanisms for
2S-PDZ. Differential scanning calorimetry (DSC) experiments discard two- and three-state models,
showing a more complex scenario. DSC experiments also rule out oligomerization during thermal
unfolding, at least at concentrations up to 2 mg/ml. Although some aggregation is detected at high
temperatures, this only seems to be associated to a minor population of 2S-PDZ and thermal
denaturation probed to be highly reversible, as a second and a third scan of DSC are completely
superimposable. Also NMR experiments suggest the existence of a minor population of 2S-PDZ that
could populate an alternative conformation.
(1) Torchio, G; Ermcora M; Sica, M. Biophysical J. 2012. p2835
(2) Oliva, F; Muoz, V. J. Am. Chem. Soc. 2004. p8596(3) Naganathan AN; Perez-Jimenez R; Sanchez-
Ruiz JM; Muoz V. Biochemistry. 2005. p7435
BPA12_Isolation and characterization of calreticulin oligomers
Moreschi A. C., Durand E. S., Montich G.G., Decca M.B.
Departamento de Qumica Biolgica-CIQUIBIC, Facultad de Ciencias Qumicas, Universidad Nacional de
Crdoba. Haya de la Torre y Medina Allende,5000, Crdoba, Argentina.
Calreticulin (CRT) oligomerization occurs in living cells as a response to stress, and leads to an
enhancement in chaperon and polypeptide binding activities of this protein. Recruitment of CRT
oligomers seems to be critical for the scaffolding of stress granules in cytoplasm, a mechanism that
regulates mRNA translation as a response to stress. Extended CRT oligomerization has been also
proposed to act as a large platform for binding of different substrates. Despite the importance of CRT
oligomers in expanding CRT functions, no structural characterization of CRT oligomers has yet been
performed.
Here we describe a method for isolation of CRT oligomers as well as its biophysical characterization.
Circular dichroism spectroscopy was used to determine structural properties of CRT oligomers in
comparison with monomeric CRT. We found that the global secondary structure of calreticulin is retained
in oligomers whereas tertiary structure is partially lost. Differential scanning calorimetry showed no
cooperative transition in the thermal denaturation of CRT oligomers, and a decreased thermal stability in
comparison with CRT monomers.
Acknowledgements: This work was supported by grants from the Agencia Nacional de Promocin
Cientfica y Tecnolgica, CONICET, SECyT Universidad Nacional de Crdoba.
BPA SAB 2013
58
BPA13_Towards regulation of peptide-DNA interaction using an allosteric molecule
Quirolo, Z. B.
a
;Mascareas, J.L.
b
;Dodero, V. I
a
a- Depto de Qumica- INQUISUR, Universidad Nacional del Sur-CONICET, Baha Blanca (AR). b-
CIQUS, Universidad de Santiago de Compostela (ES).
Cells receive a wide variety of cellular and environmental signals which must be processed to generate
specific and timely genetic responses. The first step in the expression of genetic information, namely the
synthesis of RNA from DNA template, is tightly regulated by DNA-binding proteins called Transcription
factors (TFs). One of them, is GNC4, a TFs found in yeast which recognizes it cognate DNA binding
domain as a helical dimer. The structural motif is composed by two regions: one responsible of
recognition named the basic region and the other is the leucine zipper domain responsible of
dimerization. Previously, we have used this model peptide to prove spatial and temporal control [1, 2].
Now, we envisaged the use of a truncated GNC4 peptide which possesses only the basic region
chemically modified at the N-terminal in order to promote dimerization and DNA recognition in the
presence of an allosteric molecule. This molecule possesses two -Cyclodextrins to promote peptide
dimerization and a linker based on an azobenzene moiety which is able to change its conformation upon
light irradiation. The chemical synthesis, structural characterization and the initial binding experiments are
presented.
Acknowledgements:Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Blanco J. B.; Dodero V. I.;Vzquez M. E.; et.alAngew. Chem. Int. Ed. 2006, 118, 8390.
2- Mosquera, J.; Jimnez-Balsa, A.; Dodero, V. I.; Vzquez, M. E.; Mascareas, J. L. Nature Comm,
2013, 4, 1874.
BPA14_Evaluation of 33-mer gliadin assemblies. Towards understanding its
immunomodulator role in gliadin related diseases.
Herrera, M. G.
a
; Lonez, C.
b
;Celej, S.
c
;Ritacco, H.
d
, Ruysschaert, J-M.
b
; Dodero, V. I
a
a
INQUISUR- UNS-CONICET, Baha Blanca (AR).
b
CBSB-Universit Libre de Bruxelles (BE).
c
Dto.
Qumica Biolgica, CIQUIBIC,CONICET, Fac. Ciencias Qumicas, UNC Crdoba (AR).
d
INFISUR-UNS-
CONICET, Baha Blanca (AR).
Gliadin, a protein present in wheat, rye and barley undergoes incomplete enzymatic degradation during
digestion, producing several peptides. One of them is a 33-mer peptide, LQLQPF(PQPQLPY)
3
PQPQPF,
a proline-rich fragment which elicits an immune response in susceptible individuals and it is associated
with gluten sensitivity and celiac disease [1]. Recently, our group has demonstrated that the 33-mer
gliadin peptide is able to self- assemble into nanospheres which align into fractal linear arrays above a
certain concentration under physiological conditions [2]. Herein, the evaluation of 33-mer self-assembly
system by fluorescence spectroscopy techniques, ATR-FT-IR and DLS is presented. Interestingly, 33-mer
assemblies are able to induce NFk, a transcription factor related to inflammatory reactions, in a dose
dependent manner. Our results demonstrate a relation between 33-mer assemblies and inflammation,
which can be relevant in the early steps of gliadin intolerance disorders.
Acknowledgements:Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Sapone, A. et al. BMC Medicine. 2012, 10:13.
2- Herrera, M. G., Zamarreo, F., Costabel, M., Ritacco, H., Htten, A., Sewald, N., Dodero, V. I.
Biopolymers. 2013, in press.
SAB 2013 BPA
59
BPA15_Catalytic activity of beta galactosidase entrapped in a silicate matrix. Effect of
the substrate type, porous structure and aging time.
Burgos, M.I.
a
, Carrer, D.
b
, Perillo, M.A.
a
a: Instituto de Investigaciones Biolgicas y Tecnolgicas (IIBYT, CONICET-Universidad Nacional de
Crdoba). b: Instituto de Investigacin Mdica Mercedes y Martn Ferreyra.
Protein encapsulation in solid matrixes is of interest for biotechnological purposes and it also serves as a
model of molecular crowding. We have successfully entrapped the enzyme -galactosidase (-gal) in
silicate gels via a sol-gel reaction. The catalytic activity of encapsulated -gal (E-gal) was compared with
the activity of the soluble form (S-gal) with two different substrates (ONPG and PNPG) and at different
aging times. Data were analyzed with a Michaellis-Menten model. With the substrate ONPG, we found a
Vmax value higher for E-gal than for S-gal and this difference increased at longer aging times. On the
contrary, with PNPG, Vmax was not affected neither by the enzyme condition (S-gal and E-gal) nor by
the aging time. The porous structure of the silicate matrix was also studied as a function of aging time of
the gels. Thus, fluorescence correlation spectroscopy (FCS) was applied through the analysis of
diffusional properties of fluorescent probes of different sizes, entrapped in the gel matrix. We observed an
anomalous diffusion in some conditions, suggesting a self-similar pore structure, and aging time
dependent changes in diffusion coefficients. Taken together our result supports the hypothesis that there
is a relationship between the catalytic mechanism proposed for -gal and water availability in the silicate
gel since the ratio between bulk and structured water depends on the pore size.
Acknowledgements: Authors thank CONICET, foncyt and Secyt-UNC for the financial support. MIB is a
postdoctoral fellow and MAP and DC are career members of CONICET.
BPA16_The Thermal Unfolding of 2-Glycoprotein
Soledad Bazn, Mariana Paolorossi and Guillermo Montich
Departamento de Qumica Biolgica, Facultad de Ciencias Qumicas, Universidad Nacional de Crdoba,
CIQUIBIC (CONICET)
2- Glycoprotein I (B2GPI) is an abundant glycoprotein of human plasma. It participates in blood
coagulation processes and the clearance of phosphatidylserine exposing- apoptotic cells. B2GPI is also
the major antigen involved in the aberrant immune response that characterizes the antiphos-pholipid
syndrome.
B2GPI consists of a single chain of 326 aminoacids arranged in five domains. Four of them are the so
called sushi or short consensus repeat (SCR) domain with its conserved beta sandwich- arrangement.
The fifth domain is larger and it has a lysine rich region that was shown to participate in the interaction of
B2GPI with anionic lipids. B2GPI has four N-glycosilation sites with complex bi and triantennary complex
glycans containing sialic acid that comprise ~20% of its total weight.
To study the folding behavior of this multiple domain glycoprotein we performed differential scanning
calorimetry at various pH and salts conditions. We found that at pH 7 the protein unfolding proceeds with
a pre-transition at lower temperatures. This effect is more pronounced in the absence of salt. In order to
characterize this earlier intermediate we perfomed circular dichroism studies of the thermal unfolding of
B2GPI in the far and near UV region. The far UV signal shows a two state unfolding transition centered at
the same temperature of the calorimetric one. In contrast, the near UV signal shows a more complex
behavior. These results would indicate that the folding intermediate has a different tertiary structure
protein while conserving its secondary one.
Acknowledgement: S.B. wants to thank FONCyT for it postdoctoral fellowship. We also wish to
acknowledge the following institutions for its support: CONICET, SECyT- UNC and FONCyT.
BPA SAB 2013
60
BPA17_Lactose binding kinetics of the -galatosil binding protein Galectin-1
Romero J. M.
1
, Estrin D. A.
2
, Rabinovich G. A.
3
, Di Lella S.
1,3
1
INQUIBICEN-CONICET, FCEN-UBA;
2
INQUIMAE-CONICET, DQIAyQF, FCEN-UBA,
3
Laboratorio de
Inmunopatologa, IBYME-CONICET. juanm.romero18@gmail.com
Galectin-1 is a -Galatoside binding protein involved in cell comunication and diferentiation processes.
1
It
consists of a 134-aminoacid domain which has been proved to form a homodimer at physiological
conditions.
2
In a previous work, we have studied the thermodynamics of lactose binding and the simmetry
of the homodimer applying computational simulation techniques
3
. Now we employ guided molecular
dynamics to characterise the binding and unbinding process, paying special attention to the molecular
determinants of the kinetic barrier, which turns out to be mainly the result of hydrogen bonds breaking and
forming. Furthermore, we measure the association and dissociation rate constants by stopped flow
experiments. We find both constants to be 206 M
-1
.s
-1
and 0,98 s
-1
, respectively, and correlate them with
the calculated energy barriers, while discussing results with previous reported kinetics experiments.
4
Acknowledgements: Conicet, ANPCyT, UBA, Fundacin Sales, Consejo Interuniversitario Nacional, and
Dr. Madia Trujillo
1- Rabinovich GA and Toscano MA, Nat. Rev. Immunol, 2009.
2- Lpez-Lucendo et al, J Mol Biol, 2004.
3- Romero JM and Di Lella S, Unpublished results, 2012
4- Ghler et al, Biophysical J., 2010
BPA18_Tinkering with coordination in globins
Boron, I.
1,2
, Chisari, L.
1
, Wetzler, D. E.
1
, Capece, L
2
, Marti, M
1,2
, Estrin, D
2
, Nadra, A
1
1
Departamento de Qumica Biolgica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Buenos Aires, Argentina, C1428EGA
2
Departamento de Qumica Inorgnica, Analtica y Qumica Fsica, INQUIMAE-CONICET, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, C1428EGA
Background. Human Myoglobin (Mb) and hemoglobin are relevant examples of pentacoordinated (5c)
heme proteins and many of their biological functions involve the coordination of exogenous ligands in the
sixth position of the heme group. On the other hand, in several globins direct coordination of HisE7 to the
Fe atom leads to the formation of a hexacoordinated (6c) globin, as noted in neuroglobin (Ngb) and
cytoglobin, among others. It has been suggested that this bond and the equilibrium between the 6c and
the 5c state regulate ligand affinity. Recent work by Nadra et al. has shed light on the key determinants
for globin hexacoordination, engineering Mb and Ngb by swapping their CD region. A partially 6c
myoglobin and a subtly 5c Ngb shown that each chimeric protein shifted its coordination state according
to its CD region. Goal. With the aim of further displacing the coordination equilibrium, we designed point
mutations to enhance the observed behaviour. Results. By exploring the contacts stabilizing the CD
region we have found a contact between a lysine and a glutamic acid which may account for Mb's
preference for the 5c state. Molecular dynamics simulations, equilibrium spectroscopy and ligand binding
results for a chimeric Ngb punctual mutant suggest differences in agreement with the theoretical
predictions.
SAB 2013 BPA
61
BPA19_Effect of molecular crowding on the conformation and thermal stability of -Gal
Nolan, MV and Perillo, MA
IIByT (CONICET-UNC), ICTA and Ctedra de Qumica Biolgica, Dpto. de Qumica, Facultad de
Ciencias Exactas, Fsicas y Naturales(UNC). e-mail: vnolan@efn.uncor.edu
In previous works we studied the effect of molecular crowding on beta galactosidase (-Gal) enzymatic
activity. The results showed that V
max
was just slightly affected, while the affinity of the enzyme (K
M
)
suffered a significant decrease at growing molecular crowding levels. In that opportunity we also found,
as a first approach to the study of the enzyme conformation in crowded systems that -Gal fluorescence
spectrum suffered a red shift when the crowding agent concentration was increased from 0 to 35 % W/V.
In the present work, studies on the conformation and thermal stability were carried out using both infrared
(IR) and fluorescence spectroscopy were done to investigate possible changes on the enzyme structure
due to overcrowding using polyethylene glycol molecular weight 6000 (PEG
6000
) was used as crowding
agent. The -Gal IR spectrum showed an important diminution in the main -structure band (at around
1620 cm
-1
) when the molecular crowding agent concentration was increased up to 35 % W/V. At the
same time, typical bands corresponding to disordered structures appeared. On the other hand, the effect
of crowded systems on -Gal thermal stability was noticeable, prevented the typical protein aggregation
that occurs in proteins denatured by temperature and denaturation occurred in a less cooperative way.
Taken together our results showed that in crowded conditions -Gal is thermodynamically trapped in a
conformation more open, flexible and stable than in dilute solutions.
Acknowledgements: we thank Dr Montich for the access to the IR equipment. Work financed with grants
from CONICET, Foncyt, SeCyT-UNC. MVN and MAP are career members of CONICET.
BPA20_Thermal inactivation of the NA
+
,K
+
-ATPase, ATP and Mg
2+
effects.
Placenti, M.A., Kaufman, S.B. Gonzlez Flecha, F.L. And Gonzlez-Lebrero, R.M.
IQUIFIB-Departamento de Qumica Biolgica, Facultad de Farmacia y Bioqumica, Universidad de
Buenos Aires, Junn 956, 1113, C.A.B.A., Argentina. E-mail: aguedaplacenti@gmail.com
Folding and stability are key factors for the proper biological function of proteins, therefore the importance
of its study. 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. During this catalytic cycle, the enzyme interconvert
between two conformers, E
1
and E
2
.The aim of this work is to characterize the effect of natural ligands
(ATP and Mg
2+
) on the thermal inactivation of Na
+
,K
+
-ATPase. In a typical thermal inactivation
experiment, the enzyme is incubated for different time periods at several temperatures in the presence or
absence of the ligand. The functional or structural integrity of the protein is followed by measurement of
some property, in our case, ATPase activity, Trp fluorescence and circular dichroism spectroscopy. We
observed that thermal inactivation in all conditions tested followed a first-order kinetic. The decrease of
ATPase activity is concomitant with the conformational change detected by Trp fluorescence.
Additionally, circular dichroism experiments showed a slight change in the secondary structure when the
ATPase was fully inactivated. A clear stabilization of the enzyme was observed when ATP or Mg
2+
was
present in the incubation media. Even though Na or ATP are known to displace the equilibrium of the
enzyme towards E
1
conformer, our results showed that those ligands have opposite effects in terms of
thermal stability of the Na
+
,K
+
-ATPase.
With grants from: UBACyT, CONICET and ANPCyT.
BPA SAB 2013
62
BPA21_Analysis of the interaction between dengue virus NS3 helicase and ssRNA under
equilibrium conditions
Incicco JJ
1
, Gebhard LG
2
, Gonzlez-Lebrero RM
1
, Gamarnik AV
2
, Kaufman SB
1
1
Instituto de Qumica y Fisicoqumica Biolgicas y Departamento de Qumica Biolgica, Facultad de
Farmacia y Bioqumica, Universidad de Buenos Aires, Ciudad de Buenos Aires, Argentina.
2
Fundacin
Instituto Leloir-Consejo Nacional de Investigaciones Cientficas y Tcnicas, Ciudad de Buenos Aires,
Argentina.
Dengue virus nonstructural protein 3 (NS3) is a molecular motor protein that unwinds RNA duplexes
driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This RNA-helicase was
shown to bind preferentially to single stranded (ss) RNA along which it would move with 3' 5'
directionality. Here we report results from the study of the interaction between dengue virus NS3 helicase
and ssRNA with a quantitative fluorescence titration technique.
Binding experiments were performed with a series of fluorescein-labeled ssRNA oligomers of different
lengths. Model-independent analysis of the titration curves provided estimates for the stoichiometry and
the macroscopic equilibrium constants. Experimental results were well described by a statistical
thermodynamic model that takes into account the overlap of potential binding sites and cooperative
interactions between adjacent NS3 molecules. Global fitting of this model to all titration curves gave a
binding site size of 9.1 0.1 bases per NS3h; an intrinsic association constant of 0.0048 0.0001 nM
-1
and a cooperativity factor of 3.4 0.7.
(With grants from Universidad de Buenos Aires, from CONICET and from ANPCyT, Argentina).
BPA22_GOSPEL enhances in vitro nitric oxide-induced aggregation of GAPDH
Gonzlez, M.C., Ingaramo, M.C., Romero, J.M., Curtino, J.A. and Carrizo, M.E.
CIQUIBIC-CONICET, Depto. Qumica Biolgica, Facultad de Ciencias Qumicas., U. N. de Crdoba. E-
mail: ecarrizo@fcq.unc.edu.ar
Among its many functions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a proapoptotic role
associated with oxidative and nitrosative stress. Nitric oxide stress leads to reversible S-nitrosylation of
the active site cysteine of GAPDH. When S-nitrosylated GAPDH binds to the E3-ubiquitin-ligase Siah1
which possesses a nuclear localization signal that promotes the translocation of the complex to the
nucleus, where both proteins have cytotoxic effects. This cascade is regulated by S-nitrosylated GOSPEL
(GAPDH's competitor Of Siah Protein Enhances Life) whose association with GAPDH prevents the
cytotoxic interaction GAPDH-Siah1 (1).
Oxidative stress, produced either by hydrogen peroxide or nitric oxide, can also promote the formation of
amyloid-like aggregates of GAPDH through disulfide bonds involving its active site cysteine (2). It is worth
mentioning that GAPDH has also been found deposited as insoluble aggregates in some
neurodegenerative diseases.
Here we present the analysis of the effect of GOSPEL on the in vitro aggregation of GAPDH induced by
nitric oxide. Our results indicate that, under these conditions, both proteins co-aggregate and that the
aggregation of GAPDH was enhanced in the presence of GOSPEL. Our work also includes the
characterization of the aggregates obtained and the study of the nature of the interaction between the
proteins therein.
1. Sen, N. et al. (2009) Neuron 63, 81-91.
2. Nakajima, H. et al.(2007) J. Biol. Chem. 282, 2656226574.
SAB 2013 BPA
63
BPA23_Spying into the supramolecular arrangement of -synuclein amyloid oligomers
Gallea JI and Celej MS
Dto. de Qumica Biolgica,CIQUIBIC,CONICET, Facultad de Ciencias Qumicas, Universidad Nacional
de Crdoba.
Amyloid oligomers are the most toxic species in many devastating neurodegenerative disorders, which
are characterized by the misfolding and pathological aggregation of a particular polypeptide chain. In
Parkinsons disease the protagonist is the 140-aa presynaptic protein -synuclein (AS). Very little is
known about the structure of AS oligomers and their mechanisms of toxicity.
We showed that AS oligomers adopt a distinctive antiparallel -sheet structure. In this work, we use site-
directed fluorescence-labeling to define a map of cooperatively folded units and intermolecular proximities
in AS oligomers.
Spectral analysis of Acrylodan-labeled AS oligomers showed that all the labeled positions along the
sequence are shielded from the solvent, in contrast to reported results. Complementary GdmCl
denaturation experiments revealed two distinguishable cooperatively folded units. Excimer formation in
Pyrene-labeled AS oligomers revealed close proximity of positions 9 and 76 in adjacent molecules. Our
results will contribute to model the supramolecular structure of AS oligomers, and therefore, to the
understandig of the structure-based toxicity of such species.
Acknowledgements: Drs. Bertoncini and Jovin for proving us the several Cys-AS variants. SECyT-UNC,
FONCyT, CONICET and ISN-CAEN for financial support.
1- Celej, MS et al.,.Biochem J. 2012. 719.
2- Van Rooijen, BD. J Mol Biol. 2009. 826.
BPA24_The Supramolecular structure of gliadin protein is modulated by pH: toward
understanding the molecular triggers of gliadin related pathologies.
Herrera, M.
.
G.; Veuthey, T. V.; Dodero, V.I
a
INQUISUR- UNS-CONICET, Baha Blanca (AR).
Gliadin has been described as the most toxic protein fraction of gluten related with different human
diseases, such as celiac disease, durhing disease, ataxia and gliadin sensibility and allergy, among
others [1, 2]. It has been hypothesized that gliadin can reach the intestinal lumen inducing an increase in
intestinal permeability that seem to be a critical and early event in the pathogenesis of these diseases [3].
Although it is known the ability of gliadin to form high molecular weight aggregates [4], it has not been
performed a complete characterization of those aggregates. Considering the relevance of pH in the
digestion process, we decided to carry out a supramolecular evaluation of gliadin at different pHs by a
physicochemical approach. The aim will be directed to shed light on the molecular self-organization
depending on environmental stimulus. Herein, the evaluation of gliadin aggregates by a combination of
UV-Vis, steady state fluorescence, dynamic light scattering, optical and electron microscopy is presented.
Acknowledgements:Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Rubio-Tapia, A.; Murray, J. A. Curr opin gastroenterol. 2010, 116.
2- Sapone A, Bai J.C, Ciacci C, et al. BMC Med.2012, 10, 13.
3- Hadjivassiliou, M.; Williamson, C. A.; Woodroofe, N. Trends in immunol.2004, 25, 578.
4- Kasarda D.D, Bernardin J.E, Thomas R.S. Science. 1967, 203.
BPA SAB 2013
64
BPA25_Growth Hormone Releasing Hexapeptide is able to form Nanotubes: a
complementary study by Small Angle X-Ray Scattering, Transmission Electron
Microscopy and Molecular Dynamic Simulations
Barbosa, LRS
1
, Santana, H
2
, Avila, CL
3
, Cabrera, I
4,5
, Pez, R
2
, Falcn, V
2
, Pessoa, Jr. A
6
, Ventosa, N
4,5
,
Veciana, J
4,5
, Itri, R
1
1
Inst. de Fsica, USP, Brazil.
2
Dept. of Pharmaceutical Technology, CGEB, Cuba.
3
INSIBIO, CONICET-
UNT, Argentina.
4
Dept of Molecular Nanoscience and Organic Materials (ICMAB-CSIC), Spain.
5
CIBER
de Bioingeniera, Biomateriales y Nanomedicina (CIBER-BBN), Spain.
6
Dept of Biochemical and
Pharmaceutical Sciences, School of Pharmaceutical Sciences, USP, Brazil.
Growth hormone releasing peptide belongs to a class of synthetic growth hormone secretagogues, which
stimulate growth hormone secretion from somatotrophs in several species including humans. Besides, it
was recently used in vivo in the treatment of amyotrophic lateral sclerosis (ALS) disease. In the present
study, we investigated the self-assembling properties of GHRP-6, using small angle X-ray scattering,
transmission electronic microscopy (TEM) as well as molecular dynamics simulation. The combined
results demonstrated that GHRP-6 self-assembles into very long nanotubes, with inner and outer cross-
sections of 7(1) and 13(1) nm, respectively, at 20 mg/mLin phosphate buffer solution at pH 7.4. At
concentrations > 30 mg/ml, both SAXS and cryo-TEM results indicate that the shape of the long
aggregates remains the same and a bundle of nanotubes are disposed in a hexagonal arrangement in
solution, with a very well defined center-to-center distance of circa15 nm, according to SAXS data
analysis These results indicates that GHRP-6 self-assembles like dimers in the cylinder wall. Molecular
dynamics simulations also reflect the capability of the peptides to self-assemble like amphiphilic
molecules in aqueous solutions, giving additional details about the arrangement of the peptides within the
nanotube.
BPA26_Structural characterization of heparin-induced GAPDH protofibrils preventing -
synucleinoligomeric species toxicity
Avila, CL
1
; Torres-Bugeau, CM
1
; Barbosa, LRS
4
, Morand Sales, E
4
; Ouidja, O
2.3
, Socas B
2,3
; Raisman-
Vozari, R
2
, Papy-Garcia, D
3
, Itri R
4
, Chehn R
1
1 INSIBIO and Inst.deQca Biolgica Dr Bernab Bloj (CONICET-UNT). 2 INSERM. Thrapeutique
Exprimentale de la neurodgnrescence. 3 Laboratoire CRRET, Universit Paris EstCrteil, 4 Instituto
de Fsica da Universidade de So Paulo
Oligomeric arrangement of -synuclein has been associated to neuronal cell loss in neurological
diseases. Although -SNis mainly found in the cytosol, the presence of misfolded or aggregated -
SNoutside the cell suggests that it might play a role also at the extracellular level. Glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and glycosaminoglycans (GAGs) have been found associated to -
SN amyloid aggregates in PD. However, it remained to be determined whether the interplay between
GAGs, GAPDH and -SN may exert a protective or a deleterious role on the development or evolution of
PD. We demonstrate that -synuclein oligomers toxicity on cultured human dopaminergic neurons can be
prevented by a newGAPDH specie formed in the presence of heparin. Structural characterization,
performed by SAXS shows a cylinder-like specie with average size of 22 x 12 nmcompatible with a
protofibril. Using computational modelling we obtained the first all-atom model of the GAPDH protofibril
capable to satisfy all the experimental restrictions derived from SAXS and mass spectrometry. Moreover,
we propose a pathway for GAPDH aggregation involving the loss of the tetrameric state of the enzyme
with a concomitant appearance of native-like dimeric species, which quickly assemble to protofibrils. It
seems plausible that heparansulphates from brain extracellular matrix can effectively interact with
GAPDH and induce the formation of protofibrils able to scavenge -SN displaying an important role in
cellular proteostasis.
SAB 2013 BPA
65
BPA27_Functional and structural comparison between protofibrils of rabbit muscle and
human GAPDH
Defonsi, E, Vera, C, Chaves, S, Torres-Bugeau, CM, Minahk, CJ, Bellomio, A, Avila, CL,Chehin, R.
Instituto Superior de Investigaciones Biolgicas (INSIBIO), CCT-Tucumn and Instituto de Qumica
Biolgica Dr Bernab Bloj (CONICET-UNT) Chacabuco 461 (T4000ILI) Tucumn, Argentina
GAPDH is an ubiquitous enzyme that in addition to its central role in energy production presents highly
diverse non-glycolitic functions in the intra or extracellular space. Several independent studies suggest
that it might also be related to neurodegenerative diseases. We recently reported that highly sulfated
GAGs like heparin and heparin sulphates, are able to trigger GAPDH amyloid aggregation. The GAPDH
protofibrils formed during the early stages of the aggregation process are able to efficiently accelerate -
Sinuclena (AS) aggregation. We proposed that these protofibrils could play an important role
sequestering the AS oligomeric species involved in amyloid disease spreading in the extracellular space.
Still, all the experiments were performed using commercially available GAPDH from rabbit muscle. In this
way the study of the ability of protofibrillar species from human GAPDH to neutralize AS toxic species
acquire relevance. In the present work we have expressed and purified human GAPDH and compared
the aggregation kinetics of human GAPDH vs. rabbit muscle GAPDH. Structural comparison of these
proteins as well as their aggregation intermediates and fibrils were performed by spectroscopic
techniques. The ability of the different protofibrils to neutralize the membrane perturbation capability of AS
oligomers was also compared. The relevance of the different mutation of human and rabbit GAPDH in
heparin binding and aggregation was assessed using bioinformatic tools. This study represents a
milestone in the assessment of the feasibility of using heparin-induced-GAPDH protofibrilsas a
therapeutic agent in the treatment of PD.
BPA28_Loop insertions as a tool to study folding and stability of human frataxin
Noguera, M.E., Roman, E.A., Faraj S.E. and Santos, J
Instituto de Qumica y Fisicoqumica Biolgicas (CONICET-UBA), Junn 956, Ciudad Autnoma de
Buenos Aires, Argentina.
A complete description of the protein structure requires, in addition to the average localization of all of its
atoms, the changes in time of these positions, i.e. its dynamic behavior, and ultimately the
characterization of the complete native ensemble, including low-populated conformations. The study of
protein dynamics is crucial to understand biological function. Throughout the structural study of the
human frataxin, we found that there is a relationship between the dynamics of the C-terminal region
(CTR) and loop-1, the segment linking the helix-1 with the strand-1 of this single domain protein:
sequence mutations that affect the dynamics of CTR, also alter the dynamics of loop-1.
In this work, we explored these relationships through the manipulation of protein sequence: introducing
mutations in the loop-1 region to alter its local dynamics, and assessing its effects in the dynamics of
other regions of the protein.First of all, we investigated whether the loop-1 is tolerant to the sequence
changes, without severe alterations in the stability of the native state. For this purpose, we designed two
variants: in one of them, the only two residues which establish tertiary contacts with the rest of the protein
were mutated to alanine, whereas in the second variant, substitutions and insertions were performed
resulting in a loop extension with six new glycine residues. Both variants were characterized by
spectroscopic and hydrodynamic methods, and both were shown to be well folded, stable, and
monomeric. These results will be presented along a preliminary assessment of the influence of these
mutations in conformational dynamics.
BPA SAB 2013
66
BPA29_Stability of the ATP binding domain of a hiperthermophilic Cu(I) transport
ATPase against sodium dodecyl sulfate
Alvaro A. Recoulat Angelini and F. Luis Gonzlez Flecha
Laboratorio de Biofisica Molecular. IQUIFIB Departamento de Qumica Biolgica, Universidad de
Buenos Aires - CONICET, Argentina.
In this work, we characterize the stability against the detergent sodium dodecyl sulfate (SDS) of the ATP
binding domain (ATPBD) of the Cu(I) transport ATPase from Archaeoglobus fulgidus CopA, a
hiperthermophilic -helical membrane protein that is involved in transporting Cu+ throughout biological
membranes. To accomplish this, we carry out experiments registering the intrinsic fluorescence of the
unique tryptophan resildue as a function of the detergent concentration. A decrease in the fluorescence
was observed in the range of 0.03 1.6 mM of SDS with 3 different phases in the downfall. This decrease
was proving to be reversible by dialysis and even by dilution of the sample, which recover the original
fluorescence of the protein. We also observed that intrinsic anisotropy increased in the range 0.2 - 1 mM
SDS, indicating that the SDS-denatured protein acquire a rigid structure. Finally, we explore the
interaction of CopA-ATPBD with the fluorescent probe 1-anilino-naphthalene-8-sulfonate (ANS),
observing an initial increase of the ANS fluorescence until 0.3 mM SDS followed by decrease up to a
constant value at 2-3 mM SDS. We can hypothesize that SDS-induced unfolding of CopA-ATPBD
proceeds through an intermediate state that acquire an expanded -molten globule like- globular structure,
holding the ANS in a more hydrophobic environment.
With grants from UBACyT and ANPCyT
BPA30_Quantitative analysis of local dynamics of the C-terminal region of human
frataxin
Faraj, SE;
1
Aran, M;
2
Gallo, M;
3
Santos, J.
1
(1) Instituto de Qumica y Fsicoqumica Biolgicas (UBA-CONICET). (2) Fundacin Instituto Leloir. (3)
Dipartimento di Scienze e Tecnologie Chimiche, Universit di Roma Tor Vergata, Italy.
Friedreich's ataxia is a progressive neurological hereditary disease, caused by the deficiency in the
activity of frataxin (FXN), a mitochondrial iron chaperone that regulates the transference of metal ions to
other proteins. Previous studies from our laboratory found that the C-terminal region (CTR) is a crucial
element in the stabilization of FXN. The local unfolding of the CTR may enable the protein to smoothly
modulate its dynamics and stability, acting as a conformational lock.
In an attempt to quantify the stability of the interaction between the CTR and the rest of the domain, we
replaced, one at a time, three Leu residues from the CTR which establish hydrophobic interactions with
core residues by Cys residues. Cys mutants are useful tools to analyze local dynamics through the
reactivity of their free thiol, allowing us to infer the dynamics at particular positions (in which Cys are
placed). We followed the reaction of Cys modification by DTNB (Ellmans reagent), and found that
different positions possess varying rates of modification. Our results indicate that the difference in free
energy of local unfolding of the CTR is half the global stabilization energy. Besides, molecular dynamics
simulations results are consistent with our experimental observations, and allow us to estimate average
accessibility of Cys residues for each variant, to assess the effective contribution to observed reactivity
from local unfolding events. Altogether, these results were analyzed side by side with wild-type protein
and L198R mutant NMR relaxation dispersion experiments.
Acknowledgements: ANPCyT, UBACyT and CONICET.
SAB 2013 BPA
67
BPA31_Cool adaptation: An electrostatic switch defines the signaling state of
thermosensor DesK
Inda, Mara Eugenia
1
, Michel Vandenbranden
2
, Diego de Mendoza
1
, Jean-Marie Ruysschaert
2
, Larisa
Cybulski*
1
.
1- Instituto de Biologa Molecular y Celular de Rosario (IBR)- CONICET and Departamento de
Microbiologa, Facultad de Ciencias Bioqumicas y Farmacuticas, UNR, Rosario, Argentina.
2- Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and
Bioinformatics, Universit Libre de Bruxelles, Brussels, Belgium.
DesK is a multipass transmembrane protein that allows the bacterium Bacillus subtilis to adjust the levels
of unsaturated fatty acids required to optimize membrane lipid fluidity after a cold stress. This is achieve
by its bifunctional cytoplasmic catalytic domain that alternates between kinase/phosphatase activity.
Temperature sensing, however, involves a built-in instability caused by a group of hydrophilic residues
located near the N-terminus of the first transmembrane (TM) segment. These residues detect changes in
membrane thickness that occur as a consequence of temperature variations, promoting the required
conformational change
1
. Nevertheless, the core question, still remains: how is the information sensed by
the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain that allows
switching between kinase and phosphatase states? At the inner interphase, there is a linker region that
connects the TM sensing domain with the cytoplasmic catalytic domain. A helix/random coil
conformational duality is mechanistically exploited to transmit temperature-dependent conformational
changes from the transmembrane to the intracellular region. By combining genetic, spectroscopic and
biochemical techniques the role of the linker in DesK signal transduction is studied. We also assess the
role of lipids charges variations as well as ionic force in the interaction of the linker with the membrane
inner phase as additional factors that modulate kinase to phosphatase activity ratio.
1. Cybulski, Martin, Mansilla, Fernandez, de Mendoza. Curr. Biology. 2010. 1539
BPA32_Quaternary Structure of HPRT Trypanosoma cruzi: A role for the C-terminal
region?
Valsecchi, WM
1
; Santos, J
1
; Delfino, JM
1
1
Departamento de Qumica Biolgica, IQUIFIB (UBA-CONICET). FFyB, UBA.
Hypoxanthine/guanine phosphoribosyl transferase (HPRT), a globular / protein of 221 amino acid
residues, catalyzes the recovery of hypoxanthine and guanine, and is essential for the survival of
trypanosomatids. It was described in literature that the asymmetric unit of the highest resolution crystal
contains an HPRT dimer where subunits are closely associated. However, dynamic light scattering (DLS)
experiments performed in our laboratory indicate that HPRT behaves as tetrameric structures in solution.
In this work, we introduce our first structure determined at 2.65 that, interestingly, shows that HPRT
crystallized as a tetramer. Remarkably, the experimental model obtained lacks the C-terminal region (CR)
of the protein (residues 198-229), pointing to the high mobility of this stretch of residues. Based on the 3D
structure, preliminary MDS results and controlled proteolysis experiments, we hypothesized that CR
interactions may participate in the stabilization of the quaternary structure of HPRT. In addition, we
studied the conformation by CD and multimerization state (DLS) of mutant F164W. Enzymatic activity
assays were also performed. In this mutant, a Phe side-chain located in the active site was replaced by
Trp providing a local sensor: the fluorescence quantum yield of a unique Trp residue in the protein.
Altogether these results indicate that, as well as wild-type, F164W behaves a tetramer, is active, and well-
folded. To further understand the molecular basis of the catalytic process, our main goal will be to analyze
the effect of different ligands on the stability and the activity of HPRT.
Acknowledgements: ANPCyT, UBACyT and CONICET.
BPA SAB 2013
68
BPA33_Solvent-mediated assembly of new chemically modified Phe-Phe-Cys peptides
Sequeira, M. A.; Learte, S.; Dodero, V. I
Depto de Qumica- INQUISUR, Universidad Nacional del Sur-CONICET, Baha Blanca (AR).
Self-assembly is a natural process that spontaneously and reversibly organizes molecular units into
ordered structures through non-covalent interactions. Among all biological molecules that can self-
assemble, peptides, specifically dipeptides and tripeptides, offer numerous advantages such as chemical
variation and biocompatibility therefore they can be used in various biological and non-biological
applications [1, 2]. Previous work have defined dipeptide system composed by Phe-Phe as a central motif
for the formation of ordered self-assembled tubular, spherical and two-dimensional structures at the nano-
scale [3]. Herein, we present the synthesis and supramolecular characterization of new Phe-Phe-Cys
derivatives which undergo solvent-mediated assembly. In order to establish a relation between structure
and morphology of the aggregates, it has been incorporated different functionalities at the N-terminal.
Additionally, the cysteine located at the C-terminal allows the chance to modulate aggregation based on
external stimulus, due to cysteine oxidationreduction capabilities promoted by pH and additives
changes.
Acknowledgements: Supported by UNS, CONICET, DAAD, and ME-SPU (17-16-296) grants.
1- Ozin, G. A.; Hou, K.; Lotsch, B. V.; Cademartiri, L.; Puzzo, D. P.; Scotognella, F.; Ghadimi, A.;
Thomson, J. Mater. Today2009, 12, 12.
2- Demirel, G.; Malvadkar, N.; Demirel, M, Langmuir Letter2010, 26(3), 1460.
3- Reches, M.; Gazit, E. Phys. Biol. 2006, 3, S10.
BPA34_Design and characterization of specific p38 peptide inhibitors
Bucci, H.A.
1
, Lopez, E.D.
2
, Turjanski, A.G.
2
, Wetzler, D.E.
1
1
Departamento de Qumica Biolgica, IQUIBICEN, FCEN UBA
2
INQUIMAE, FCEN 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 study of
the specificity of peptide binding to MAPKs is relevant not only for the design of specific inhibitors but also
for understanding the mechanisms of protein-protein recognition in this signaling network. This work
consists on designing specific p38 peptide inhibitors by computational methods to be tested in vitro
using biophysical techniques. Based on the crystal structure of p38 complexed to the docking site of the
transcription factor MEF2A, we calculated the binding energy of wild-type and point mutation peptides.
We propose four potential inhibitors with higher binding energy than the wt peptide. We obtained highly
purified recombinant p38. We present here in silico calculations and a detailed biophysical
characterization of the protein that will be used to test the proposed inhibitors that we are currently
synthesizing.
SAB 2013 BPA
69
BPA35_Studying structure and aggregation of amyloid proteins using small organic
compounds as molecular probes
Valiente Gabioud A.A.
1
, Bertoncini C.
1,2
, Outeiro T.F.
3
,Griesinger C.
4
, Fernndez C.O.
1,2
1
Instituto de Biologa Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina,
2
Laboratorio
Max Planck de Biologa Estructural, Qumica y Biofsica Molecular de Rosario (LMPbioR), UNR.
3
Dept.of
Neurodegeneration and Restaurative Research, University of Gttingen, Gttingen, Germany.
4
Dept. of
NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Gttingen, Germany
The misfolding of proteins into a toxic conformation is proposed to be at the molecular foundation of a
number of neurodegenerative disorders including Alzheimer and Parkinsons disease. One common and
defining feature of protein misfolding diseases is the formation and deposition of amyloid-like fibrils.
Currently, no preventive therapy is available for Parkinson and Alzheimer diseases. Identification of
therapeutic drugs is not only complicated by a lack of understanding of many of the key aspects of the
pathogenesis of these diseases but also by the multifactorial etiology of them. The aggregation pathway
of the proteins linked to these disorders represents then an obvious target for therapeutic intervention.
The detailed understanding of the phenomenon of amyloid fibrillation and its inhibition is therefore highly
clinically important. Our work paves the road for the high resolution characterization of the modulatory
effect of small compounds on amyloidogenic intrinsically disordered proteins, which is critical for the
rational design of efficient anti-amyloid agents to treat Parkinson and Alzheimers disease.
Acknowledgements: ANPCyT, CONICET, Fundacin Bunge y Born, Max Planck Society, Alexander von
Humboldt Foundation.
BPA36_28-mer peptide from enterocin CRL35 displays bacericidal activity
E. Masias
1
, P.R da Silva Sanches
3
, L. Saavedra
2
, A. Bellomio
1
, E. Cilli
3
, C. Minahk
1
1
Instituto Superior de Investigaciones Biolgicas, Chacabuco 461. San Miguel de Tucumn, Argentina.
2
Centro de Referencia para Lactobacilos, Chacabuco 145. San Miguel de Tucumn, Argentina.
3
Instituto
de Qumica de Araraquara UNESP. Rua Prof. Francisco Degni, 55 Araraquara - So Paulo. Brasil.
Email: emilmas@hotmail.com
Enterocin CRL35 is a pediocin-like bacteriocin produced by Enterococcus mundtii CRL35. This
antimicrobial peptide is active against food-borne pathogen Listeria monocytogenes. In this work we used
the following synthetic peptides: the full-length bacteriocin (43- mer) and short derived peptides from the
C-terminus and N-terminus of enterocin CRL35 (28-mer and 15-mer respectively). Peptide binding to
target cells was analyzed by fluorescence polarization. The Ka values calculated from experimental data
indicated that the 3 peptides could bind similarly to the sensitive cells. Dissipation of membrane potential
assays showed that although all 3 peptides were able to dissipate the transmembrane electrical potential,
the 15-mer peptide produced a slower and incomplete dissipation. 43-mer has a MIC of 100 nM while 28-
mer and 15-mer have a much higher values of 10 and 20 M, respectively. However, only 43-mer and 28-
mer peptides displayed bactericidal effect against target cells. 15-mer peptide showed a bacteriostatic
effect since no cells were killed by this peptide in HEPES buffer. These results open up the possibility of
designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a
promising approach for combating Listeria.
BPA SAB 2013
70
BPA37_Labatidin interaction study using lipid monolayer
Barbosa, S.C.
1
, Cilli, E.M.
2
, Stabeli, R.G.
3
, Itri,R.
4
and Ciancaglini, P.
1
1
Departamento de Qumica, FFCLRP-USP, Ribeiro Preto, SP, Brasil;
2
Departamento de Bioqumica e
Biotecnologia, IQ-UNESP-Univ. Estadual Paulista, Araraquara, SP, Brasil.
3
Universidade Federal de
Rondnia (UNIR); Fundao Oswaldo Cruz - Fundao Oswaldo Cruz - Rondnia (FIOCRUZ), 76812-
245 Porto Velho, RO, Brasil.
4
Instituto de Fsica, IF-USP, So Paulo, SP, Brasil
Labaditin (VWTVWGTIAG) is a hydrophobic cyclic peptide from Jatropha Multifida and is composed of 10
residues. It has been studied due to its rigid conformation, which is considered significant for biologic
activity. The Labaditin (Lo) peptide and its open chain analogs (L
1
) were synthesized and its adsorption
on the monolayers of dipalmitoylphosphatidylcholine (DPPC), DPPC/cholesterol and DPPC/dipalmitoyl
phosphatidylserine (DPPS) mixtures was studied. The monolayers were obtained by spreading the lipid,
solubilized in chloroform, on the aqueous surphace (20L, 1mM) of a Langmuir system. The
measurements of A isotherms were done using the Wilhelmy (10 mm/min) method, varying the
concentrations of peptide from 0.0117 to 0.0704M. The results indicated that Lo changed the packing
density of the structures in DPPC. The increase of the initial pressure and a more expanded curve led to
the interpretation that the highest peptide interaction was with the DPPC isotherm. The neutral
phosphocholine groups were in direct contact with Lo peptide, thereby increasing peptide adsorption by
hydrophobic interaction. In DPPC/cholesterol, the monolayer was highly expanded. In the presence of the
Lo and L
1
peptide the adsorption was lower than in pure DPPC. This is probably due to highly the packed
DPPC molecules in the presence of cholesterol. Therefore, the peptide adsorption depends highly on the
condensing effect of cholesterol in the DPPC monolayer. In DPPC/DPPS mixtures both peptides showed
similar interaction with this monolayer, but less than pure DPPC, probably due to the anionic membrane.
So, the cyclic peptide showed more interaction with the DPPC monolayer, as already has been observed
by fluorescence spectrometry. This has probably occurred because of hydrophobic interaction. Although
the L
1
peptide has charged terminals, it had a stronger interaction with the anionic membrane.
Financial Supports: CAPES, CNPq and FAPESP.
BPA38_A lectin and phospholipases A
2
from Bothrops diporus venom: identification and
sequencing by mass spectrometry
Yunes Quartino, Pablo J.
a
, Portela, M.
b
, Lima A.
b
, Durn R.
b
and Fidelio G.D.
a
a
CIQUIBIC, CONICET, Universidad Nacional de Crdoba, Argentina.
b
Institut Pasteur de Montevideo,
Uruguay.
We report protein sequences from lipolitic fractions of venom of Bothrops diporus, a viper from Argentina
known as Yarar Chica. We have previously cloned two phospholipases and produce them via
heterologous expression and subsequent renaturation (1). By similarity and identity to public database
sequences, we classified these as type A
2
(PLA
2
). In the present work we used conventional
chromatographic techniques coupled to MALDI-TOF-TOF mass spectrometry analysis to prove the
existence at the protein level of the cloned sequences and portions of new PLA
2
isospecies. Along this
work we also found a lectin dimer for which it is proposed the disulfide intermolecular connectivity.
Acknowledgements
CONICET, FONCYT, SECYT-UNC (Argentina) and Unidad de Protemica y Bioqumica Analticas,
Institut Pasteur de Montevideo (Uruguay).
1- Yunes Quartino P.J., Barra J.L., Fidelio G.D. Biochemical and Biophysical Research
Communications.2012.321.
SAB 2013 BPA
71
BPA39_pH effect on the folding mechanism of frataxin from Psychromonas ingrahamii
Roman EA, Santos J, Craig PO
Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of
Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Frataxin is a protein that participates in iron binding and delivery to other protein partners. Its absence in
humans yields Friedreich's Ataxia. In our laboratory we study the iron binding and folding mechanism of
human and Psychromonas ingrahamii frataxin (pFXN). We previously reported that pFXN stability is
highly modulated by pH in the 6-8 pH range. In this work, we studied the folding reaction of pFXN as a
function of pH by computational techniques. We used a coarse grained structure based model
supplemented with a general electrostatic potential to extensively sample the folding and unfolding
transitions and evaluate pFXN conformational stability. Residues were reduced to 3 atoms over which the
bonded and non bonded forces were projected. In this model only the residues that make contact in the
native state interact favorably whereas all the others interact only repulsively through excluded volume
effect. The model was supplemented with a coulombic electrostatic potential to account for the effect of
pH on the stability of the protein. Preliminary results show that protonation of histidine residues highly
modulate protein stability. The protonation of key residues and their contribution to the overall effect was
also evaluated.
Acknowledgements. We thank Diego Ferreiro and Rodolfo Gonzalez Lebrero for fruitful discussions.
BPA40_Characterization of iron binding to frataxin from Psychromonas ingrahamii
Rigal JB, Noguera M, Santos J, Roman EA.
Instituto de Qumica y Fisicoqumica Biolgicas, Universidad de Buenos Aires, Buenos Aires, Argentina
Frataxin is a protein highly conserved protein among biology kingdoms. In humans its absence yields
Friedreich's ataxia. Its biological function is to bind and deliver iron to other protein partners. In our
laboratory we study iron binding and its effect on stability and dynamics of different frataxin variants. In
this work, we characterized iron binding to frataxin from Psychromonas ingrahamii at different metal and
protein concentrations. The resulting binding isotherm will define the metal affinity and stoichiometry to
this protein. Previous results reported for other frataxin variants suggest that metal binding could move
protein equilibrium towards oligomeric species
1
. This fact will be studied by light scattering and native gel
techniques.
1- Bou-Abdallah, F., S. J. Mol. Biol. 2004 (341):605.
BPA SAB 2013
72
BPA41_Stability and folding kinetics of frataxin from Psychromonas ingrahamii are
highly modulated by pH
Roman EA, Gonzalez-Lebrero RM, Santos J.
Instituto de Qumica y Fisicoqumica Biolgicas, Universidad de Buenos Aires, Buenos Aires, Argentina
Frataxin is a highly conserved protein among biology kingdoms. In humans its absence yields Friedreich's
ataxia. In our laboratory we study iron binding and its effect on stability and dynamics of different frataxin
variants. In this work, we explored folding kinetics of frataxin from Psychromonas ingrahamii (pFXN) at
different pHs. Previous results from our laboratory revealed that pFXN stability is highly modulated by pH
in the 6 to 8 range
1
. Molecular dynamics simulations also suggested that histidine residues could be
participating in this effect. Here we introduce stopped-flow experiments studying pFXN folding and
unfolding reaction by monitoring Trp-fluorescence and circular dichroism at different pHs. Results show
that both signals reproduce well our previous equilibrium measurements by means of equilibrium constant
and total signal change. Moreover, observed rate constants obtained by both kinetic measurements yield
similar results. Our experiments indicate that as the pH becomes more acid, the rate constant of folding
becomes higher and unfolding rate constant lower. Future experiments with histidine point mutations
would shed light in the role of these residues and pH in general in the folding reaction.
Acknowledgements. We thank Diego Ferreiro and Ignacio Sanchez for fruitful discussions.
1- Roman, E., A. Biochimica et Biophysica Acta. 2013 (1834):1168-80.
BPA42_Conformation coalescence of -barrel proteins at sub-aggregating
concentrations of TFE mimics the formation of aggregation prone species.
CR Angelani
1
, JJ Caramelo
2
, JM Delfino
1
, LM Curto
1
1
Instituto de Qumica y Fisico-qumica Biolgicas Prof. Alejandro Paladini, BA, Argentina
2
Instituto de investigaciones Bioqumicas de Buenos Aires, Fundacin Instituto Leloir. BA, Argentina
98 and 78 are two functional all- sheet variants of IFABP (intestinal fatty acid binding protein),
being useful models to study the determinants related to aggregation of -barrel proteins. Albeit
displaying increased conformational plasticity, these variants exhibit a native-like -barrel topology and
are able to support a cooperative folding. Interestingly, while IFABP and 98 are monomers, 78 is
dimeric. These proteins share a common nucleation-elongation mechanism of aggregation, where the
rate-limiting step is the formation of dimeric nuclei. A straightforward correlation between their intrinsic
stability (IFABP78>98) with their aggregation propensity (78>IFABP98) cannot be
established. This observation appears at odds with the established notion that perturbations of the native
fold necessarily favor the population of aggregation-prone species. To understand the early events
leading to the formation of the critical nucleus, the initial conformational changes occurring after the
addition of an aggregating inducing co-solvent were examined (25 % v/v TFE). The spectral changes in
the far and near UV CD were measured immediately after diluting the protein in TFE. Interestingly, the CD
spectra for each of the proteins revealed the coalescence of all three proteins into conformations richer in
content. The same behavior was observed when incubating at sub-aggregating concentrations of TFE
( 10%v/v). In this scenario, low concentrations of co-solvent might produce conformational changes
similar to those leading to the aggregation prone species. This approach might help in understanding the
early conformational changes controlling the onset of aggregation.
SAB 2013 BPA
73
BPA43_What is the information component in sequences of repeat-proteins ?
Espada R*, Mora, T
, Walczak, AM
, Ferreiro DU*
*QB, FCEN, UBA IQUIBICEN, CONICET
*; Agudelo, WA
; Aran, M
; Gallo, M
; Gonzlez, Flecha, FL
; Gonzlez Lebrero, MC
; and
Santos, J
.,Rodriguez-Fris A
., Appignanesi G.A
., Fernndez A.S
)
in the same model. Transient species were
introduced as chemical intermediates at each step; they decay to intermediate enzyme species
accordingly with the mass law, at 6.56x10
12
s
-1
. Other rate constants are as in (1). The transient evolution
of the model was followed with a computer program. The evolution of the ligands concentrations agree
with experimental results. Enzymatic species concentrations lies within 10
-10
10
-6
M, transient species
lies within 10
-19
-10
-17
M, at 0,1 s., when the reaction is in a cuasi-steady-state. These large differences
preclude the use of the standard (G
o
) or basic (G
basic
) free energies to describe G fall along the cycle.
Forward and backward G
are shown for 10 and 100 ms. of simulated reactions; both are negative in the
exergonic forward cycles, at any reaction time, in sealed and permeable membranes, and partial sums
equals each partial G. The results show the absence of any activation energy hill previously proposed
(2)- to be surmounted upon the reaction advance. Similar results were obtained for the binding of 2 Ca
ions to the enzyme through (+)cooperative reactions. The results do not disagree with the modern theory
of the transient-state intermediates (3), since we used rate constants obtained from enzyme catalyzed
reactions, already including transition coefficients which reduce partial activation energies.
(1) Alonso and Hecht, J. theor. Biol (1990) 147:161-176. (2)Pickart and Jencks (1984) J. Biol. Chem. 259:
1629-1643. (3)Hammer-Shiffer (2013) Biochemistry 52:2012-2020.
TMSB4_Molecular Dynamics Studies of Trypanosoma cruzi Trans Sialidase and T.
rangeli Sialidase Mutants at the Covalent Intermediate Stage.
Diego J. Alonso de Armio
a
, Guy Lezama
a
, Daro Estrin
b
, R. M. S. Alvarez
a
, Adrian E. Roitberg
c
a. Instituto Superior de Investigaciones Biolgicas, INSIBIO (CONICET-UNT). Chacabuco 461, San
Miguel de Tucumn, Tucumn, Argentina.
b. Instituto de Quimica Fisica de los Materiales, Medioambiente y Energa, INQUIMAE-CONICET,
Ciudad Universitaria, Pab 2, C1428EHA, Buenos Aires, Argentina.
c. Quantum Theory Project and Departament of Chemistry, University of Florida, Gainesville, Florida,
United Status of America.
Trypanosoma cruzi is the causative agent of Chagas' disease. T. cruzi trans-sialidase (TcTS) was shown
to be an important factor in the microorganism's virulence, and has been proposed as a target for drug
design, a goal that requires a thorough understanding of the enzyme's mechanism. Evidence indicates
that the catalytic mechanism involves a long-lived covalent intermediate (CI), which is later attacked by an
acceptor glycoconjugate, completing the sialic acid transfer. A key question is thus, how does TcTS
protect the CI from hydrolysis until the acceptor glycoconjugate can position itself in the active site for the
transfer reaction to take place. Previous works suggested the existence of a new switch mechanism
sensitive to lactose, which deactivates the enzyme in abscence of acceptor ligand at the CI stage. Here
we present new in silico studies in which we discuss the structural and sequence differences between
TcTS and TrSA responsible for it.
SAB 2013 TMSB
83
TMSB5_Study of the interaction of L-cysteine ethyl ester with DPPC membrane
Marcelo Puiatti,
Mara E. Tuttolomondo,
Sonia B. Daz,
and
Adriana B. Pierini
*
Diaz, C.
1,2,3
, Min, A.
1
, Pissinis, D.
1
, Schilardi, P.
1,3
, Pietrasanta, L.I.
2,3,4
1
Instituto de Investigaciones Fisicoqumicas Tericas y Aplicadas, CONICET -UNLP.
2
Centro de
Microscopas Avanzadas, FCEyN-UBA.
3
Consejo de Investigaciones Cientficas y Tcnicas, Argentina.
4
Departamento de Fsica, FCEyN-UBA. *cdiaz@df.uba.ar
The design, development and characterization of substrates with different physical, chemical and
mechanical properties are critical for the study of biologically active surfaces and biointerfaces
1
. These
kinds of devices involve the interaction of synthetic materials with biological systems and require a
complete understanding of the physical properties of molecules, composites and nanostructures. Since its
development, AFM is a powerful diagnostic and investigation tool. Nowadays, Peak Force QNM
2
allows
quantitative nanomechanical mapping of material properties, including modulus, adhesion and
dissipation, while simultaneously imaging sample topography at high resolution. In order to study cellular
mechanostransduction, we characterize interfacial arquitectures and micro-nano structured platforms
based on molecular recognition.
Acknoledgments: UBA, ANPCyT, UNLP. Min and Pissinis have fellowships from CONICET.
1- Agheli, H., J. Malmstrm, et al. "Nanostructured biointerfaces." Materials Science and Engineering: C
2006. 26(5-7): 911-917.
2- Bede Pittenger, Natalia Erina, Chanmin Su. Quantitative Mechanical Property Mapping at the
Nanoscale with PeakForce QNM. 2010. Veeco. Solutions for a nanoscale world.
BBA10_Atomic Force Spectroscopy of single adhesin on living Bordetella pertussiss
surface
L.Arnal
1
, Giovanni Longo
3
, Federico Castez
2
, Osvaldo M. Yantorno
1
, Sandor Kassas
3
and Maria E. Vela
2
.
1
INIFTA-CONICET-UNLP.Suc. 4 CC 16, (1900) La Plata Argentina
2
CINDEFI-CONICET-50 y 115 CP (1900). La Plata-Argentina.
3
IPSB, colePolytechniqueFdrale de Lausanne, Lausanne, Switzerland.
Pertussis is a humans respiratory tract disease caused by Bordetella pertussis (Bp), a Gram
negative bacterium. Recent publications indicate that Bp might grow as biofilm attached to the upper
respiratory tract as a mean to persist in the host. Filamentous haemaglutinin (FHA) is the mayor adhesin
of Bp and is involved in different steps of biofilm formation.
Here, by using Atomic Force Spectroscopy, we studied single interactions between purified FHA and
a specific antibody attached to Si
3
Ni
4
cantilevers and we also detected single FHA molecules in the
surface of living BpTohama I(reference strain) cells. A scan rate of 500nm/s was used for single cell
spectroscopy measurements. We used BpTohama living cells, several individual cells were mapped and
the recorded Force Volume (fv) images were analysed. Force interaction maps were built, showing
specific interactions on bacterial surface for wild type cells but not for FHA- strain (FHA defective mutant
strain used as control). The analysis of the results allowed us to observe that the localization on the
bacteriums surface of FHA proteins is not homogeneous and dynamic; proteins seem to be localized into
nanodomains or clusters of adhesins. This distribution into nanodomains could mean that the bacterium
clusters FHA in certain regions of the membrane reaching biggest and strongest domains of interactions
which could be advantageous for cell-substrate and cell- cell interactions during biofilm development.
Serra, D. O.et all. PLoS ONE 2011,28811.
Yersin et all, PNAS, 2003, 8736.
BBA SAB 2013
116
BBA11_Study of the adsorptive performance of novel foam-based chromatographic
columns.
Mirna L. Snchez
[1,2]
, Leandro Martnez
[1]
, Marcelo Fernndez-Lahore
[2]
and Mariano Grasselli
[1]
mi.sanchez@jacobs-university.de
[1]
Universidad Nacional de Quilmes - IMBICE (CONICET).
[2]
Jacobs University Bremen.
Modern biotechnology heavily depends on the availability of efficient processes, which have to generate
competitive products in terms of quality and cost. Most commonly, the purification of bioproducts is based
on chromatographic platforms. However, the low productivity imposed by diffusional limitations, the need
for multiple separation steps, and the requirement of extensive solids separation, drives to a technological
situation characterised by low global bioproduct yields.
1
In a previous work, we fabricated reticulated open-pore polyurethane foams supporting a functional
hydrogel layer. Preliminary studies showed that the adsorptive properties of these materials allow the
reversibly binding of proteins by ion exchange mechanisms.
Dynamic binding capacity tests (DBC) were performed employing Lysozyme (HEWL) on a cation
exchanger foam. DBC experiments were carried out as a function column length, compression degree
and flow velocity. Furthermore, bed hydrodynamics was studied via residence time experiments (Pe Nr)
and correlated to the observed amount of protein captured.
Experimental data indicated that optimum performance, as judged by Pe and DBC, was obtained for the
following conditions: 30 cm column length, non-compaction, and 210 cm/h linear flow rate. The novel
cation exchange foams show as promise matrix material for the direct capture and purification of
biotechnology-derived products.
Acknowledgements: M.L.S. thanks to CONICET and DAAD for the fellowship. M.G. is member of
CONICET.
1- DePalma A. Genetic Engineering News 29 (2009) 8
BBA12_Physical evidence justifying experimental biological results in Photodynamic
Therapy of Cancer
Etcheverry, ME
1
, Pasquale, MA
2
y Garavaglia, M
1,3
1
Departamento de Fsica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP)
2
Instituto de Investigaciones Fisicoqumicas Tericas y Aplicadas (INIFTA) (CCT CONICET La Plata,
UNLP y CIC)
3
Centro de Investigaciones pticas (CIOp) (CCT CONICET La Plata, y CIC)
In order to verify the efficiency of absorption of radiation emitted by an LED lamp at 637 nm with an
average width of 8 nm in certain concentrations of drug, in this paper we present results recorded with a
spectrophotometer (Ocean Optics HR2000 + ES) coupled to an optical fiber.
From preliminary results performed on cultured HeLa cells (human cervical carcinoma), using a
temoporfirin as photosensitizer and the lamp was obtained the proportion of cell death for different drug
concentrations (0.05 mg / ml, 0.25 mg / ml and 1g/ml) and illumination times (3min, 6min and 12min).
The absorption spectrum obtained for temoporfirin showed its characteristic peak centered at 650nm.
In order to justify the biological results, spectrophotometric measurements were performed which showed
that in the area of drug absorption differences are significant when using two concentrations and four light
intensities obtained varying the distance of the lamp to the detector. Indeed, the higher the drug
concentration is lower spectrophotometric record, which corresponds to a greater number of photons
absorbed by HeLa cells.
SAB 2013 BBA
117
BBA13_Adhesion Kinetics of Escherichia coli onto surface-modified HDPE: Looking for
Answers in DLVO theories.
Quiroga, F.Y, & Grasselli, M.
Laboratorio de Materiales Biotecnolgicos (LaMaBio), UNQ-IMBICE-CONICET, Quilmes, Argentina.
This work studies the interactions between recombinant Escherichia coli cells and high density
polyethylene (HDPE) surface-modified with different concentrations of cationic branches. The adhesion
kinetics of E. coli on cationic HDPE films was measured in situ by fluorescence detection of green
fluorescence protein (GFP) expressed by the bacteria attached to the film. The overall interactions
between cells and surface were determined by contact angle and zeta potential measurements and
quantified using the classical and extended Derjaguin-Landau-Verwey-Overbeek theories (DLVO and
xDLVO respectively) [1]. According to those theories, interfacial interaction between a colloid (bacteria)
and a surface in aqueous media correspond to Lifsithz.van der Waals interactions, Lewis acid-base
interactions, and electrostatic interactions [2]. The adhesion capacity of E. coli was found higher for those
polymers with the highest cationic and hydrophobic values.
Acknowledgements
FYQ thanks to CONICET for their fellowship. MG is a CONICET researcher.
1- M. Hermansson. Colloid and Surfaces B. 1999. 105
2- C. J. van Oss. Colloids and Surfaces B. 2007. 2
BBA14_Latex colored particles: synthesis and BSA absorption for potential applications
in immunochromatography
Femia, L*; Gonzalez, V.*; Minari, R.*; Alonso S. del V**; Gugliotta L.*
*INTEC (UNL-CONICET), Santa Fe, Argentina.
**Laboratorio de Biomembranas, Depto de Ciencia y Tecnologa, UNQ, Quilmes, Bs As, Argentina.
Colored latexes of polystyrene have acquired much interest in biological fields, since they are applicable
in many topics [1]. Several authors had reported the synthesis of these materials, and its interaction with
proteins [2]. There are several potential applications like immunochromatography, immunoagglutination
assays, particle designs for flux cytometry, protein-latex detection kits, etc. In view of the increasing
interest in the use of these particles, we have synthesized a new polymerizable colorant from basic
fuchsine (BF). The functionalized colorant was characterized by NMR, FTIR and ESI-mass spectrometry.
Latex colored particles were prepared with this color-monomer and with the non-functionalized colorant,
BF. The obtained particles were characterized in terms of size, color incorporation, conversion efficiency
in the synthesis process and protein adsorption (BSA). We have obtained purple colored latex particles
with a sized range from 80-110 nm, which were stable in the presence of emulsifier. The monomer
conversion was higher in the styrene/colored-monomer reaction than styrene/BF reaction. Color
incorporation was not significantly different but, color leakage was greater for polystyrene/BF system.
Acknowledgements: To CONICET, MinCyT, and UNL for the financial supports.
1. Piskin E, Tuncel A, Denizli A, Ayhan H (1994) J Biomat Sci Pol. 451.
2. Luck M, Pistel KF, Li YX, Blunk T, Muller RH, Kissel T (1998) J Control Release. 107.
BBA SAB 2013
118
Sab2013
119
ndice por autores / Author index
Abriata, L.A. BPA3 53
Adamo, H.P.A. TRC18 102
Aguayo, R. TRC15 101
Agudelo, W.A. BPA44, TMSB11 73, 86
Ainciart, N. NTB2 108
Alancay, M.M. NTB3 109
Alarcon, L. BLM15 36
Albeirtman, C. BBA3 112
Ale, N.M. BLM33 45
Alleva, K. TRC5, TRC6, TRC8, TRC10 96, 97, 98
Alonso de Armio, D.J. TMSB4, TMSB7 82, 84
Alonso, G.L. TMSB3 82
Alonso, S. del V.
BLM10, BLM17, BLM37, BLM40,
BBA1, BBA2, BBA4, BBA14
33, 37, 47, 48, 111,
112, 117
Altabef, A.B. BLM33, BLM34, TMSB5 45, 83
lvarez, R.M.S. BLM18, BLM19, TMSB4, TMSB7 37, 38, 82, 84
lvarez, Y.D. NTB6 110
Ambroggio, E. BLM8, BLM35 32, 46
Ambroggio, X. S4.1 23
Amodeo, G. TRC5, TRC6, TRC8 96, 97
Amundarain, M.J. TMSB13, TMSB22 87, 91
Andrade, X. BTE4 80
Angelani, C.R. BPA42 72
Angiolini, J.F. SDI7, SDI8 106
Antollini, S.S. BLM22, BLM23 39, 40
Appignanesi, G.A. BLM15, ENZ6 36, 76
Aptekmann, A. BPA1 52
Aramenda, P. F. P7 13
Arn, M. S2.3, BPA11, BPA30, BPA44 20, 57, 66, 73
Arcn, J.P. TMSB18 89
Argello, J.M. S1.4 18
Arias, J.M. BLM34, TMSB5 45, 83
Arnal, L. BBA10 115
Auzmendi, J.A. S3.2 21
Aveldao, M.I. BLM13, BLM22, BLM23 35, 39, 40
Avila, C.L. BPA25, BPA26, BPA27 64, 65
Ayub, N. TRC6 96
Azzoni, A. S5.2 25
SAB 2013
120
Bagatolli, L.A. P4 10
Baks, L. TRC11 99
Balbino, T. S5.2 25
Barbosa, L.R.S. BPA25, BPA26 64
Barbosa, S.C. BPA37 70
Barrantes, F.J. BLM22 39
Basaez, G. S2.2 19
Bazn, S. BPA16 59
Bea, E.A. BTE4 80
Bellomio, A. BLM19, BPA27, BPA36, BTE3 38, 65, 69, 80
Belluzo, B.S. BPA5, BPA7 53, 55
Beltramo, D. BLM46 51
Beltrandi, M. S2.1 19
Benedini, L. BLM12, BLM30 34, 43
Benseor, L. SDI8 107
Berberin, M.V. BLM42 49
Berlin, J.S. S3.4 22
Bernar, E.M. NTB1 108
Bertoncini, C. BPA35 69
Bianchi, M. SDI3, SDI4, NTB6 105, 110
Bisch,P.M. S2.4 20
Blackledge, M. S2.1 19
Blangy, S S2.1 19
Blocquel, D. S2.1 19
Bocco Gianello, M.D. BLM43 50
Boeris, V. BPA9 56
Boffi, A. BPA8 55
Bolean, M. BLM28 42
Bonafe, C.F.S. ENZ10 78
Bonamore, A. BPA8 55
Borioli, G. BLM44 50
Boron, I. BPA18 60
Bosich, W. TRC16 101
Bouchet, A.M. BLM39, ENZ2 48, 74
Bougis, K. BLM20 38
Bouzat, C. TRC14 100
Bragas, A.V. NTB4 109
Brandelli, A. BPA9 56
Brandenburg, K. BLM3 30
Bredeston, L.M. TRC16, TRC17, TRC19 101, 102, 103
Brito, T. TMSB9 85
Bruch, M.B. SDI5 106
Bruno, L. SDI1, SDI7, SDI8 104, 107
Sab2013
121
Bucci, H.A. BPA34 68
Burgos, I. S2.3, BPA11, BPA15 20, 57, 59
Bustillo, I. S2.2 19
Bustos, M. TMSB1, TMSB2 80
Cababie, L.A. ENZ5 76
Cabrera, I. BPA25 64
Calabr, V. BLM17, BLM40 37, 40
Caldarola, M. NTB4 109
Calzetta, N.L. BBA1 111
Capece, L. BPA18 60
Caramelo, J.J. BPA42, NTB5 72, 110
Carrasquel-Ursulaez, W. TRC15 101
Carrire, F. P2 8
Carnovale, C.E. MLB7 32
Carrer, D. BPA15 59
Carrizo, F. TRC11 99
Carrizo, M.E. BPA22, ENZ1 62, 74
Caruso, B. BLM4, BLM6, BLM35 30, 31, 46
Carvalho, R.V. S4.3 24
Castez, F. BBA10 115
Cattoni, D.I. S1.4 18
Cavalcanti, L. S5.2 25
Celej, M.S. BPA14, BPA23 58, 63
Centeno, M. TRC12 99
Ceoln, M. S5.4 26
Chaln, M.C. BTE3 80
Chaves, S. BPA27 65
Chehn, R. BPA26, BPA27 64, 65
Chemes, D.M. BLM18 37
Chaillou, L.L. ENZ3 75
Chiaramoni, N.S. BLM17, BLM37, BLM40, BBA4 37, 47, 48, 112
Chisari, L. BPA18 60
Ciancaglini, P. BLM28, BPA37, ENZ7 42, 70, 77
Cilli, E. BPA36, BPA37 69, 70
Ciocco Aloia, F. BLM42 49
Clop, E.M. ENZ10 78
Clop, P.D. BBA6 113
Colque, A. BLM1 29
Communi, G. S2.1 19
Contreras, G.F. TRC15 101
Cooper, A. S1.1 17
Crsico, B. S1.1, TMSB15 17, 88
Corradi, J. TRC14, TRC18 100, 102
SAB 2013
122
Correa, W. BLM3 30
Cossy Isasi, S. TMSB10 85
Costabel, M.D. TMSB13, TMSB15, TMSB22 87, 88, 91
Craig, P.O. BPA39, NTB5 71, 110
Crosetti, D. BLM7 32
Cunha, K.C. TMSB24 92
Curtino, J.A. BPA22, ENZ1 62, 74
Curto, L.M. BPA42 72
Cutro, A.C. BLM31, BLM32 44
Cybulski, L S1.2, BPA31 17, 67
Czysezon, N.A. TRC18 102
da Silva Sanches, P.R. BPA36 69
de Athayde Moncorvo
Collado, A. BLM25 41
de Azevedo Delou, J.M. TRC20 103
de La Torre, L. S5.2 25
de Mendoza, D. BPA31 67
De Rossi, M.C. SDI1 104
De Sautu, M. TRC3 95
Decca, M.B. S1.3, BPA12 18, 57
Defelipe, L.A. TMSB16, TMSB18 88, 89
Defonsi, E. BPA27 65
Del Boca, M. BLM44 50
Del Popolo, M.G. BLM42 49
Delfino, J.M. BPA32, BPA42, NTB1, NTB2 67, 72, 108
Despsito, M.A. SDI8 107
Dewhirst, M.W. BBA7 114
Di Lella, S. BPA17 60
Diaz, C. BBA9 115
Daz, S.B. BLM33, BLM34, TMSB5 45, 83
Diaz-Franulic, I S3.1 21
Disalvo, E. A.
BLM15, BLM31, BLM32, BLM36,
BLM39, ENZ2 36, 44, 46, 48, 74
Dodero, V.I.
BLM12, BLM30, BPA13, BPA14,
BPA24, BPA33, TMSB13
34, 43, 58, 63, 68,
87
Dodes Traian, M.M. BLM27, TRC7 42, 97
Dominighini, A. BLM7 32
Donnamaria, M.C. TMSB21 91
Dosnon, M. S2.1 19
Dumas, V.G. TMSB18 89
Dupuy, F. BLM45, BLM46 51
Durn, R. BPA38 70
Durand, E.S. BPA12 57
Elso-Berberin, G. TRC9 98
Sab2013
123
Emperador, A. TMSB26 93
Enoki, T.A. BLM41 49
Erales, J. S2.1 19
Ermcora, M. S2.3, S5.3, BPA2, BPA10, BPA11 20, 26, 52, 56, 57
Esersky, I. TRC19 103
Espada, R. BPA43 73
Espejo, F. BLM3 30
Espinosa, Y.R. BLM26 41
Esposito, G. BBA3 112
Estrin, D.A.
BPA8, BPA17, BPA18, TMSB4,
TMSB7, TMSB14 55, 60, 82, 84, 87
Etcheverry, M.E. BBA5, BBA12 112, 116
Etienne, W. BBA7 114
Falcn, V. BPA25 64
Fanani, M.L. BLM9, BLM12, BLM13, BLM23, BLM24 33, 34, 35, 40
Faraj, S.E. BPA28, BPA30 65, 66
Farinola, J. BBA1, BBA2 111
Favarolo, B. TRC19 103
Femia, L. BBA14 117
Fernandez Ruocco, M.J. BLM37, BBA4 47, 112
Fernndez, A.S. ENZ6 76
Fernndez, C.O. BPA35 69
Fernndez-Lahore, M. BBA11 116
Ferreira-Gomes, M. TRC1, TRC2, TRC3, TRC4, TRC12 94, 95, 99
Ferreiro, D.U. BPA43 73
Ferreyra, R.G. BPA2 52
Fidelio, G.
S2.3, BLM8, BLM35, BPA11, BPA38,
ENZ9
20, 32, 46, 57, 70,
78
Flores, S.S. ENZ4 75
Flores-Romero, H. S2.2 19
Folmer, A.P. BPA9 56
Franchini, G.R. S1.1 17
Fras, M.A. BLM31, BLM32, BLM36, BLM39, BBA8 44, 46, 48, 114
Frigini, E. BLM21 39
Fuentealba, J. TRC19 103
Futerman A. P6 12
Gaggiotti, M.C. BLM44 50
Galassi, V.V. BTE1 79
Galizia, L. TRC13 100
Gallea, J.I. BPA23 63
Galliano, S. MLB7 32
Gallo, M. S2.3, BPA11, BPA30, BPA44 20, 57, 66, 73
Galvn, A.E. BTE3 80
Gamarnik, A.V. BPA21, ENZ5 62, 76
SAB 2013
124
Garavaglia, M. BBA5, BBA12 113, 116
Garay, A.S. BLM29 43
Garbarino, E. TMSB6 83
Garca, G. BLM7 32
Garcia-Valero, J. S2.2 19
Gasperini, A. S5.2 25
Gastaldi, L. SDI3, SDI4 105
Gauto, D.F. TMSB18 89
Gebhard, L.G. BPA21, ENZ5 62, 76
Gennaro, A.M. BLM43 50
Gennis, R. BTE3 80
Gerbelli, B.B. BLM14, BLM20 35, 38
Germano, R. TMSB9 85
Giordani, C. BLM2 29
Giraldo, M. BLM2 29
Giudice, F. BLM9 33
Glisoni, R. TMSB17 89
Goldbaum, F.A. S5.1 25
Goldman, R.P. TRC10 98
Gmez Zavaglia, A. BLM33 45
Gmez, G.E. NTB1, NTB2 108
Gonalves da Silva, A.M.P.S. P6 12
Gonzlez Flecha, F.L.
S1.4, BLM27, BPA20, BPA29, BPA44,
TMSB11, TMSB12, TRC7, TRC16
18, 42, 61, 66, 73,
86, 97, 101
Gonzalez Montoro, M.A. BLM1 29
Gonzlez, C. TRC15 101
Gonzlez, D.A. TMSB3 82
Gonzlez, M.C. BPA22 62
Gonzalez, V. BBA14 117
Gonzlez-Lebrero, R.M.
BPA20, BPA21, BPA41, BPA44, ENZ5,
TMSB11, TMSB12
61, 62, 72, 73,76,
86
Gonzlez-Nilo, F. S3.1, TRC15 21, 101
Gonzalvez, J. BLM7 32
Granados, A. BLM5 31
Grasselli, M. BBA2, BBA3, BBA4, BBA11, BBA13 111, 112, 116, 117
Grasso, E.J. BPA4 53
Griesinger, C. BPA35 69
Grigera, J.R. BLM26, TMSB21 41, 91
Gualtieri, A.F. TMSB19 90
Gurin Aguilar, D.M. TMSB22 91
Gugliotta, L. BBA14 117
Guido, M.E. TMSB6 83
Habchi, J. S2.1 19
Hecht, J.P. TMSB19 90
Sab2013
125
Henriques, V.B. TMSB9 85
Heredia, V. BLM46 51
Herlax, V. TRC11 99
Herrera, F.E. BLM29, TMSB20 43, 90
Herrera, M.G. BPA14, BPA24, TMSB13 58, 63, 87
Hbarnter, C. BPA6 54
Hoylaerts, M.F. BLM28 42
Hoyos de Rossi, R. BML16 36
Hugo, M. TMSB14 87
Ibez-Shimabukuro, M. TMSB15 88
Incicco, J.J. BPA21, ENZ5 62, 76
Inda, M.E. BPA31 67
Ingaramo, M.C. BPA22 62
Inman, B. BBA7 114
Issoglio, F.M. ENZ1 74
Itri, R. BPA25, BPA26, BPA37 64, 70
Iturriaga, L.B. NTB3, BBA8 109, 114
Jozefkowicz, C. TRC6, TRC8, TRC10 96, 97, 98
Juarez, A.C. BLM18, BLM19 37, 38
Kassas, S. BBA10 115
Kaufman, S.B. BPA20, BPA21, ENZ5 61, 62, 76
Kaur, G. SDI7 107
Kennedy, M.W. S1.1 17
Kiffer-Moreira, T. BLM28 42
Klinke, S. S5.1 25
Kotsias, B.S. TRC13 100
Krause, R. BLM5 31
Labanda, M.S. NTB5 110
Lamy, M.T. BLM41 49
Landajuela, A. S2.2 19
Landeta, O. S2.2 19
Landon, C.D. BBA7 114
Lanterna, A. BLM5 31
Latorre, R. TRC15 101
Learte, S. BPA33 68
Lee, C.T. BBA7 114
Leiva, E.P.M. TMSB26 93
Lenilson, TMSB9 85
Leupen, S. TMSB2 81
Levalle, A. TRC17 102
Levi, V. BLM27, SDI1, SDI7, SDI8 42, 104, 107
Levstein, F. TMSB1 81
Lezama, G. TMSB4 82
SAB 2013
126
Lichtig, P.L. TMSB14 87
Lima, A. BPA38 70
Lins, R.D. S4.3, TMSB24, TMSB25 24, 92, 93
Llarrull, L.I. C1, BPA5, BPA7, ENZ8 14, 54, 55, 77
Llovera, R. BPA10 56
Lobo, M.O. NTB3 109
Lombardi, J. BPA9 56
London, N. S4.1 23
Lonez, C. BPA14 58
Longhi, S. S2.1 19
Longo, G. BBA10 115
Lopes da Silva,T.S. TRC20 103
Lopez, E.D. BPA34, TMSB16 68, 88
Lpez, L. SDI2 104
Luquita, A. MLB7 32
Maccarini, P.F. BBA7 114
Madoery, R. ENZ9 78
Maggio, B.
BLM5, BLM9, BLM12, BLM13, BLM16,
BLM23, BLM45, BLM46, BPA4
31, 33, 34, 35, 36,
40, 51, 53
Mangialavori, I.C. TRC1, TRC2, TRC3, TRC4, TRC7 94, 95, 97
Mangiarotti, A. BLM4, BLM6 30, 31
Manrique-Moreno, M. BLM3 30
Maranzana, E.S.B. BBA1 111
Marchesini, G.R. BBA6 113
Mariani, M.E. ENZ9 78
Mari, I. BLM30 43
Marino, G.I. TRC13 100
Marques Capella, M.A. TRC20 103
Marquez, M. TRC5 96
Marsanasco, M. BLM17, BLM40 37, 48
Marti, M.A. BPA8, BPA18, TMSB18 55, 60, 89
Martiarena, J. TRC2 94
Martin, O.A. BLM21 39
Martnez, L. BBA11 116
Martnez, S. S1.4 18
Martini, M.F. TMSB17 89
Mascareas, J.L. BPA13 58
Mashal, A. BBA7 114
Masias, E. BPA36 69
Mat, S. TRC11 99
Mayorga, L.S. BLM42 49
McNerny, K.L. BBA7 114
Medina, A.V. ENZ2, ENZ3 74, 75
Sab2013
127
Menegon Arantes, G. BTE1 79
Meneses, K. BLM2 29
Menzaque, A. BLM5 31
Miguel, V. TMSB8 84
Milln, J.L. BLM28 42
Minahk, C.J. BLM25, BPA27, BPA36, BTE3 41, 65, 69, 80
Min, A. BBA9 115
Minari, R. BBA14 117
Mocskos, E. SDI7 107
Moffatt, L. S3.2 21
Moglioni, A. TMSB17 89
Monachesi, S.N. TMSB21 91
Montanari, J. BLM10 33
Montes de Oca, J.M. ENZ6 76
Montes, M.R. REC12 99
Monti, J. MLB7, NB1 32, 108
Montich, G.G. BPA12, BPA16 57, 59
Mora, T. BPA43 73
Morand Sales, E. BPA26 64
Morero, R.D. BLM25 41
Moreschi, A.C. BPA12 57
Morgada, M.N. BPA3 53
Morini, M. BLM15 36
Mottola, M. BLM24 40
Nadra, A. BPA1, BPA8, BPA18 52, 55, 60
Nallet, F. BLM14, BLM20 35, 38
Naranjo, D. S3.1 21
Navailles, L. BLM14, BLM20 35, 38
Navarro, N. S3.1 21
Nazareno, M.A. BLM32, BLM39, ENZ2, ENZ3 44, 48, 74, 75
Nieto, P.S. TMSB6 83
Noguera, M.E. BPA2, BPA28, BPA40 52, 65, 71
Nolan, M.V. BPA19 61
Nomura, D.A. BLM41 49
Oliveira, C. S5.2 25
Oliveira, C.L.P. BLM14, BLM20 35, 38
Oliveira, E.A. BLM14, BLM20 35, 38
Oliveira, R.G. BLM11, BLM45, BPA4 34, 51, 53
Oliveira, T.R. BBA7 114
Oate, J. BLM3 30
Ontiveros, M.Q. TRC2 94
Orozco, M. TMSB26 93
Ouidja, O. BPA26 64
SAB 2013
128
Outeiro, T.F. BPA35 69
Ozu, M. TRC10 98
Pez, R. BPA25 64
Palacios, A.R. ENZ8 77
Pli, T. P5 11
Pallavicini, C. SDI8 107
Palma, A. TRC13 100
Pantano, S. S4.2 23
Paolorossi, M. BPA16 59
Papageorgiou, N. S2.1 19
Papy-Garcia, D. BPA26 64
Paris, G. S5.1 25
Pasquale, M.A. BBA5, BBA12 113, 116
Patio, E. BLM3 30
Pedroni, V. BLM25 36
Peluffo, R.D. S3.3 22
Pealva, D.A. BLM13, BLM22, BLM23 35, 39, 40
Peppe, S. TRC19 103
Perillo, M.A.
BPA15, BPA19, ENZ4, ENA10,
TMSB8, BBA6
59, 61, 75, 78, 84,
113
Perillo, V.L. BLM22 39
Peris, D. BPA5 53
Perrota, R. BLM37 47
Pessoa, Jr. A. BPA25 64
Piccinni, F.E. BPA8 55
Pickholz, M. TMSB17, TMSB23 89, 92
Piegari, E. SDI2 104
Pierini, A.B. TMSB5 83
Pietrasanta, L.I.
TRC5, TRC8, SDI3, SDI4, NTB4,
NTB6, NTB9, BBA9
96, 97, 105, 109,
110, 115
Pignataro, M.F. BLM27, TRC7 42, 97
Pinto, O.A. BLM36 46
Pinto, S.N. P6 12
Pinzn Barrantes, J. BLM16 36
Piotrkowski, B. BLM17, BLM40 37, 48
Pissinis, D. BBA9 115
Placenti, M.A. BPA20 61
Plachta, N. SDI7 107
Politi, M.T. TRC10 98
Ponce Dawson, S. SDI2, SDI6 104, 106
Ponce, M.L. SDI6 106
Porasso, R.D. BLM21 39
Portela, M. BPA38 70
Prieto M. P6 12
Sab2013
129
Primo, E. BPA10 56
Puiatti, M. TMSB5 83
Pusterla, J.M. BLM11 34
Quinzio, C. BBA8 114
Quiroga, F.Y. BBA13 117
Quirolo, Z.B. BPA13 58
Rabinovich, G.A. BPA17 60
Raimunda, D. TRC9 98
Raisman-Vozari, R. BPA26 64
Rakes, C. TMSB2 81
Rasia, R.M. BPA6 54
Recoulat Angelini, A.A. BPA29 66
Revelli, J.A. TMSB6 83
Rey, F S1.1 17
Rico, M. BTE2 79
Rigal, J.B. BPA40 71
Rinaldi, J.J. S5.1 25
Ringkjobing-Jensen, M. S2.1 19
Ros, A. BPA10 56
Risso, P. BPA9 56
Risso, V. BBA4 112
Ritacco, H. BPA14 58
Rodi, P. BLM43 50
Rodrigues, D. E. BLM29 43
Rodriguez, D. SDI1 104
Rodriguez-Fris, A. ENZ6 76
Roitberg, A.E. TMSB4, TMSB7 82, 84
Romn, E.A.
S4.4, BPA28, BPA39, BPA40, BPA41,
TMSB12 24, 65, 71, 72, 86,
Romn, M.D. TMSB6 83
Romero, J.M. BPA17 60
Romero, J.M. BPA22 62
Ronco, M.T. MLB7 32
Rossi, J.P.F.C. TRC1, TRC2, TRC3, TRC4, TRC7 94, 95, 97
Rossi, R.C. TRC12 99
Roveri, O.A. BTE2 79
Rubim, R.L. BLM14, BLM20 35, 38
Rubio, A. BTE4 80
Ruigrok, R.W.H. S2.1 19
Rusu, V.H. TMSB24, TMSB25 92, 93
Ruysschaert, J.M. BPA14, BPA31 58, 67
Saavedra, L. BPA36 69
Sabeckis, M.L. TMSB12 86
SAB 2013
130
Saffioti, N.A. TRC1 94
Salcedo, C.L. BLM31, BLM32, BLM39 44, 48
Saldaa, C. TRC19 103
Samus, I. BPA2 52
Sanchez, J.M. ENZ4 75
Snchez, M.L. BBA11 116
Santana, H. BPA25 64
Santos, J.
BPA28, BPA30, BPA32, BPA39,
BPA40, BPA41, BPA44, TMSB11
65, 66, 76, 71, 72,
73, 86
Schilardi, P. BBA9 115
Schurig-Briccio, L. BTE3 80
Scochera, F. TRC6, TRC8 96, 97
Seddon, J. M. P1 7
Seplveda, R. S3.1, TRC15 21, 101
Sequeira, M.A. BLM12, BLM30, BPA33 34, 43, 68
Sferco, S.J. TMSB20 90
Sfriso, P. TMSB26 93
Shimabukuro, M.I. S1.1, TMSB15 17, 88
Sica, M. S2.3, BPA11 20, 57
Siciliano, N. TRC4 95
Sierra, M.B. BLM15 36
Sigaut, L. TRC5, TRC8, SDI3, SDI4, SDI6, NBT6
96, 97, 105, 106,
110
Silva L.C. P6 12
Silva, E.R. BLM14, BLM20 35, 38
Silveira, N.P. BLM41 49
Simo, A.M.S. BLM28 42
Siri, M. BLM37, BBA4 47, 112
Smith, B.O. S1.1 17
Soba, A. BTE4 80
Socas, B. BPA26 64
Sorre, B P3 9
Sosa Morales, M.C. BLM18, BLM19 37, 38
Sosnik, A. TMSB17 89
Soto Espinoza, S.L. BBA3 112
Soto, G. TRC6 96
Stabeli, R.G. BPA37 70
Stauffer, P.R. BBA7 114
Stefani, F. NTB6 110
Suarez, I.P. BPA6 54
Sued, M. SDI1 104
Sutka, M. TRC10 98
Sycz, G. S5.1 25
Tabares LC C2, SDI5 15, 106
Sab2013
131
Takara, D. TRC3 95
Tamarit, F.A. TMSB6 83
Tarkowski, N. NTB6 110
Terrones, O. S2.2 19
Tvez, N. NTB6 110
Toledo, P. BPA10 56
Torchio, G. S2.3, BPA11 20, 57
Torres-Bugeau, C.M. BPA26, BPA27 64, 65
Toscanini, M.A. TRC17 102
Trujillo, M. TMSB14 87
Turjanski, A.G. BPA34, TMSB16, TMSB18 68, 88, 89
Tuttolomondo, M.E. BLM34, TMSB5 45, 83
Un, S. SDI5 106
Urli, L. MLB7 32
Valdez-Taubas, J. BLM1 29
Valiente Gabioud, A.A. BPA35 69
Valsecchi, W.M. BPA32 67
Vandenbranden, M. BPA31 67
Varela A.R. P6 12
Vasques Camargo da Silva,
J.P. BTE1 79
Vazquez, D.S. BPA44, TMSB11 73, 86
Vazquez, R. TRC11 99
Veciana, J. BPA25 64
Vela, M.E. BBA10 115
Ventosa, N. BPA25 64
Vera, C. BPA27 65
Veuthey, T.V. BPA24 63
Viana, I.F.T. S4.3 24
Vianna Jorge, R. TRC20 103
Vico, R. BLM5, BLM16, BLM24 31, 36, 40
Vila, A.J. BPA3, ENZ8 53, 77
Villanueva, G. BLM3 30
Villanueva, M. BLM24 40
Villarreal, M.A. TMSB8, TMSB26 84, 93
Villarruel, C. SDI6 106
Viso, J.F. TMSB13, TMSB22 87, 91
Vitale, A.J. BLM22 39
von Bilderling, C. SDI3, SDI4, NTB4 105, 109
Wetzler, D.E. BPA18, BPA34, SDI1, SDI8 60, 68, 104, 107
Whitworth, K. TMSB2 81
Wilke, N. BLM1, BLM4, BLM6, BLM13, BLM35 29, 30, 31, 35, 46
Wood, I. TMSB23 92
SAB 2013
132
Yaneff, A. TRC5 96
Yantorno, O.M. BBA10 115
Yoneda, J.S. ENZ7 77
Yunes Quartino, P.J. BPA38 70
Zamarreo, F. TMSB13, TMSB15, TMSB22 87, 88, 91
Zarycki, M. TRC19 103
Zeida, A. TMSB14 87
Sab2013
133
NOTAS/NOTES
SAB 2013
134
NOTAS/NOTES