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Sociedad Argentina de Biofsica

XLII Reunin Anual

2 - 4 de Diciembre 2013

Villa Carlos Paz, Crdoba

ARGENTINA

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Fanani, LauraXLII Reunin Anual de la Sociedad Argentina de Biofsica / Laura Fanani ; Natalia

Wilke ; Gerardo Fidelio. - 1a ed. - Buenos Aires : SAB - Sociedad Argentina deBiofsica, 2013.

146 p. ; 30x21 cm.

ISBN 978-987-27591-2-4

1. Biologa.Investigacin. I. Wilke, Natalia II. Fidelio, Gerardo III. TtuloCDD 570.711

Fecha de catalogacin: 07/11/2013

Quedan prohibidos, dentro de los lmites establecidos en la ley y bajo apercibimiento

legalmente previsto, la reproduccin total o parcial de esta obra por cualquier medio o

procedimientos ya sea electrnico o mecnico, el tratamiento informtico, el alquiler o

cualquier otra forma de cesin de la obra sin la autorizacin previa y por escrito de los

titulares del copyright.

Diagramacin y Edicin: LauraFanani, Natalia Wilke.

Diseo de Tapa: Agustin Mangiarotti, Gerardo Fidelio

Asistencia tcnica web: Marcos Solovey

Impreso en Crdoba, Argentina, Noviembre 2013.

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Comisin Directiva de la Sociedad Argentina de Biofsica Ao 2013

Presidente Gerardo FidelioUniversidad Nacional de Crdoba

Vicepresidente Gabriela AmodeoUniversidad de Buenos Aires

Presidente saliente Luis Gonzlez FlechaUniversidad de Buenos Aires

Secretario Mauricio SicaCentro Atmico Bariloche

Tesorera La PietrasantaUniversidad de Buenos Aires

Vocales Titulares Karina Alleva

Universidad de Buenos Aires

Rosana ChehnUniversidad Nacional de Tucumn

Vocales suplentes Rodolfo RassiaUniversidad Nacional de Rosario

Florencia MartiniUniversidad de Buenos Aires

Comit Cientfico SAB 2013 Comit Organizador y Logstica SAB 2013

Dr. Gerardo D Fidelio (UNC) Dr. Gerardo D Fidelio (UNC)

Dr. Guillermo Montich (UNC) Dr. Ernesto Ambroggio (UNC)

Dra. Laura Fanani (UNC) Dr. Benjamn Caruso (UNC)

Dra. Graciela Borioli (UNC) Dr. Ernesto Grasso (UNC)

Dr. Rafael Oliveira (UNC) Dra. Soledad Bazn (UNC)

Dr. Ernesto Ambroggio (UNC) Lic. Pablo Yunes Quartino (UNC)

Dr. Marcos Villarreal (UNC) Lic. Maria Elisa Mariani (UNC)

Dra. Natalia Wilke (UNC) Lic. Julio Pusterla (UNC)

Dra. Karina Alleva (UBA) Lic. Ignacio Gallea (UNC)

Dr. Rodolfo Gonzlez Lebrero (UBA) Lic. Agustin Mangiarotti (UNC)

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Palabras de Agradecimientos / Words of Ac knowledgements

Desde su fundacin, hace 42

aos, SAB organiza su reunin anual en

forma ininterrumpida, congregando a

cientficos nacionales e internacionales

que trabajan el rea de la Biofsica.

En nombre de SAB y como

responsables organizadores de la

Reunin Anual 2013 de la Sociedad, es

para nosotros un honor dar la bienvenida

en Crdoba a los participantes de la XLII

Reunin Anual de SAB. Hacemos

extensivos estos saludos de bienvenida a

los alumnos y docentes/investigadores

asistentes al VI Curso POSLATAM co-

organizado con LAFeBS y auspiciado por

IUPAB.

Queremos agradecer muy

especialmente a los conferencistas y

simposistas que aceptaron venir a

Crdoba y honramos su muestra de

gratitud. Al grupo de Logstica del

Congreso que puso todo de s para una

buena organizacin.Queremos incluir en los

agradecimientos a las autoridades de

LAFeBs que depositaron su confianza

para organizar el VI Curso POSLATAM

de la regin en Crdoba para 2013.

Por ltimo, agradecemos a la

Facultad de Ciencias Qumicas y a la

Universidad Nacional por su ayuda en

infraestructura y financiacin; a CONICET

y a FONCYT de Agencia Nacional dePromocin Cientfica y Tecnolgica por

su ayuda financiera. A IUPAB que, a

travs de LAFeBS, ha financiado con

becas el traslado de doctorandos para el

VI Curso POSLATAM. Tengan todos

Ustedes una grata estada en Crdoba.

Since its founding, 42 years ago,

SAB organizes its annual meeting without

interruption, bringing together national

and international scientists working in the

field of biophysics.

On behalf of SAB and as

organizers of the 2013 Annual Meeting of

the Society is an honor to welcome to the

participants of the XLII Annual Meeting of

the SAB held in Crdoba. We want to

extend these greetings to welcome also to

students and teachers/researchers

attending the VI POSLATAM Course co-

organized with LAFeBS and sponsored by

IUPAB.

We want to especially thank to the

speakers who agreed to come to Cordoba

and we honor their selfless displays of

gratitude, and to the Congress Logistics

group for its great labor in the

organization of the Meeting.

In the acknowledgments we want

to include to LAFeBs authorities whoplaced their trust in us to organize the VI

POSLATAM Course of the region in 2013.

Finally, we thank to the School of

Chemical Sciences and the National

University of Crdoba for their

infrastructure and financial help; to both

CONICET and the National Agency for

Promoting Science and Technology for

financial assistance. To IUPAB, that

throughout LAFeBS has financed thetransfer of doctoral students to assist to VI

POSLATAM Course.

We wish to all of you a pleasant

stay in Crdoba.

Organizadores / Organizers SAB 2013

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400 palabras sobre la historia de la UNC (http://www.400.unc.edu.ar/)

El origen de la Universidad Nacional de Crdoba fue la promesa de un capitaldel Obispo Trejo y Sanabria al padre jesuita Diego de Torres en 1613; independiente

de la autoridad real, aquella Universidad, se gobern y se financi a s misma. LaCompaa de Jess opt por formar lites y clero en un modelo tradicional deconocimiento, alterado cuando la Orden Franciscana se hizo cargo de la Universidad eintrodujo obras de Descartes, Newton y Leibnitz.

Durante la disolucin del Estado nacional, la Universidad qued en la rbitaprovincial hasta su nacionalizacin en 1856. La Ley Universitaria data de 1885. En esaltima poca, la UNC se moderniz: elimin la Teologa e incorpor las Facultades deFsico-Matemticas y Medicina.

Para 1918, la UNC acumulaba las tensiones de una sociedad entransformacin y los estudiantes plantearon un conjunto de demandas que fueron

paulatinamente atendidas por el gobierno de H. Yrigoyen.

La UNC no fue ajena a los vaivenes de la poltica: en 1930 como en cada unode los cinco golpes militares posteriores padeci intervenciones y restricciones a laautonoma.

Durante las presidencias de Juan D. Pern las universidades ampliaron sumatrcula y jerarquizaron nuevas reas de conocimiento, que en Crdoba incluyCiencias Econmicas, y Filosofa y Humanidades. Luego se incorporaron nuevoscampos como Arquitectura y Urbanismo, Trabajo Social, Matemtica, Astronoma yFsica, Agronoma y Comunicacin.

Durante la primavera democrtica de 1973 las expectativas se concentraron enla renovacin curricular y el compromiso intelectual con el cambio social pero elproceso se trunc en 1975, cuando la presidenta Pern envi la intervencin a lasuniversidades nacionales. Desde 1976, la dictadura militar profundiz un proyecto deuniversidad elitista y funcional a sus objetivos restringiendo el ingreso, la libertad dectedra y anulando el co-gobierno.

El retorno a la democracia en 1983 permiti recuperar la institucionalidad de la UNCrestituyendo el co-gobierno y la autonoma, y renovando la idea de Universidadcomprometida con la sociedad.

La Ley de Educacin Superior de 1995, modific la vida universitaria en aspectoscomo la produccin de conocimientos y el rol de las Universidades en las sociedades.

El siglo XXI conforma un escenario diferente en el que hay nuevos horizontes paradisear e implementar polticas de educacin superior que jerarquicen a lasuniversidades como productoras de ciencia y tecnologa atentas a las demandas deldesarrollo productivo y social en los valores ciudadanos propios de una sociedaddemocrtica.

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Indice/Index

VI Curso POSLATAM / VI POSLATAM Course. i

Programa del Congreso / Meeting Program........ 1

Conferencias Plenarias/ Plenary Lectures... 7

Mini-conferencias/ Short lectures.. 14

Simposios/ Symposia.......... 17

Resumenes de posters/ Poster abstracts: ......... 27

Biofsica de lpidos y membranas / Biophysics of lipid and membranes (BLM)... 29

Biofsica de protenas y cidos nuclicos / Biophysics of proteins and nucleic acids

(BPA)... 52

Enzimologa/ Enzymology (ENZ).. 74

Bioenergtica y transferencia electrnica / Bioenergetics and transfer (BTE)

79

Teora y modelado de sistemas biolgicos / Theory and modeling of biological

systems (TMSB) 81

Transportadores, receptores y canales / Transporters, receptors and channels

(TRC)... 94

Sealizacin y Dinmica Intracelular / Signaling and Intracellular dynamics (SDI).. 104

Nuevas tcnicas en biofsica / New techniques in biophysics (NTB) 108

Biofsica: Aplicaciones biotecnolgicas / Biophysics: Biotechnological applications

(BBA)... 111

Indice por Autores / Authors index.. 119
http://sab2013.fcq.unc.edu.ar/images/1_RESUMENES%20SAB%202013_BLM.pdfhttp://sab2013.fcq.unc.edu.ar/images/2_RESUMENES%20SAB%202013_BPA.pdfhttp://sab2013.fcq.unc.edu.ar/images/2_RESUMENES%20SAB%202013_BPA.pdfhttp://sab2013.fcq.unc.edu.ar/images/2_RESUMENES%20SAB%202013_BPA.pdfhttp://sab2013.fcq.unc.edu.ar/images/3_RESUMENES%20SAB%202013_ENZ.pdfhttp://sab2013.fcq.unc.edu.ar/images/4_RESUMENES%20SAB%202013_BTE.pdfhttp://sab2013.fcq.unc.edu.ar/images/5_RESUMENES%20SAB%202013_TMSB.pdfhttp://sab2013.fcq.unc.edu.ar/images/5_RESUMENES%20SAB%202013_TMSB.pdfhttp://sab2013.fcq.unc.edu.ar/images/5_RESUMENES%20SAB%202013_TMSB.pdfhttp://sab2013.fcq.unc.edu.ar/images/6_RESUMENES%20SAB%202013_TRC.pdfhttp://sab2013.fcq.unc.edu.ar/images/6_RESUMENES%20SAB%202013_TRC.pdfhttp://sab2013.fcq.unc.edu.ar/images/6_RESUMENES%20SAB%202013_TRC.pdfhttp://sab2013.fcq.unc.edu.ar/images/7_RESUMENES%20SAB%202013_SDI.pdfhttp://sab2013.fcq.unc.edu.ar/images/8_RESUMENES%20SAB%202013_NTD.pdfhttp://sab2013.fcq.unc.edu.ar/images/8_RESUMENES%20SAB%202013_NTD.pdfhttp://sab2013.fcq.unc.edu.ar/images/9_RESUMENES%20SAB%202013_BBA.pdfhttp://sab2013.fcq.unc.edu.ar/images/9_RESUMENES%20SAB%202013_BBA.pdfhttp://sab2013.fcq.unc.edu.ar/images/9_RESUMENES%20SAB%202013_BBA.pdfhttp://sab2013.fcq.unc.edu.ar/images/9_RESUMENES%20SAB%202013_BBA.pdfhttp://sab2013.fcq.unc.edu.ar/images/9_RESUMENES%20SAB%202013_BBA.pdfhttp://sab2013.fcq.unc.edu.ar/images/8_RESUMENES%20SAB%202013_NTD.pdfhttp://sab2013.fcq.unc.edu.ar/images/7_RESUMENES%20SAB%202013_SDI.pdfhttp://sab2013.fcq.unc.edu.ar/images/6_RESUMENES%20SAB%202013_TRC.pdfhttp://sab2013.fcq.unc.edu.ar/images/6_RESUMENES%20SAB%202013_TRC.pdfhttp://sab2013.fcq.unc.edu.ar/images/5_RESUMENES%20SAB%202013_TMSB.pdfhttp://sab2013.fcq.unc.edu.ar/images/5_RESUMENES%20SAB%202013_TMSB.pdfhttp://sab2013.fcq.unc.edu.ar/images/4_RESUMENES%20SAB%202013_BTE.pdfhttp://sab2013.fcq.unc.edu.ar/images/3_RESUMENES%20SAB%202013_ENZ.pdfhttp://sab2013.fcq.unc.edu.ar/images/2_RESUMENES%20SAB%202013_BPA.pdfhttp://sab2013.fcq.unc.edu.ar/images/2_RESUMENES%20SAB%202013_BPA.pdfhttp://sab2013.fcq.unc.edu.ar/images/1_RESUMENES%20SAB%202013_BLM.pdf

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LATIN AMERICAN FEDERATION OF BIOPHYSICAL SOCIETIESVI POSLATAM COURSE SCHEDULE

Bi ophysical approaches to study systems of biological in terest.28thNovember to 4thDecember 2013, Crdoba, Ar genti na

Dia /DayTiempo/Time

Profesor/ProcedenciaProfessor/Affiliations

Tpicos/Topics

Mircoles 27 de Noviembre / Wednesday 27 th November LLEGADA DE ESTUDIANTES/ARRIVAL OF STUDENTS.

Lugar de desarrollo del Curso del 28 de noviembre al 1 de Diciembre de 2013 en la Facultad de Ciencias Qumicas, Ciudad Universitaria, Universidad Nacional de Crdoba

(www.fcq.unc.edu.ar).Las conferencias relacionadas al Meeting del 2 al 4 de diciembre 2013 se llevaran a cabo en el hotel sede en Villa Carlos Paz, Crdoba (www.portaldelago.com.ar).Course development from November 28th to December 1st2013 will be held at the School of Chemical Sciences, Campus, National University of Cordoba, Crdoba (400thAnniversary)

Meeting-related conferences from 2nd-4thDecember 2013 will be held at the Congress Hotel in Villa Carlos Paz, Crdoba (www.portaldelago.com.ar).

Coordinador/Coordinator: Dr. Gerardo Fidelio (mails:[email protected] ;[email protected])web:http://sab2013.fcq.unc.edu.ar/

Jueves 28 de Noviembre/

Thursday 28thNovember

Total Tiempo estimado/Estimated Total time: 6 hs

Maana/Morning (Primera Parte/First Part of Dr. Disalvo 3 hs)

Primera Clase/First Lecture: 9.00-10.30

Caf/Coffe Break: 10.30-10.55

Segunda Clase/Second Lecture: 11.00-12.30

Dr. Anbal DisalvoUni versidad Nacional de Santiago del

Estero. Ar gentina

MEMBRANE HYDRATION AND BIOLOGICAL FUNCTIONS (First Part)Thermodynamics of lipid self-assembly in water.Structural and physicochemical

properties of water. Swelling processes: area, thickness and interlamellar space.Methods and criteria. Hydration Water. Excluded volume and hydration forces.Densities and distribution of water and hydration centers in membranes. Influence ofthe topology on hydration and water states.

The membrane-solution interphase. Criteria for a new model for membranes.Surface potentials. Determination of zeta potential. Limitations. Electrophoreticmobility. Isotherms of adsorption of charged amino acids and peptides.

Synergism and cooperativity. Partition of amino acids and polyols: Correctionsto the Wiener -White and Butler-Barclay rules in relation to the water content.

Almuerzo/Lunch 13.00/14.30Jueves 28 de Noviembre/Thursday 28 thNovember

Tarde/Afternoon Maana/Morning (Segunda Parte/Second Partof Dr. Disalvo 3 hs)




MEMBRANE HYDRATION AND BIOLOGICAL FUNCTIONS (Second Part)Water activity in membranes. Water activity and surface pressure. Defay-

Prigogine model. Effect of hydrogen bonding compounds on water activity inmembranes. Relationship between dipolar potential and hydration water.Aqueous domains. Hydration water and confined water. Water species and

phase states. Effect of lipid composition. Lipidomics and aquaomics.Lyotropic phenomena.Expansion and contraction of lipid membranes. Water

penetration and dielectric properties. Generation of defects by osmosis andelectrical fields. Surface changes in hypertonic and hypotonic processes.Kinetics of dehydration and rehydration. Relaxation processes in the insertion

of peptides. Influence of the order parameter and fluctuations. Chemicalpotential of water and water activity by peptide insertion. Kinks and water
http://www.portaldelago.com.ar/http://www.portaldelago.com.ar/mailto:[email protected]:[email protected]://sab2013.fcq.unc.edu.ar/http://sab2013.fcq.unc.edu.ar/http://sab2013.fcq.unc.edu.ar/mailto:[email protected]:[email protected]://www.portaldelago.com.ar/

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species. Translocons and waterons.Tiempo Libre. Free time

Viernes 29 de Noviembre/Friday 29thNovember

Maana/Morning

Tiempo Estimado/Estimated Time: 3 hs.




Dra. Natalia WilkeUni versidad Nacional de Crdoba.

Argentina

MEMBRANE RHEOLOGY AND ELECTROSTATICS.Membranes with phase coexistence:Distribution of the phases at the plane of the

membrane: domain size, domain shape, number of domains. Domain growth, Oswaldripening, equilibrium and non-equilibrium domain distributions and sizes. Linetension. Intra and inter-domains long-range interactions.

Membrane rheology and electrostatics:Shear in membranes: free diffusion, solidobstacles, interacting obstacles. Bending and compression of membranes.Experi mental approaches:Model membranes.

Active and pasive methods in membrane rheology. Manipulation of membranes usingelectric fields and optical tweezers.

Almuerzo/Lunch 13.00/14.30

Viernes 29 de Noviembre/Friday 29thNovember

Tarde/Afternoon





Dr. Luis BAGATOLLIMembrane Biophysics and Biophotoni cs

Group/MEM PHYS - Center for

Biomembrane Physics, Department of

Biochemistry and Molecular Biology,

Uni versity of Southern Denmar k,

Denmark

FLUORESCENCE MICROSCOPY FOR BIOPHYSICAL STUDIES INBIOMEMBRANES

Modern Fluorescence microscopy instrumentation. Spectral properties of more popularprobes. Epi-, confocal and two photon excitation fluorescence microscopy. Giantunilamellar vesicles (GUVs) as tool to study lateral lipid organization and membrane

perturbation: lipid-lipid interaction, peptide, protein and lipolytic enzymes interactingwith organized lipids.

Tiempo Libre. Free timeSbado 30 de Noviembre/Saturday 30 thNovember

Maana/Morning





Dr. Benoit SORRECenter for Studies in Physics and

Biol ogy, The Rockefeller U niversity,

New York, NY., USA

BIOPHYSICS OF MEMBRANE CURVATURE

Fundamentals of membrame curvature. The mean -field theoretical description ofmembrane mechanics (Helfrich Hamiltionian). Artificial systems to study membranes

mechanical properties, (GUVs, micropipette aspiration, membrane fluctuationsmethods). Ways to pull membrane nanotubes (optical tweezers) to study biologyinspired questions like lipid sorting and membrane deformation by proteins

(amphiphysin, dynamin).

Almuerzo/Lunch 13.30/15.00Tarde/Afternoon 3/4 hs

Tarde/Afternoon





Dr. Frederic CARRI RELabor atory of Enzymology at Interf aces

and Physiology of Lipolysis E.I .P.L

C.N.R.S., M ARSEILL E, Fr ance

INTERFACIAL ENZYMOLOGY: THE CASE STUDY OF LIPOLYTICENZYMESFundamentals of interfacial enzymology. Modes of action of lipolyticenzymes (lipases and phospholipases) and kinetic models. Monomolecular films as

model interfaces for studying lipase-lipid interactions (adsorption/penetration) andinterfacial activity (the "zero-order" trough and the barostat technique). Structure-function relationships deduced from X-ray crystallography. Probing conformational

changes using site-directed spin-labeling coupled to EPR spectroscopy, kinectics.Surface spectroscopy for studying lipase adsorption at various interfaces (TIRF, ATR-FTIR).

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Tiempo Libre. Free Time

Domingo 1ro Diciembre / Sunday 1 stDecember

Maana/Morning





Dra. Maria Elena CarrizoUni versidad Nacional de

Crdoba. Argentina

PROTEIN CRYSTALLOGRAPHYProtein crystallization.Principles and techniques. Diffraction data collection.Fundamentals of the theory of X-ray diffraction by a crystal. X-ray sources anddetectors. Facilities at the Brazilian Synchrotron Light Laboratory.From diffractiondata to electron density. Electron density as a function of intensities and phases.Phase determination and improvement. Electron density maps.From electron densitymaps to molecular models.Electron density map interpretation. Model building.Structure refinement. PDB files.

Almuerzo/Lunch 13.30/15.00

Tarde/Afternoon

Tiempo Estimado/Estimated Time: 3/4 hs.




Dr . Paulo Mascarello Bisch

Laboratrio de Fsica Biol gica

Uni dade Multi disciplinar de Genmica

I nstit uto de Biof sica Carl os Chagas

Fi lhoU niversidade Federal do Rio de

Janeiro, Brazil

PROTEIN FOLDING AND STRUCTURE MODELING

1. Protein FoldingGeneral Concepts; 2. Protein folding and misfolding, the

funel theory; 3. Molecular Dynamic Simulations of Protein folding; 4.

Comparative Molecular Modeling.

Tiempo Libre. Free timeLunes 2 de Diciembre/Monday 2ndDecember

Maana/Morning

Traslado de Estudiantes del Curso al Hotel del Meeting / Transfer of the students to the Hotel's Meeting

MAIN LECTURES FROM THE XLII ARGENTINIAN BIOPHYSICAL SOCIETY MEETING ASSOCIATED TO VI POSLATAM COURSE

From 2nd

-4th

December 2013. (Place: Meeting Hotel)

Lunes 2 de Diciembre / Monday 2nd

December

15.00-16.00 CONFERENCE 1 Dr. John SEDDONHydrostatic Pressure Effects on the Structure and Stability of Lipid Membranes and

Lyotropic Mesophases. Membrane Biophysics group, Department of Chemistry,Imperial College London, Imperial College, London. UK.

19.00-20.00 CONFERENCE 2 Dr. Frdric CARRIREI nterf acial enzymology: in vestigating the mode of action of lipases requires a combination

of vari ous biophysical approaches. Laboratory of Enzymology at Interfaces andPhysiology of Lipolysis E.I.P.L, C.N.R.S. MARSEILLE, FRANCE FRANCIA.

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Martes 3 de Diciembre / Tuesday 3 rdDecember

8.30-9.30 CONFERENCE 3 Dr. Benoit SORRE" Dynamics of TGF-beta signaling: h ow positional i nformation can be learned from achanging morphogen gradient". Center for Studies in Physics and Biology, TheRockefeller University, New York, NY, USA.

9.30-10.30 CONFERENCE 4 Dr. Luis BAGATOLLI " Do liposomes penetrate skin" . Membrane Biophysics and Biophotonics Group/MEMPHYS- Center for B iomembrane Physics, Department of Bi ochemistry and Molecular Bi ology,

Uni versity of Southern Denmar k, Denmark

17.30-18.30 CONFERENCE 5 Dr. Tibor Lszl PLI

" On the r otary mechanism of the vacuolar proton-ATPase". Institute of Biophysics,

Biological Research Centre, P.O. Box 521, H-6701 Szeged, HUNGARY.

19.30-20.30 CONFERENCE 6 Dr. Manuel PRIETOCeramide and Glucosylcerami de impact on membrane biophysical pr operties: f rom model

to cell membranes.Instituto Superior Tcnico, Universidade Tcnica de Lisboa, Lisboa,PORTUGAL.

Mircoles 4 de Diciembre / Wednesday 4 thDecember

17.30-18.30CONFERENCE 7 Dr. Pedro ARAMENDA Single molecule and single nanoparticle fluorescence microscopy. Fac. Ciencias Exactasy Naturales, UBA. CIBION-CONICET, ARGENTINA.

19.30-20.30CONFERENCE 8 Dr. Flix GOI Membrane properties of the simple sphingolipids. Unidad de Biofsica (CSIC-UPV/EHU), Pas Vasco, ESPAA, SPAIN.

Fin de las Actividades / END OF ACTIVITIES

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PROGRAMA/PROGRAM

Lun es 2 de Diciemb re / Monday, December 2

nd

8.30-13.00 Llegada de Participantes y Registro/Arriv al of Part icipants and Registrat ion

9.00-12.30 Reunin de Representantes del Ncleo Disciplinar de Biofsica AUGM(Asociacin Universidades Grupo Montevideo) / Meeting ofRepresentat ives of B iophysics Discip l ine f rom AUGM (Associa t ion ofUniversi t ies of Montevideo Group )

13.00-14.30 Almuerzo. Colocacin de posters impares de todas las secciones/L unch .Placing of posters. Odd boards: A l l Topics

14.45-15.00 Apertura del Congreso/ Open ing Cer em ony.Bienvenida / Welcom e by Dr. Gerardo Fidelio

15.00-16.00 CONFERENCIA DE APERTURA/ OPENING LECTURE.

Dr. John Seddon: Hydrostatic Pressure Effects on the Structure and Stability of LipidMembranes and Lyotropic Mesophases. Membrane Biophysics Group,Department of Chemistry, Imperial College London, Imperial College, London.UK.

Chair : Brun o Maggio.

16.00-18.00 SIMPOSIO 1 / SYMPOSIUM 1: BIOFSICA DE BIOMEMBRANAS EINTERACCIN LPIDO-PROTENA / LIPID-PROTEIN INTERACTION ANDMEMBRANE BIOPHYSICS.

Chairs: Ernesto Am brogg io and Natalia Wilke

16:00-16:30: Betina Crsico.Novel lipid binding proteins from helminth parasites. Structuraland functional analysis. INIBIOLP-La Plata. Argentina

16:30-17:00: Larisa Cybulski The power of being at the interface: mechanism of DesKthermosensing.IBR-CONICET-Rosario. Argentina

17:00-17:30: Beln Decca. Conformation of peripherally bound membrane proteins: theinfluence of the lipid phase state.CIQUIBIC-Crdoba. Argentina

17:30-18:00: Luis Gonzalez Flecha.Phospholipid modulation of membrane protein thermalstability. IQUIFIB-Buenos Aires. Argentina

18.00-18.25 Caf / Coffee Break

18.30-19.00 SECCIN JOVEN I / YOUNG SECTION I

Mini-conferencia I /Short- lectureIPREMIO MEJOR TESIS SAB /BEST THESIS AWARD SAB

Dra. Leticia Llarrul:Insights on the Molecular Events that unleash resistance to b-lactam

antibiotics in Staphylococcus aureus. IBR- CONICET-Rosario, Argentina.Chair: Mar io Ermco ra

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19.00-20.00 CONFERENCIA PLENARIA 2/ PLENARY LECTURE 2

Dr. Frdric Carrire: Interfacial Enzymology: investigating the mode of action of lipasesrequires a combination of various biophysical approaches. Laboratory ofEnzymology at Interfaces and Physiology of Lipolysis E.I.P.L,C.N.R.S.MARSEILLE, FRANCE.

Chair: Gerardo Fidel io

20.30-23.00 SECCIN DE POSTERS (Posters impares: Todas las Secciones) /POSTER SECTION (Odd bo ards: A l l Topics )

(cena, snack / dinner, snack)

Martes 3 de Diciembre / Tuesday, December 3rd

8.30-9.30 CONFERENCIA PLENARIA 3 / PLENARY LECTURE 3

Dr. Benoit Sorre: Dynamics of TGF-beta signaling: how positional information canbe learned from a changing morphogen gradient. Center for Studies inPhysics and Biology, The Rockefeller University, New York, NY

Chair : Ernesto Am broggio

9.30-10.30 CONFERENCIA PLENARIA 4 / PLENARY LECTURE 4

Dr. Luis Bagattoli: Do liposomes penetrate skin?Membrane Biophysics and Biophotonics

group. MEMPHYS Center for biomembrane Physics, Department of

Biochemistry and Molecular Biology, University of Southern Denmark.

Chair : Fe li x Goi

10.30-10.55 Caf/ Coffee Break

11.00-13.00 SIMPOSIO 2/ SYMPOSIUM2. ESTRUCTURA Y FUNCIN DE PROTENAS /PROTEIN STRUCTURE AND FUNCTION.

Chair: Gerardo Fidel io

11:00-11:30 Sonia Longhi.Structural disoder and induced folding in the nucleoproteins

and phosphoproteins of paramyxoviruses.Group Leader "Structural Disorderand molecular Recognition" Architecture et Fonction des MacromoleculesBiologiques (AFMB) UMR 7257 CNRS et Universitd'Aix-Marseille, FRANCE.

11:30-12:00 Gorka Basaez. Elucidating the mechanisms of action of BCL-2 familyproteins in apoptosis using in vitro reconstituted systems.Unidad de Biofsica(CSIC-UPV/EHU), Pas Vasco, Espaa, Spain.

12:00-12:30 Mauricio Sica. Equilibrium Unfolding of the PDZ Domain of b2-SyntrophinDepartamento de Ciencia y Tecnologa, UNQ, Buenos Aires, y Laboratorio deBioenergas, IEDS, CONICET, Centro Atmico Bariloche, Ro Negro,Argentina.

12:30-13:00 Paulo Mascarello Bisch: "A large scale search for protein sequence-structure-

function relationship". Laboratrio de Fsica Biolgica, Unidade

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Multidisciplinar e Genmica, Instituto de Biofsica Carlos Chagas FilhoUniversidade Federal do Rio de Janeiro, Brazil.

13.00-14.30 Almuerzo. Colocacin de posters pares de todas las secciones/ Lunch.Placing of posters. Even boards: Al l Topics

15.00-17.00 SIMPOSIO 3 / SYMPOSIUM 3: TRANSPORTADORES Y CANALES DEMEMBRANA/ TRANSPORTERS AND CHANNELS IN MEMBRANES.

Chairs : Rodo lfo Gonzlez Lebrero and K arina Alleva.

15:00-15:30 David Naranjo.Small and Large conductance postassium channels: Where isthe difference? Centro Interdisciplinario de Neurociencias de Valparaso,Universidad de Chile

15:30-16:00 Luciano Moffatt.Achieving maximal speed of solution exchange for patchclamp experiments in purinergic receptors. INQUIMAE, UBA, Argentina.

16:00-16:30 Daniel Peluffo.Cationic Amino Acid Transporters: insights from a non-transportable enantiomer. Universidad de la Repblica, Regional Norte,

Uruguay.

16:30-17:00 Josh Berlin."Conformational changes in transmembrane alpha helices of theH-ATPase, AHA2". Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, USA

17.00-17.25 Caf/ Coffee Break

17.30-18.30 CONFERENCIA PLENARIA 5 / PLENARY LECTURE 5Dr. Tibor Lszl Pli: On the rotary mechanism of the vacuolar proton-ATPase.Institute

of Biophysics, Biological Research Centre, Szeged, HungaryChair: Gui l lermo Montich

18.30 a 19.30 CONFERENCIA PLENARIA 6 / PLENARY LECTURE 6

Dr. Manuel Prieto: Ceramide and Glucosylceramide impact on membrane biophysicalproperties: from model to cell membranes Instituto Superior Tcnico,Universidade Tcnica de Lisboa, Lisboa, Portugal.

Chair: Laura Fanani

19.30-20.00 Reunin LAFeBS-POSLATAM / LAFeBS-POSLATAM Meeting

20.00 Asamblea Anual de Socios de SAB / Annual meet ing of SAB m embers

20.30-23.00 SECCIN DE POSTERS (Posters pares: Todas las Secciones) / POSTERSECTION (even boards : Al l Topics)

(cena, snack / dinner, snack)

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Miercoles 4 de diciembre/ Wed nes day , Dec em ber 4th

8.30-10.30 SIMPOSIO 4 / SYMPOSIUM 4: MODELADO BIOMOLECULAR /

BIOMOLECULAR MODELINGChair: Marco s Vil larreal.

8:309:00 Xavier Ambroggio. Strategies for the de novo design of protein-proteininteractions. Rosetta Design Group LLC, Virginia, USA.

9:009:30 Sergio Pantano. Botulinum neurotoxins and SNARE complexes: A newstructural view from modeling and simulations.Institut Pasteur, Montevideo,Uruguay.

9:3010:00 Roberto Lins.PredictiveBiomolecular Modeling Applied to Protein Engineeringand Proteomics.Department of Fundamental Chemistry, Federal University ofPernambuco, Recife, PE, Brazil

10:0010:30 Ernesto A. Romn. Study of Frataxin folding. IQUIFIB-Buenos Aires.Argentina

10.30-10.55 Caf /Coffee Break

11.00-13.00 SECCIN JOVEN II/ YOUNG SECTION II

11.00-11.30 Mini-conferencia II /Short- lectureII

Leandro C. Tabares: One enzyme two pathways: Single molecules studies on NitriteReductase". CEA Saclay, France.

Chair: Rodolfo Rasia

11.30-13.00 Exposicin Oral de Posters Seleccionados /Oral Presentat ion of SelectedPosters.

Chairs : Laura Fanani and Natalia Wilk e

11.30-11.50 BLM39_Surface and hysteresis properties of lipid interphases composed byhead group substituted phosphatidylethanolamines. Salcedo, C.L., Bouchet,

A.M., Nazareno, M.A., Disalvo, E.A., Frias, M.A.

11.50-12.10 BPA25_Growth Hormone Releasing Hexapeptide is able to form Nanotubes: acomplementary study by Small Angle X-Ray Scattering, Transmission

Electron Microscopy and Molecular Dynamic Simulations. Barbosa, LRS,

Santana, H, Avila, CL, Cabrera, I, Pez, R, Falcn, V, Pessoa, Jr. A, Ventosa, N,Veciana, J, Itri, R.

12.10-12.30 TMSB1_Stochastic and Algebraic Methods for Modeling the Binding ofTranscription Factor Cohorts to Cis-acting Gene Regulatory Modules.MauricioBustos and Fernando Levstein.

12.30-12.50 TRC15_The S6 transmembrane segment modulates channel opening in BKchannels. Carrasquel-Ursulaez, W., Contreras, G. F., Seplveda, R., Aguayo, D.,Gonzlez-Nilo, F., Gonzlez, C. and Latorre, R.

13.00-14.30 Almuerzo /Lunch

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15.00-17.00 SIMPOSIO 5 / SYMPOSIUM 5: DIFRACCIN DE RAYOS X Y SAXS ENBIOESTRUCTURAS / X-RAYS DIFFRACTION AND SAXS TO STUDYBIOSTRUCTURES.

Chairs : Rafael Oliveira and Graciela Bo riol i .

15:00-15:30 Sebastin Klinke. Structural studies on a two-component system activated byblue light in Brucellaabortus, FundacinInstituto Leloir, Buenos Aires,Argentina.

15:30-16:00 Leide Pasos Cavalcanti."Small Angle X ray Scattering to study liposomes forgene therapy", Laboratorio Nacional de Luz Sincrotron, Campinas, Brazil.

16:00-16:30 Mario Ermcora.Structure and function of ICA2, a receptor involved in insulinsecretion Structural Biology and Biotechnology Group, IMBICE, UNQ-Conicet, Argentina.

16:30-17:00 Marcelo Ceolin. "Synchrotron radiation experiments on the biomineralizationof ferritin", INIFTA, Univ. Nac. de La Plata, Argentina.

17.00-17.30 Caf / Coffee Break

17.30-18.30 CONFERENCIA PLENARIA 7 CONFERENCIA GREGORIO WEBER/GREGORIO WEBER CONFERENCEPLENARY LECTURE 7.

Pedro Aramenda: Single molecule and single nanoparticle fluorescence microscopy.Fac. Ciencias Exactas y Naturales, UBA. CIBION-CONICET, Argentina.

Chair: Luis Bagatol l i .

19.30-20.30 CONFERENCIA DE CLAUSURA / CLOSING LECTURE

Flix Goi: Membrane properties ofthe simple sphingolipidsUnidad de Biofsica (CSIC-UPV/EHU), Pas Vasco, Espaa, Spain.

Chair: Gerard o D. Fidelio.

20:30 Anuncio del Premio al Mejor Poster/ Announcem en t o f Bes t Poster Awar d

20:40 Palabras sobre PosLatam y LAFeBS / Words about PosLatam and LAFeBS.Dr. Silvia Alonso, Dr. Pietro Ciancaglini, Dr. Marcelo Morales.

20:55 Palabras de Cierre por el Dr. G. Fidelio /Closing w ords by Dr. G. Fidel io21.00 Cena de Cierre/ Clos ing Di nner

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7

CONFERENCIAS PLENARIAS/ PLENARY LECTURES

P1_Hydrostatic Pressure Effects on the Structure and Stability of Lipid Membranes and

Lyotropic MesophasesSeddon, J. M.

Membrane Biophysics Group, Chemistry Department and Institute of Chemical Biology, Imperial College

London, Exhibition Road, London SW7 2AZ, UK. Lyotropic liquid crystals of 1-, 2-, or 3-dimensional periodicity spontaneously assemble when lipids are

mixed with solvent under various conditions of temperature, pressure and hydration. The mesophases

formed include the 1-D fluid lamellar (L), 2-D hexagonal (HI/HII) and 3-D cubic phases (Q I/QII). Although

the flat fluid lamellar phase is the structure on which biomembranes are generally based, there is

increasing evidence that curved structures such as the inverse cubic phases may be present in cell

membranes, and/or may facilitate various cellular processes such as endo- and exocytosis, membranebudding, and fusion, as these all involve changes in membrane topology. Previous studies of lyotropic

phase transitions have mainly concentrated on transformations between lamellar phases and from

lamellar to inverse hexagonal structures, with little work done on transitions involving cubic phases.

However, a complete understanding of the physical processes governing such transitions, including the

nature of any intermediates formed, and the mechanistic routes taken, is essential if we are to further our

knowledge of their possible roles in fundamental cellular processes involving membranes. We have

therefore been using high pressure and pressure-jump X-ray diffraction to investigate lyotropic phases

and transitions in a range of lipid systems. The use of pressure to trigger transitions has several

advantages: 1) the solvent properties are not significantly altered; 2) pressure propagates rapidly meaning

that equilibrium is achieved rapidly; and 3) pressure-jumps can be both in the pressurisation and

depressurisation directions. We have studied the effects of pressure on the gel-fluid transition in

sphingomyelin bilayer membranes, and have found that the ordering of the chains and the development of

the ripples on forming the gel phase occur on different timescales. We have previously shown that by

addition of weakly-polar amphiphiles such as diacylglycerols to phospholipids, one can tune the interfacial

curvature to be strongly inverse, leading to the formation of a discontinuous cubic phase of spacegroup

Fd3m, with a structure based upon a complex close packing of two types of inverse micelle of different

diameters. We have recently investigated the effect of hydrostatic pressure on the structure and stability of

this phase, and have discovered a number of novel effects. We discovered a lyotropic liquid crystal phase

of space group P63/mmc, whose structure is based upon a hexagonal close packing of identical quasi-

spherical inverse micelles. The system consists of a hydrated mixture of dioleoylphosphatidylcholine,

dioleoylglycerol, and cholesterol. This novel phase has a number of unique features which may render it

useful for a wide range of applications. We have studied the effect of chain branching on glycolipid

thermotropic and lyotropic phases for a series of synthetic -D-glucosides derived from Guerbet alcohols,whose total hydrocarbon chain length ranged from C8to C24. A wide range of liquid-crystalline phases was

observed, with the C16Guerbet glucoside (i.e. -Glc-C10C6) forming an inverse bicontinuous cubic phase

of space group Ia3din excess water, which is very unusual behaviour.

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P2_Interfacial enzymology: investigating the mode of action of lipases

requires a combination of various biophysical approaches

Carrire, F.

CNRS, Aix Marseille Universit, UMR7282 Enzymology at Intefaces and Physiology of Lipolysis,

Marseille, France

Many enzymes are active at interfaces in the living world (such as in signaling processes at the surface of

cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental

enzymology remains largely focused on the interactions between enzymes and soluble substrates. The

biochemical and kinetic characterization of lipases has opened up however new paths of research in the

field of interfacial enzymology and specific interfacial kinetic models have been developed (1). In order to

hydrolyze their insoluble substrate, triglycerides, lipases have first to bind the lipid-water interface(adsorption step) before forming an enzyme-substrate complex in a two dimensional space. This process

is often associated with conformational changes allowing lipases to interact with lipids/amphiphiles while

preserving these enzymes from total unfolding. It is still difficult to characterize the successive steps of

enzymatic lipolysis in a single experimental set-up and access to kinetic constants. Individual steps can

however be studied separately using various biophysical methods. Conformational changes have been

studied using first X-ray crystallography, and more recently site-directed spin labelling coupled to electron

paramagnetic resonance spectroscopy has allowed monitoring the opening of the amphiphilic lid covering

the active site of pancreatic lipase in the presence of amphiphiles and lipids (2, 3). The adsorption step or

lipase-lipid interactions are usually approached using other means such as monomolecular films and

surface pressure measurements (4, 5) or surface spectroscopy like total internal fluorescence

spectroscopy (TIRF; (6)). Examples taken from structure-function studies of pancreatic and gastric

lipases, as well as some microbial lipases, will be presented here to illustrate the complex mode of action

of lipolytic enzymes.

1Aloulou et al.: Biochim. Biophys. Acta - Molecular and Cell Biology of Lipids. 2006. 995.

2Belle et al.: Biochemistry. 2007. 2205.

3Ranaldi et al.: Biochemistry. 2010.2140.

4Point et al. Biochimie. 2013. 51.

5Bnarouche et al.: Colloids and Surfaces B: Biointerfaces. 2013. 306.

6Chahinian et al.: Biochemistry. 2006. 993.

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P3_Dynamics of TGF-beta signaling: how positional information can be learned from achanging morphogen gradient.

Benoit Sorre1,2

, Aryeh Warmflash1,2

, Ali H. Brivanlou1& Eric D. Siggia

2

1 - Laboratory of Molecular Vertebrate Embryology

The Rockefeller University. New York, USA.

2 - Laboratory of Theoretical Condensed Matter Physics

The Rockefeller University. New York, USA.

Genetics and biochemistry have defined the components and wiring of the signaling pathways that patternthe embryo. Many of these pathways have the potential to behave as morphogens: in vitro experimentshave clearly established that these molecules can dictate cell fate in a concentration dependent manner.How morphogens convey positional information in a developing embryo, where signal levels are changingwith time, is less understood. Recently we showed that the evolutionarily conserved TGF-beta pathwayresponds transiently and adaptively to a step in ligand stimulation. Building on previous work, here we useintegrated microfluidic cell culture to stimulate the cells with well-defined temporal profile of morphogen(TGF-) and timelapse microscopy to record their response in real-time, we demonstrate that the speed ofligand presentation has a key and previously unexpected influence on signaling outcomes. Slowly

increasing the ligand concentration diminishes the response while well-spaced pulses of ligand combineadditively resulting in greater pathway output than is possible with constant stimulation. Our resultssuggest that in an embryonic context, an adaptive pathway can naturally extract positional information asligand spreads dynamically from a fixed source, thereby providing an alternative to the static morphogenmodel where the rate of change of ligand concentration, rather than its level, is the meaningful signal forpatterning.

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P5_On the rotary mechanism of the vacuolar proton-ATPase

Pli, T.

Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged,

Hungary

The internal compartments of eukaryotic cells are more acidic than the cytoplasm. The transport protein

complex that is responsible for the acidification is Nature's most universal proton pump, the vacuolar

proton-ATPase (V-ATPase). The V-ATPase is a membrane-bound molecular rotary engine, which

converts the chemical energy from ATP hydrolysis to the rotation of the rotor domain via a torque between

specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and

rotor domains. We have estimated the rate of rotation of the rotor in the yeast V-ATPase, relative to the

stator or steady parts of the enzyme, in native vacuolar membrane vesicles from Saccharomyces

cerevisiae under standardised conditions, in two ways:

(A) The fraction of the total ATPase activity originating from the V-ATPase was determined by using the

potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in

the vacuolar membrane vesicle system was assayed spectrophotometrically for different concanamycin A

concentrations. A fit of the quadratic binding equation to the inhibitor titration curve determined the

concentration of the enzyme. Combining this data with the known ATP/rotation stoichiometry of the V-

ATPase has led to an average rate of ~10 Hz for full 360 rotation, as a lower-limit estimate (1).

(B) We have tested the effect of alternating electric (AC) field on V-ATPase activity in the same yeast

vacuolar vesicle system. This was the first of its kind of experiment on V-ATPase, and we got strikingly

different results from previous studies on other proteins: both low and high frequency AC field reduced

ATPase activity in a wide frequency range, and a sharp resonance was seen at ~88 Hz, where the

ATPase activity reached or exceeded the control (no AC) level. Assuming that the AC field interacts with

the proton movements, and considering the estimated geometry of the proton binding sites and the

hydrophilic proton channels, we conclude that the resonance frequency corresponds to that of the 60

rotor steps. Therefore the rotation rate of the rotor is ~15 Hz, which agrees very well with the above lower-

limit estimate.

To our knowledge, we are the first to report the rotation rate in a V-ATPase that is not subjected to genetic

or chemical modification and is not fixed to a solid support, instead it is functioning in its native membrane

environment.

Acknowledgement: This work was supported by Hungarian National Science Fund (OTKA) grants K68804and K101633.

1Ferencz, C., Petrovszki, P., Kota, Z., Fodor-Ayaydin, E., Haracska, L., Bota, A., Varga, Z., Der, A.,

Marsh, D. and Pali, T. European Biophysics Journal 42(2-3). 2003. 147.

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P6_ Ceramide and Glucosylceramide impact on membrane biophysical properties: from

model to cell membranes

Varela A.R.a,b,c

, Pinto, S.N.c

, Gonalves da Silva, A.M.P.S.d

, Futerman A.b

, Silva L.C.a

and Prieto M.c

aiMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Faculdade de Farmcia,

Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, PortugalbDepartment of Biological Chemistry, Weizmann Institute of Sciences, Rehovot 76100, Israel

cCentro de Qumica-Fsica Molecular & Institute of Nanoscience and Nanotechnology and

dCentro de

Qumica Estrutural, Instituto Superior Tcnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001

Lisboa, Portugal

Sphingolipids (SLs) have emerged as an important class of lipids due to their bioactive role in a number of

cellular events and in disease. The evidence that several SL species participate in the formation of lipid

domains and that this might underlie their biological mechanism of action has fostered research in the

biophysical aspects of bioactive SLs. This has been one of the aims being pursued in my research group.

In this talk I will focus on two important SLs ceramide and glucosylceramide and their interplay with

other lipid components in simple and complex membrane models. Using a combination of biophysical

methodologies that include fluorescence spectroscopy, confocal and two-photon microscopy, surface

pressure-area measurements, allowed elucidating their effects on the biophysical properties of

membranes composed of a variety of lipids and displaying different phase properties. In addition, I will

describe how these interactions can be modulated by alterations in the membrane environment, such as

changes in pH. It will be highlighted how the small structural differences of these lipids influence theirpacking properties, membrane shaping and lateral organization. Inferences will be made regarding the

importance of the headgroup, acyl chain length and unsaturation on the modulation of membrane

properties.

Finally, I will emphasize the significance of these model membrane studies to predict the biophysical and

biological implications of these lipids in cellular membranes and will show examples of how the

observations obtained from model membranes are translated into the cell level.

Supported by FCT (Portugal) grants PTDC/BBB-BQB/0506/2012, PTDC/QUI-BIQ/111411/2009,

SFRH/BD/69982/2010 to ARV, SFRH/BD/46296/2008 to SNP, Compromisso para a Cincia 2008 to LCS.

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P7_Single molecule and single nanoparticle fluorescence microscopy

Aramenda, P. F.

Dept. Qumica Inorgnica. FCEN. Univ. Buenos Aires and CIBION-CONICET. Godoy Cruz 2390. 1425

Ciudad de Buenos Aires. [email protected].

Single molecule fluorescence detection has found many applications in materials science and biology

since its first report in 1990. It offers unique possibilities because of its ultimate detection limit, the ability to

detect and follow in time single events, and the access to the distribution of behaviors versus the average

value of conventional bulk detection methods. At the same time, it attains a time resolution in the

milliseconds range and a spatial resolution of hundreds of nanometers, in conventional detection, and

tens of nanometers in super resolution techniques. More recently, metallic nanoparticles (MNP) have been

extensively used to enhance the performance of molecular fluorescence in bulk and single molecule

applications. The interaction between MNP and fluorophores opens new perspectives in fluorescence

microscopy, based on the interaction of the plasmonic band of the nanostructure and the molecular

electronic states. These interactions allow to detect low intrinsic fluorescent molecules, by an

enhancement in emission brightness, and they also provide an increase in the total number of emitted

photons and in the monitoring time before photo bleaching. In the last six years our laboratory has been

performing research in single molecule techniques, including the use of gold NP (AuNP). In this lecture I

will illustrate experiments in fluorescence microscopy using AuNP to detect low emission quantum yieldmolecules, to increase the monitoring time in cellular environments, to provide protection against

photobleaching, and to enhance the performance of a fluorescent photochromic system in super

resolution localization.
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]

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14

Mini-conferencia I / Short con ference I: Premio a la mejor tesis SAB / best thesis

award

Insights on the Molecular Events that Unleash Resistance to -lactam antibiotics in

Staphylococc us aureus

Llarrull, L.I.

Instituto de Biologa Molecular y Celular de Rosario (IBR), Ocampo y Esmeralda, Predio CONICET,

2000, Rosario.

Staphylococcus aureus is the main cause of hospital- and community-associated infections.1The

expression of the BlaZ-lactamase and of the PBP2a DD-transpeptidase renders S. aureus

resistant to -lactam antibiotics. The expression of these gens is regulated by two related systems,

composed of a membrane associated sensor protein (BlaR1 or MecR1, respectively) and a

repressor (BlaI or MecI, respectively). The sensor proteins and PBP2a itself are promising targets

for the design of inhibitors that would restore the efficiency of -lactam antibiotics. We have used a

combination of spectroscopic techniques, biochemical techniques, molecular modeling and organic

chemistry to characterize different aspects of these two systems. We have documented a lysine N -

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 vivoconcentrations account for the basal level transcription of the resistance genes.4We

have also presented evidence that support the hypothesis that BlaR1 fragmentation is a means for

turnover,5a process required for recovery from induction of resistance in S. aureusin the absence

of the antibiotic challenge, and that BlaR1 is indeed a metallo-protease that degrades the generepressor 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.7In 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

1Llarrull LI, Fisher JF, Mobashery S. Antimicrob. Agents Chemother.2009. 4051.

2Borbulevych O, Kumarasiri M, Wilson B, Llarrull LI, et al. J. Biol. Chem. 2011. 31466

3Kumarasiri M, Llarrull LI, et al. Journal of Biological Chemistry. 2012. 8232.

4Llarrull LI, Prorok M, Mobashery S. Biochemistry. 2010. 7975

5Llarrull LI, Toth M, Champion MM, Mobashery S. Journal of Biological Chemistry. 2011. 38148

6Llarrull LI, Mobashery S. Biochemistry, 2012. 4642.

7Otero LH, Rojas-Altuve A, Llarrull LI, et al. Proc. Natl. Acad. Sci. USA. 2013. 16808.

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S2: Estructura y funcin de protenas (protein structure and function)

17

SIMPOSIOS / SYMPOSIA

S1: Biofsica de biomembranas e interaccin lpido-protena / l ip id-protein

interaction and membrane biophy sics

S1.1_ Novel lipid binding proteins from helminth parasites. Structural and functionalanalysis.

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 drugtargets.

S1.2_The power of being at the interface: mechanism of DesK thermosensing

Cybulski LInstituto de Biologa Molecular y Celular de Rosario (IBR)- CONICET and Departamento deMicrobiologa, 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 theTM 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.

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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 underconditions 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. Cattoni1,2

, Jos M Argello3and F. Luis Gonzlez Flecha

1

1Laboratorio de Biofisica Molecular. IQUIFIB , Universidad de Buenos Aires-CONICET, Argentina

2Centre de Biochimie Structurale, INSERM, Universit de Montpellier, France.

3Department 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 theenthalpic 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

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19

S2: Estructura y funcin de protenas / protein structure and functio n

S2.1_Structural disorder and induced folding in the nucleoproteins and

phosphoproteins of paramyxoviruses

Longhi, S1, Habchi, J

1, Blocquel, D

1, Beltrandi, M

1, Erales, J

1, Dosnon, M

1, Papageorgiou, N

1,

Blangy, S1, Communi, G2,3, Ringkjobing-Jensen M2, Blackledge, M2, Ruigrok, RWH31AFMB, UMR 7257, CNRS and Aix-Marseille University, Marseille, France,

2Institut de Biologie

Structurale, Grenoble, France,3UVHCI, 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 to230 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 reconstitutedsystems 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.

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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 state1and 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 pipette2. 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)

1Hattoriet al.Nature 2012: 207

2Auzmendi et al. PLoS ONE 2012: e42275

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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 (VM)-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 VMdependence 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 Ca2+

-ATPase (SERCA) have led to

detailed mechanistic proposals for how biochemical reactions, ATP binding, enzyme

phosphorylation, and Pihydrolysis drive vectorial transport of Ca2+

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 Ca2+

transport by SERCA. Cysteine scanning mutagenesis experiments were performed with AHA2expressed 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 Ca2+

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.

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S4: Modelado molecular (Biomolecular modeling)

23

S4: Modelado molecular / Biom olecular mo deling

S4.1_Strategies for the de novodesign of protein-protein interactions

Nir Londona,b,

Xavier Ambroggioa

aRosetta Design Group LLC, Burlington, Vermont, USA

bDepartment 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 novodesign 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. [email protected]

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 andeven 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.

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S4: Modelado molecular (Biomolecular modeling)

24

S4.3_Predictive Biomolecular Modeling Applied to Protein Engineering andProteomics

Isabelle F. T. Viana1,2

, Ranieri V. Carvalho3and Roberto D. Lins

1

1Department 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;

3Center 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. Thispathology 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.

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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 difract ion and SAXS to

study biostruc tures

S5.1_Structural studies on a two-component system activated by blue light in

Bruc el la 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:[email protected]

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 responseregulator 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

1Univ. Campinas,

2Brazilian Synchrotron Light Lab (LNLS),

3 , 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 multiplebilayers, 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. 1Balbino et al, Langmuir (2012) 28:11535
mailto:[email protected]:[email protected]

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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 oftheir 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.Ce

libro sab 2013

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