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SAB 2013

Sociedad Argentina de Biofísica

SAB 2013 Sociedad Argentina de Biofísica XLII Reunión Anual 2 - 4 de Diciembre 2013 Villa

XLII Reunión Anual

2 - 4 de Diciembre 2013 Villa Carlos Paz, Córdoba ARGENTINA

SAB 2013

Fanani, Laura XLII Reunión Anual de la Sociedad Argentina de Biofísica / Laura Fanani ; Natalia Wilke ; Gerardo Fidelio. - 1a ed. - Buenos Aires : SAB - Sociedad Argentina de Biofísica, 2013. 146 p. ; 30x21 cm.

ISBN 978-987-27591-2-4

1. Biología.Investigación. I. Wilke, Natalia II. Fidelio, Gerardo III. Título CDD 570.711

Fecha de catalogación: 07/11/2013

Quedan prohibidos, dentro de los límites establecidos en la ley y bajo apercibimiento legalmente previsto, la reproducción total o parcial de esta obra por cualquier medio o procedimientos ya sea electrónico o mecánico, el tratamiento informático, el alquiler o cualquier otra forma de cesión de la obra sin la autorización previa y por escrito de los titulares del copyright.

Diagramación y Edición: Laura Fanani, Natalia Wilke.

Diseño de Tapa: Agustin Mangiarotti, Gerardo Fidelio

Asistencia técnica web: Marcos Solovey

Impreso en Córdoba, Argentina, Noviembre 2013.

SAB 2013

Comisión Directiva de la Sociedad Argentina de Biofísica Año 2013

Presidente

Gerardo Fidelio

Universidad Nacional de Córdoba

Vicepresidente

Gabriela Amodeo

Presidente saliente

Universidad de Buenos Aires

Luis González Flecha

Universidad de Buenos Aires

Secretario

Mauricio Sica

Centro Atómico Bariloche

Tesorera

Lía Pietrasanta

Universidad de Buenos Aires

Vocales Titulares

Karina Alleva

Universidad de Buenos Aires

Rosana Chehín

Universidad Nacional de Tucumán

Vocales suplentes

Rodolfo Rassia

Universidad Nacional de Rosario

Florencia Martini

Universidad de Buenos Aires

Comité Científico SAB 2013

Dr. Gerardo D Fidelio (UNC)

Dr. Guillermo Montich (UNC)

Dra. Laura Fanani (UNC)

Dra. Graciela Borioli (UNC)

Dr. Rafael Oliveira (UNC)

Dr. Ernesto Ambroggio (UNC)

Dr. Marcos Villarreal (UNC)

Dra. Natalia Wilke (UNC)

Dra. Karina Alleva (UBA)

Dr. Rodolfo González Lebrero (UBA)

Comité Organizador y Logística SAB 2013

Dr. Gerardo D Fidelio (UNC)

Dr. Ernesto Ambroggio (UNC)

Dr. Benjamín Caruso (UNC)

Dr. Ernesto Grasso (UNC)

Dra. Soledad Bazán (UNC)

Lic. Pablo Yunes Quartino (UNC)

Lic. Maria Elisa Mariani (UNC)

Lic. Julio Pusterla (UNC)

Lic. Ignacio Gallea (UNC)

Lic. Agustin Mangiarotti (UNC)

SAB 2013

Agradecimientos por financiamiento / Acknowledgements for funding

SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I

C I Q U I B I C

C O N I C E T
C O N I C E T

U

N

C

SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I
SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I
SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I
SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I
SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I
SAB 2013 Agradecimientos por financiamiento / Acknowledgements for funding C I Q U I B I

SAB 2013

Palabras de Agradecimientos / Words of Acknowledgements

Desde su fundación, hace 42 años, SAB organiza su reunión anual en forma ininterrumpida, congregando a

científicos nacionales e internacionales

que trabajan el área de la Biofísica.

En nombre de SAB y como responsables organizadores de la Reunión Anual 2013 de la Sociedad, es para nosotros un honor dar la bienvenida en Córdoba a los participantes de la XLII Reunión 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 Córdoba y honramos su muestra de gratitud. Al grupo de Logística del Congreso que puso todo de sí para una buena organización. Queremos incluir en los agradecimientos a las autoridades de LAFeBs que depositaron su confianza para organizar el VI Curso POSLATAM de la región en Córdoba para 2013.

Por último, agradecemos a la Facultad de Ciencias Químicas y a la Universidad Nacional por su ayuda en infraestructura y financiación; a CONICET y a FONCYT de Agencia Nacional de Promoción Científica y Tecnológica por

su ayuda financiera. A IUPAB que, a

través de LAFeBS, ha financiado con becas el traslado de doctorandos para el

VI Curso POSLATAM. Tengan todos

Ustedes una grata estadía en Córdoba.

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 Córdoba. 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 who placed 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 Córdoba 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 the transfer of doctoral students to assist to VI POSLATAM Course. We wish to all of you a pleasant stay in Córdoba.

Organizadores / Organizers SAB 2013

SAB 2013

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

El origen de la Universidad Nacional de Córdoba fue la promesa de un capital del Obispo Trejo y Sanabria al padre jesuita Diego de Torres en 1613; independiente de la autoridad real, aquella Universidad, se gobernó y se financió a sí misma. La Compañía de Jesús optó por formar élites y clero en un modelo tradicional de conocimiento, alterado cuando la Orden Franciscana se hizo cargo de la Universidad e introdujo obras de Descartes, Newton y Leibnitz.

Durante la disolución del Estado nacional, la Universidad quedó en la órbita provincial hasta su nacionalización en 1856. La Ley Universitaria data de 1885. En esa última época, la UNC se modernizó: eliminó la Teología e incorporó las Facultades de Físico-Matemáticas y Medicina.

Para 1918, la UNC acumulaba las tensiones de una sociedad en transformación 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 política: en 1930 como en cada uno de los cinco golpes militares posteriores padeció intervenciones y restricciones a la autonomía.

Durante las presidencias de Juan D. Perón las universidades ampliaron su matrícula y jerarquizaron nuevas áreas de conocimiento, que en Córdoba incluyó Ciencias Económicas, y Filosofía y Humanidades. Luego se incorporaron nuevos campos como Arquitectura y Urbanismo, Trabajo Social, Matemática, Astronomía y Física, Agronomía y Comunicación.

Durante la primavera democrática de 1973 las expectativas se concentraron en la renovación curricular y el compromiso intelectual con el cambio social pero el proceso se truncó en 1975, cuando la presidenta Perón envió la intervención a las universidades nacionales. Desde 1976, la dictadura militar profundizó un proyecto de universidad elitista y funcional a sus objetivos restringiendo el ingreso, la libertad de cátedra y anulando el co-gobierno.

El retorno a la democracia en 1983 permitió recuperar la institucionalidad de la UNC restituyendo el co-gobierno y la autonomía, y renovando la idea de Universidad comprometida con la sociedad.

La Ley de Educación Superior de 1995, modificó la vida universitaria en aspectos como la producción de conocimientos y el rol de las Universidades en las sociedades.

El siglo XXI conforma un escenario diferente en el que hay nuevos horizontes para diseñar e implementar políticas de educación superior que jerarquicen a las universidades como productoras de ciencia y tecnología atentas a las demandas del desarrollo productivo y social en los valores ciudadanos propios de una sociedad democrática.

SAB 2013

Indice/Index

VI Curso POSLATAM / VI POSLATAM Course……………………………….

Programa del Congreso / Meeting Program………………………………

Conferencias Plenarias / Plenary Lectures……………………………………

Mini-conferencias / Short lectures………………………………………………

Simposios / Symposia………………………………………………………

Resumenes de posters / Poster abstracts: ………………………………

(BPA) ………………………………………………………………………………

Enzimología/ Enzymology (ENZ)…………………………………………………………………………

systems (TMSB) ……………………………………………………………………

(TRC) ………………………………………………………………………………

(BBA) ………………………………………………………………………………

Indice por Autores / Authors index…………………………………………………………

i

1

7

14

17

27

29

52

74

79

81

94

104

108

111

119

SAB 2013

LATIN AMERICAN FEDERATION OF BIOPHYSICAL SOCIETIES VI POSLATAM COURSE SCHEDULE

Biophysical approaches to study systems of biological interest.

28 th November to 4 th December 2013, Córdoba, Argentina

Dia /Day

 

Profesor/Procedencia

Tópicos/Topics

Tiempo/Time

Professor/Affiliations

Miércoles 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 Químicas, Ciudad Universitaria, Universidad Nacional de Córdoba (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, Córdoba (www.portaldelago.com.ar). Course development from November 28 th to December 1 st 2013 will be held at the School of Chemical Sciences, Campus, National University of Cordoba, Córdoba (400 th Anniversary) Meeting-related conferences from 2 nd - 4 th December 2013 will be held at the Congress Hotel in Villa Carlos Paz, Córdoba (www.portaldelago.com.ar). Coordinador/Coordinator: Dr. Gerardo Fidelio (mails: gfidelio@mail.fcq.unc.edu.ar ; gerardo.fidelio@gmail.com) web: http://sab2013.fcq.unc.edu.ar/

Jueves 28 de Noviembre/ Thursday 28 th November

Dr. Aníbal Disalvo Universidad Nacional de Santiago del Estero. Argentina

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 of the topology on hydration and water states. The membrane-solution interphase. Criteria for a new model for membranes. Surface potentials. Determination of zeta potential. Limitations. Electrophoretic mobility. Isotherms of adsorption of charged amino acids and peptides. Synergism and cooperativity. Partition of amino acids and polyols: Corrections to the Wiener -White and Butler-Barclay rules in relation to the water content.

Total Tiempo estimado/Estimated Total time: 6 hs

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

 

Primera Clase/First Lecture:

9.00-10.30

Café/Coffe Break:

10.30-10.55

Segunda Clase/Second Lecture:

11.00-12.30

 

Almuerzo/Lunch 13.00/14.30

Jueves 28 de Noviembre/Thursday 28 th November

   

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 in membranes. 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 and electrical 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. Chemical potential of water and water activity by peptide insertion. Kinks and water

Tarde/Afternoon Mañana/Morning (Segunda Parte/Second Part of Dr. Disalvo 3 hs)

Primera Clase/First Lecture:

15.00-16.30

Café/Coffe Break:

16.30-16.55

Segunda Clase/Second Lecture:

17.00-18.30

SAB 2013

   

species. Translocons and waterons.

 

Tiempo Libre. Free time

Viernes 29 de Noviembre/Friday 29 th November

Dra. Natalia Wilke Universidad Nacional de Córdoba. 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, Oswald ripening, equilibrium and non-equilibrium domain distributions and sizes. Line tension. Intra and inter-domains long-range interactions. Membrane rheology and electrostatics: Shear in membranes: free diffusion, solid obstacles, interacting obstacles. Bending and compression of membranes. Experimental approaches: Model membranes. Active and pasive methods in membrane rheology. Manipulation of membranes using electric fields and optical tweezers.

Mañana/Morning Tiempo Estimado/Estimated Time: 3 hs.

Primera Clase/First Lecture:

9.00-10.30

 

Café/Coffe Break:

10.30-10.55

Segunda Clase/Second Lecture:

11.00-12.30

Almuerzo/Lunch 13.00/14.30

   

Viernes 29 de Noviembre/Friday 29 th November

Dr. Luis BAGATOLLI Membrane Biophysics and Biophotonics Group/MEMPHYS - Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark

FLUORESCENCE MICROSCOPY FOR BIOPHYSICAL STUDIES IN BIOMEMBRANES

Tarde/Afternoon

Tiempo Estimado/Estimated Time: 3 hs.

Modern Fluorescence microscopy instrumentation. Spectral properties of more popular probes. Epi-, confocal and two photon excitation fluorescence microscopy. Giant unilamellar vesicles (GUVs) as tool to study lateral lipid organization and membrane perturbation: lipid-lipid interaction, peptide, protein and lipolytic enzymes interacting with organized lipids.

Primera Clase/First Lecture:

15.00-16.30

Café/Coffe Break:

16.30-16.55

Segunda Clase/Second Lecture:

17.00-18.30

 

Tiempo Libre. Free time

Sábado 30 de Noviembre/Saturday 30 th November

Dr. Benoit SORRE Center for Studies in Physics and Biology, The Rockefeller University, New York, NY., USA

BIOPHYSICS OF MEMBRANE CURVATURE

Mañana/Morning

Fundamentals of membrame curvature. The mean-field theoretical description of membrane mechanics (Helfrich Hamiltionian). Artificial systems to study membranes mechanical properties, (GUVs, micropipette aspiration, membrane fluctuations methods). Ways to pull membrane nanotubes (optical tweezers) to study biology inspired questions like lipid sorting and membrane deformation by proteins (amphiphysin, dynamin).

Tiempo Estimado/Estimated Time: 3 hs.

Primera Clase/First Lecture:

9.00-10.30

 

Café/Coffe Break:

10.30-10.55

Segunda Clase/Second Lecture:

11.00-12.30

 

Almuerzo/Lunch 13.30/15.00

Tarde/Afternoon 3/4 hs

Dr. Frederic CARRIÉRE Laboratory of Enzymology at Interfaces and Physiology of Lipolysis E.I.P.L C.N.R.S., MARSEILLE, France

INTERFACIAL ENZYMOLOGY: THE CASE STUDY OF LIPOLYTIC ENZYMESFundamentals of interfacial enzymology. Modes of action of lipolytic enzymes (lipases and phospholipases) and kinetic models. Monomolecular films as model interfaces for studying lipase-lipid interactions (adsorption/penetration) and interfacial 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).

Tarde/Afternoon Tiempo Estimado/Estimated Time: 3 hs.

Primera Clase/First Lecture:

15.00-16.30

 

Café/Coffe Break:

16.30-16.55

Segunda Clase/Second Lecture:

17.00-19.00

SAB 2013

 

Tiempo Libre. Free Time

 

Domingo 1ro Diciembre / Sunday 1 st December

 

Dra. Maria Elena Carrizo Universidad Nacional de Córdoba. Argentina

 

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

Mañana/Morning Tiempo Estimado/Estimated Time: 3 hs.

Primera Clase/First Lecture:

9.00-10.30

 

Café/Coffe Break:

10.30-10.55

Segunda Clase/Second Lecture:

11.00-12.30

 
 

Almuerzo/Lunch 13.30/15.00

 
 

Dr. Paulo Mascarello Bisch Laboratório de Física Biológica Unidade Multidisciplinar de Genômica Instituto de Biofísica Carlos Chagas FilhoUniversidade Federal do Rio de Janeiro, Brazil

PROTEIN FOLDING AND STRUCTURE MODELING

Tarde/Afternoon Tiempo Estimado/Estimated Time: 3/4 hs.

 
 

1. Protein Folding General Concepts; 2. Protein folding and misfolding, the

Primera Clase/First Lecture:

15.00-16.30

Café/Coffe Break:

16.30-16.55

Segunda Clase/Second Lecture:

17.00-19.00

 

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

 

Tiempo Libre. Free time

 

Lunes 2 de Diciembre/Monday 2 nd December Mañana/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 2 nd -4 th December 2013. (Place: Meeting Hotel)

Lunes 2 de Diciembre / Monday 2 nd December

 

15.00-16.00 CONFERENCE 1

 

Dr. John SEDDON

“Hydrostatic 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. Frédéric CARRIÈRE

Interfacial enzymology: investigating the mode of action of lipases requires a combination of various biophysical approaches. Laboratory of Enzymology at Interfaces and Physiology of Lipolysis E.I.P.L, C.N.R.S. MARSEILLE, FRANCE FRANCIA.

SAB 2013

Martes 3 de Diciembre / Tuesday 3 rd December

 

8.30-9.30

CONFERENCE 3

Dr. Benoit SORRE

"Dynamics of TGF-beta signaling: how positional information can be learned from a changing morphogen gradient". Center for Studies in Physics and Biology, The Rockefeller 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 Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark

17.30-18.30 CONFERENCE 5

Dr. Tibor László PÁLI

"On the rotary 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 PRIETO

Ceramide and Glucosylceramide impact on membrane biophysical properties: from model to cell membranes”. Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, PORTUGAL.

Miércoles 4 de Diciembre / Wednesday 4 th December

 

17.30-18.30 CONFERENCE 7

Dr. Pedro ARAMENDÍA

Single molecule and single nanoparticle fluorescence microscopy”. Fac. Ciencias Exactas y Naturales, UBA. CIBION-CONICET, ARGENTINA.

19.30-20.30 CONFERENCE 8

Dr. Félix GOÑI

“Membrane properties of the “simple” sphingolipids”. Unidad de Biofísica (CSIC- UPV/EHU), País Vasco, ESPAÑA, SPAIN.

 

Fin de las Actividades / END OF ACTIVITIES

SAB 2013

PROGRAMA/PROGRAM

Lunes 2 de Diciembre / Monday, December 2 nd

8.30-13.00 Llegada de Participantes y Registro/Arrival of Participants and Registration

9.00-12.30 Reunión de Representantes del Núcleo Disciplinar de Biofísica AUGM (Asociación Universidades Grupo Montevideo) / Meeting of Representatives of Biophysics Discipline from AUGM (Association of Universities of Montevideo Group)

13.00-14.30 Almuerzo. Colocación de posters impares de todas las secciones/Lunch. Placing of posters. Odd boards: All Topics

14.45-15.00 Apertura del Congreso / Opening Ceremony. Bienvenida / Welcome 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 Lipid Membranes and Lyotropic Mesophases”. Membrane Biophysics Group, Department of Chemistry, Imperial College London, Imperial College, London. UK. Chair: Bruno Maggio.

16.00-18.00

SIMPOSIO

1

/

SYMPOSIUM

1:

BIOFÍSICA

DE

BIOMEMBRANAS

E

INTERACCIÓN LÍPIDO-PROTEÍNA / LIPID-PROTEIN INTERACTION AND

MEMBRANE BIOPHYSICS. Chairs: Ernesto Ambroggio and Natalia Wilke

16:00-16:30: Betina Córsico.Novel lipid binding proteins from helminth parasites. Structural and functional analysis”. INIBIOLP-La Plata. Argentina 16:30-17:00: Larisa Cybulski “The power of being at the interface: mechanism of DesK thermosensing”. IBR-CONICET-Rosario. Argentina 17:00-17:30: Belén Decca. Conformation of peripherally bound membrane proteins: the influence of the lipid phase state”.CIQUIBIC-Córdoba. Argentina 17:30-18:00: Luis Gonzalez Flecha.Phospholipid modulation of membrane protein thermal stability”. IQUIFIB-Buenos Aires. Argentina

18.00-18.25 Café / Coffee Break

18.30-19.00 SECCIÓN JOVEN I / YOUNG SECTION I

Mini-conferencia I / Short-lecture I PREMIO 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: Mario Ermácora

1

SAB 2013

19.00-20.00 CONFERENCIA PLENARIA 2 / PLENARY LECTURE 2

Dr. Frédéric Carrière: Interfacial Enzymology: investigating the mode of action of lipases requires a combination of various biophysical approaches. Laboratory of Enzymology at Interfaces and Physiology of Lipolysis E.I.P.L, C.N.R.S.MARSEILLE, FRANCE. Chair: Gerardo Fidelio

20.30-23.00

SECCIÓN DE POSTERS (Posters impares: Todas las Secciones) / POSTER SECTION (Odd boards: All Topics)

(cena, snack / dinner, snack)

Martes 3 de Diciembre / Tuesday, December 3 rd

8.30-9.30 CONFERENCIA PLENARIA 3 / PLENARY LECTURE 3

Dr. Benoit Sorre: Dynamics of TGF-beta signaling: how positional information can be learned from a changing morphogen gradient”. Center for Studies in Physics and Biology, The Rockefeller University, New York, NY Chair: Ernesto Ambroggio

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: Felix Goñi

10.30-10.55 Café/ Coffee Break

11.00-13.00 SIMPOSIO 2 / SYMPOSIUM 2. ESTRUCTURA Y FUNCIÓN DE PROTEÍNAS / PROTEIN STRUCTURE AND FUNCTION. Chair: Gerardo Fidelio

11:00-11:30 Sonia Longhi. Structural disoder and induced folding in the nucleoproteins and phosphoproteins of paramyxoviruses”. Group Leader "Structural Disorder and molecular Recognition" Architecture et Fonction des Macromolecules Biologiques (AFMB) UMR 7257 CNRS et Universitéd'Aix-Marseille, FRANCE. 11:30-12:00 Gorka Basañez. Elucidating the mechanisms of action of BCL-2 family proteins in apoptosis using in vitro reconstituted systems”. Unidad de Biofísica (CSIC-UPV/EHU), País Vasco, España, Spain. 12:00-12:30 Mauricio Sica. Equilibrium Unfolding of the PDZ Domain of b2-Syntrophin” Departamento de Ciencia y Tecnología, UNQ, Buenos Aires, y Laboratorio de Bioenergías, IEDS, CONICET, Centro Atómico Bariloche, Río Negro, Argentina. 12:30-13:00 Paulo Mascarello Bisch: "A large scale search for protein sequence-structure- function relationship". Laboratório de Física Biológica, Unidade

2

SAB 2013

Multidisciplinar e Genômica, Instituto de Biofísica Carlos Chagas Filho Universidade Federal do Rio de Janeiro, Brazil.

13.00-14.30 Almuerzo. Colocación de posters pares de todas las secciones / Lunch. Placing of posters. Even boards: All Topics

15.00-17.00 SIMPOSIO 3 / SYMPOSIUM 3: TRANSPORTADORES Y CANALES DE MEMBRANA / TRANSPORTERS AND CHANNELS IN MEMBRANES. Chairs: Rodolfo González Lebrero and Karina Alleva.

15:00-15:30 David Naranjo.“Small and Large conductance postassium channels: Where is the difference? Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Chile 15:30-16:00 Luciano Moffatt.Achieving maximal speed of solution exchange for patch clamp 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 República, Regional Norte, Uruguay. 16:30-17:00 Josh Berlin."Conformational changes in transmembrane alpha helices of the H-ATPase, AHA2". Department of Pharmacology and Physiology, UMDNJ- New Jersey Medical School, USA

17.00-17.25 Café/ Coffee Break

17.30-18.30 CONFERENCIA PLENARIA 5 / PLENARY LECTURE 5

Dr. Tibor László Páli: On the rotary mechanism of the vacuolar proton-ATPase”. Institute of Biophysics, Biological Research Centre, Szeged, Hungary Chair: Guillermo Montich

18.30 a 19.30 CONFERENCIA PLENARIA 6 / PLENARY LECTURE 6

Dr. Manuel Prieto: Ceramide and Glucosylceramide impact on membrane biophysical properties: from model to cell membranesInstituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa, Portugal. Chair: Laura Fanani

19.30-20.00 Reunión LAFeBS-POSLATAM / LAFeBS-POSLATAM Meeting

20.00 Asamblea Anual de Socios de SAB / Annual meeting of SAB members

20.30-23.00

SECCIÓN DE POSTERS (Posters pares: Todas las Secciones) / POSTER SECTION (even boards: All Topics)

(cena, snack / dinner, snack)

3

SAB 2013

Miercoles 4 de diciembre / Wednesday, December 4 th

8.30-10.30 SIMPOSIO 4 / SYMPOSIUM 4: MODELADO BIOMOLECULAR / BIOMOLECULAR MODELING Chair: Marcos Villarreal.

8:309:00 Xavier Ambroggio. Strategies for the de novo design of protein-protein interactions”. Rosetta Design Group LLC, Virginia, USA. 9:009:30 Sergio Pantano. “Botulinum neurotoxins and SNARE complexes: A new structural view from modeling and simulations”.Institut Pasteur, Montevideo, Uruguay. 9:3010:00 Roberto Lins.“Predictive Biomolecular Modeling Applied to Protein Engineering and Proteomics”.Department of Fundamental Chemistry, Federal University of Pernambuco, Recife, PE, Brazil 10:0010:30 Ernesto A. Román. “Study of Frataxin folding”. IQUIFIB-Buenos Aires. Argentina

10.30-10.55 Café /Coffee Break

11.00-13.00 SECCIÓN JOVEN II / YOUNG SECTION II

11.00-11.30 Mini-conferencia II / Short-lecture II

Leandro C. Tabares: One enzyme two pathways: Single molecules studies on Nitrite Reductase". CEA Saclay, France. Chair: Rodolfo Rasia

11.30-13.00 Exposición Oral de Posters Seleccionados /Oral Presentation of Selected Posters. Chairs: Laura Fanani and Natalia Wilke

BLM39_Surface and hysteresis properties of lipid interphases composed by head 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: a complementary study by Small Angle X-Ray Scattering, Transmission

11.30-11.50

12.10-12.30

Electron Microscopy and Molecular Dynamic Simulations.

Barbosa,

LRS,

Ventosa, N,

Santana, H, Avila, CL, Cabrera, I, Páez, R, Falcón, V, Pessoa, Jr. A, Veciana, J, Itri, R.

TMSB1_Stochastic

and

Algebraic

Methods

for

Modeling

the

Binding

of

Transcription Factor Cohorts to Cis-acting Gene Regulatory Modules. Mauricio

Bustos and Fernando Levstein.

12.30-12.50 TRC15_The S6 transmembrane segment modulates channel opening in BK

channels. Carrasquel-Ursulaez, W., Contreras, G. F., Sepúlveda, R., Aguayo, González-Nilo, F., González, C. and Latorre, R.

13.00-14.30 Almuerzo /Lunch

4

D.,

SAB 2013

15.00-17.00 SIMPOSIO 5 / SYMPOSIUM

5: DIFRACCIÓN DE RAYOS X Y SAXS EN

BIOESTRUCTURAS / X-RAYS DIFFRACTION AND SAXS TO STUDY

BIOSTRUCTURES. Chairs: Rafael Oliveira and Graciela Borioli.

15:00-15:30 Sebastián Klinke. Structural studies on a two-component system activated by blue light in Brucellaabortus”, FundaciónInstituto Leloir, Buenos Aires, Argentina. 15:30-16:00 Leide Pasos Cavalcanti."Small Angle X ray Scattering to study liposomes for gene therapy", Laboratorio Nacional de Luz Sincrotron, Campinas, Brazil. 16:00-16:30 Mario Ermácora.Structure and function of ICA2, a receptor involved in insulin secretion” Structural Biology and Biotechnology Group, IMBICE, UNQ- Conicet, Argentina. 16:30-17:00 Marcelo Ceolin. "Synchrotron radiation experiments on the biomineralization of ferritin", INIFTA, Univ. Nac. de La Plata, Argentina.

17.00-17.30 Café / Coffee Break

17.30-18.30 CONFERENCIA PLENARIA 7 CONFERENCIA GREGORIO WEBER” / “GREGORIO WEBER CONFERENCE” PLENARY LECTURE 7.

Pedro Aramendía: Single molecule and single nanoparticle fluorescence microscopy”. Fac. Ciencias Exactas y Naturales, UBA. CIBION-CONICET, Argentina. Chair: Luis Bagatolli.

19.30-20.30 CONFERENCIA DE CLAUSURA / CLOSING LECTURE

Félix Goñi: “Membrane properties of the “simple” sphingolipids” Unidad de Biofísica (CSIC- UPV/EHU), País Vasco, España, Spain. Chair: Gerardo D. Fidelio.

20:30 Anuncio del Premio al Mejor Poster / Announcement of Best Poster Award

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 words by Dr. G. Fidelio 21.00 Cena de Cierre / Closing Dinner

5

SAB 2013

6

SAB2013

Conferencias Plenarias (plenary lectures)

CONFERENCIAS PLENARIAS / PLENARY LECTURES

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

Seddon, 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 (H I /H II ) and 3-D cubic phases (Q I /Q II ). 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, membrane budding, 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 P6 3 /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 β-D-glucosides derived from Guerbet alcohols, whose total hydrocarbon chain length ranged from C 8 to C 24 . A wide range of liquid-crystalline phases was observed, with the C 16 Guerbet glucoside (i.e. -Glc-C 10 C 6 ) forming an inverse bicontinuous cubic phase of space group Ia3d in excess water, which is very unusual behaviour.

7

Conferencias Plenarias (plenary lectures)

SAB2013

P2_Interfacial enzymology: investigating the mode of action of lipases requires a combination of various biophysical approaches

Carrière, F.

CNRS, Aix Marseille Université, 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.

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

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

3 Ranaldi et al.: Biochemistry. 2010. 2140. 4 Point et al. Biochimie. 2013. 51.

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

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

8

SAB2013

Conferencias Plenarias (plenary lectures)

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

Benoit Sorre 1,2 , Aryeh Warmflash 1,2 , Ali H. Brivanlou 1 & 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 pattern the embryo. Many of these pathways have the potential to behave as morphogens: in vitro experiments have 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 changing with time, is less understood. Recently we showed that the evolutionarily conserved TGF-beta pathway responds transiently and adaptively to a step in ligand stimulation. Building on previous work, here we use integrated microfluidic cell culture to stimulate the cells with well-defined temporal profile of morphogen (TGF-β) and timelapse microscopy to record their response in real-time, we demonstrate that the speed of ligand 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 combine additively resulting in greater pathway output than is possible with constant stimulation. Our results suggest that in an embryonic context, an adaptive pathway can naturally extract positional information as ligand spreads dynamically from a fixed source, thereby providing an alternative to the static morphogen model where the rate of change of ligand concentration, rather than its level, is the meaningful signal for patterning.

9

Conferencias Plenarias (plenary lectures)

SAB2013

P4_Do liposomes penetrate skin?

Bagatolli Luis A.

Membrane Biophysics and Biophotonics group/MEMPHYS Center for biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark. Campusvej 55, DK 5230, Odense, Denmark. E-mail bagatolli@bmb.sdu.dk

A multiphoton excitation based fluorescence fluctuation spectroscopy method, Raster Image Correlation Spectroscopy (RICS), was used to measure the local diffusion coefficients of distinct model fluorescent substances in excised human skin. In combination with structural information obtained by multiphoton excitation fluorescence microscopy imaging, the acquired diffusion information was processed to construct spatially resolved diffusion maps at different depths of the stratum corneum (SC). Experiments using amphiphilic and hydrophilic fluorescently labeled molecules show that their diffusion in SC is very heterogeneous on a microscopic scale. This diffusion-based strategy was further exploited to investigate the integrity of liposomes during transdermal penetration. Specifically, the diffusion of dual color fluorescently labeled liposomes -containing an amphiphilic fluorophore in the lipid bilayer and a hydrophilic fluorophore encapsulated in the liposome lumen- was measured using cross correlation RICS. This type of experiment allows discrimination between separate (uncorrelated) and joint (correlated) diffusion of the two different fluorescent probes, giving information about liposome integrity. Independent of the liposome composition (phospholipids or transfersomes), our results show a clear lack of cross- correlation below the skin surface, indicating that the penetration of intact liposomes is highly compromised by the skin barrier.

1 J. Brewer, J. Kubiak J, M. Bloksgaard, J. A. Sørensen and L.A. Bagatolli. J. Investig.

Dermatol.(2013).133:2601268

10

SAB2013

Conferencias Plenarias (plenary lectures)

P5_On the rotary mechanism of the vacuolar proton-ATPase

Páli, 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 K68804 and K101633.

1 Ferencz, 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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Conferencias Plenarias (plenary lectures)

SAB2013

P6_ Ceramide and Glucosylceramide impact on membrane biophysical properties: from model to cell membranes

Varela A.R. a,b,c , Pinto, S.N. c , Gonçalves da Silva, A.M.P.S. d , Futerman A. b , Silva L.C. a and Prieto M. c

a iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal

b Department of Biological Chemistry, Weizmann Institute of Sciences, Rehovot 76100, Israel

c Centro de Química-Física Molecular & Institute of Nanoscience and Nanotechnology and d Centro de Química Estrutural, Instituto Superior Técnico, 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 their packing 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 Ciência 2008 to LCS.

12

SAB2013

Conferencias Plenarias (plenary lectures)

P7_Single molecule and single nanoparticle fluorescence microscopy

Aramendía, P. F.

Dept. Química Inorgánica. FCEN. Univ. Buenos Aires and CIBION-CONICET. Godoy Cruz 2390. 1425 Ciudad de Buenos Aires. Argentina. pedro@qi.fcen.uba.ar.

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 yield molecules, 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.

13

SAB2013

Conferencias (lectures)

Mini-conferencia I / Short conference I: Premio a la mejor tesis SAB / best thesis award

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

Llarrull, L.I.

Instituto de Biología 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. 1 The 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 ζ - 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.

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Conferencias (lectures)

SAB2013

Mini-conferencia II / Short conference II

One enzyme two pathways: Single molecules studies on Nitrite Reductase

Tabares LC

Service de BioénergétiqueBiologieStructuraleetMécanismes, 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.

15

SAB2013

16

S2: Estructura y función de proteínas (protein structure and function)

SIMPOSIOS / SYMPOSIA

S1: Biofìsica de biomembranas e interacción lípido-proteína / lipid-protein interaction and membrane biophysics

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

Marina Ibáñez Shimabukuro*, Florencia Rey*, Gisela R. Franchini*, Malcolm W. Kennedy # , Alan Cooper # , Brian O. Smith # and Betina Córsico* * Instituto de Investigaciones Bioquímicas de La Plata (CONICET-UNLP), Facultad de de Ciencias Médicas, 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 helminth’s limited lipid metabolism, LBP’s 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 Biología Molecular y Celular de Rosario (IBR)- CONICET and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, 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 función de proteínas (protein structure and function)

S1.3_ Conformation of peripherally bound membrane proteins: the influence of the lipid phase state

María Belén Decca Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Haya de la Torre y Medina Allende,5000, Córdoba, 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 Martínez, Diego I. Cattoni 1,2 , José M Argüello 3 and F. Luis González Flecha 1

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

2 Centre de Biochimie Structurale, INSERM, Université 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. ΔS # values were similar in membranes and mixed micelles, but higher than those obtained for CopA reconstituted in detergent micelles, whereas ΔH # 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 función de proteínas (protein structure and function)

S2: Estructura y función de proteínas / protein structure and function

S2.1_Structural disorder and induced folding in the nucleoproteins and phosphoproteins of paramyxoviruses

Longhi, S 1 , Habchi,

Blangy, S 1 , Communié, 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.

Papageorgiou, N 1 ,

J 1 ,

Blocquel, D 1 ,

Beltrandi, M 1 ,

Erales, J 1 , Dosnon, M 1 ,

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, Basañez, G Unidad de Biofísica, Consejo Superior de Investigaciones Científicas - Universidad del País 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 función de proteínas (protein structure and function)

S2.3_Equilibrium Unfolding of the PDZ Domain of b2-Syntrophin

Torchio, G 1,2 ; Burgos, I 3,2 ; Fidelio, G 3,2 ; Arán, M 4,2 ; Gallo, M 4,2 ; Ermácora, M 1,2 ; Sica, M 5,2 . 1 .Laboratorio de Plegado y Expresión de Proteínas, Univ. Nac. de Quilmes-IMBICECIC, Argentina. 2 .CONICET, Argentina. 3 .Centro de Investigaciones de Química Biológica,Univ. Nac. de Córdoba, Argentina. 4. Fundación Instituto Leloir, Argentina. 5 .Laboratorio de Bioenergías. IEDS. Centro Atómico Bariloche, Argentina.

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in the insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in a complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. We have investigated the thermal unfolding of β2S-PDZ at different conditions and applying various techniques. Our results indicate that, unlike other PDZ domains, β2S-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 β2S-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 Biofísica 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 Maranhão, AG et al.: Infect Genet Evol

3 Lery, LMS et al.: BMC Genomics. 2010. S7.

2012. 1397.

S3: Transportadores y canales de membrana (Transporters and channels in membranes)

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), González-Nilo, F.(2), Sepúlveda, R.(2), and Naranjo, D:(1). (1) Centro Interdisciplinario de Neurociencia, Universidad de Valparaíso, Valparaíso, Chile, (2) Center for Bioinformatics and Integrative Biology (CBIB), Universidad Andrés 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 2Auzmendi et al. PLoS ONE 2012: e42275

S3: Transportadores y canales de membrana (Transporters and channels in membranes)

S3.3_Cationic Amino Acid Transporters: insights from a non-transportable enantiomer

Peluffo, R. D.

Universidad de la República, 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)

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 naïve 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)

S4.3_Predictive

Proteomics

Isabelle F. T. Viana 1,2 , Ranieri V. Carvalho 3 and Roberto D. Lins 1

Biomolecular

Modeling

Applied

to

Protein

Engineering

and

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

Román 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: Difracción de rayos X y SAXS en bioestructuras (X-Ray difraction and SAXS to study biostructures)

S5: Difracción 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. Fundación 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

, 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: Difracción de rayos X y SAXS en bioestructuras (X-Ray difraction and SAXS to study biostructures)

S5.3_Structure and function of ICA2, a receptor involved in insulin secretion

Ermácora 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 β–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

Ceolín, M. Instituto de Investigaciones físico-Químicas Teóricas 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 Alzheimer’s 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

SAB 2013

Biofísica de lípidos y membranas (Biophysics of lipid and membranes)

BLM1_Mechanical properties of membranes from spheroplast using optical tweezers.

Colque, A., Gonzalez Montoro, M.A., Valdez-Taubas, J. y Wilke, N. Instituto de Química Biológica de Córdoba (CIQUIBIC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad nacional de Córdoba.

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 µL 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 10µL-drop of an aqueous solution of micro-spheres (anionic, 3 µm diameter or cationic, 1 µm 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 Microscopìa, 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 Investigación Biofísica (GIBF), Instituto de Física, Universidad de Antioquia, Medellín, Antioquia, Colombia. § Departimento de Tecnologie e Salute, Istituto Superiore di Sanitá, 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

BLM3_Studies of the Interaction of a Galleria mellonella Peptide with Molecular Models of Gram-Negative Bacteria Membranes

Villanueva G. 1 , Correa W. 1 , Oñate J. 1 , Patiño E 1 ., Espejo. F. 2 , Brandenburg K. 3 Manrique-Moreno M 1 .

1 Instituto de Química, Universidad de Antioquia, Medellín, Colombia. e-mail: mmanrique@exactas.udea.edu.co, mmanriqm@hotmail.com

2

Escuela de Química, Facultad de Ciencias, Universidad Nacional, Medellín, 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 Föster 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 Química Biológica de Córdoba (CIQUIBIC), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Pabellón Argentina, Ciudad Universitaria. X5000HUA Córdoba. 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.

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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,

de

Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. c Medicinal

Organic and Nanomaterials Chemistry, Department of Chemistry, Rhodes University.

Departamento

de

Química

Orgánica,

b CIQUIBIC-CONICET,

Departamento

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 Química Biológica de Córdoba (CIQUIBIC), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Pabellón Argentina, Ciudad Universitaria. X5000HUA Córdoba.

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.

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BLM7_Study of hemorheological properties of hypercholesterolemic rats treated with proantocianidine extract of Ligaria cuneifolia (Lc)

García 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 Biofísica, Fac. Cs. Médicas, UNR, 2 Fisiología, Fac. Cs. Bioq. y Farm.-IFISE-UNR-CONICET, 3 Farmacobotánica, 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: 119.74±1.26, E: 68.50±1.86*, HDLCho: C: 28.80±0.62, E:23.17±0.86*; LDLCho: C.25.30±0.84, E:18.16±0.59*. Blood: Morphological Index (light microscopy): C:-2.35±0.48, E:-1.98 ±0.29*; SIII(%): C:40.12±14.23, E:27.26±11.74**; Rigidity Index (nucleopore membrane filter): C:8.01±0.96; E:6.30±0.92**; Membrane erytrocyte Cho (mg%):

C:1.05±0.15, E: 0.77±0.23*.(**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 Químicas, Universidad Nacional de Córdoba. Ciudad Universitaria, X5000HUA-Córdoba, 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.

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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 Química Biológica de Córdoba (CIQUIBIC), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA, Córdoba, 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 Peña 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.

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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 Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, X5000HUA- Córdoba, 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-

Hénon, 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, Bahía Blanca, Buenos Aires.b. CIQUIBIC-CONICET, Córdoba, Córdoba.

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

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BLM13_Surface behaviour of sphingomyelines with very long chains (C28-C32) PUFAs and their interaction with premixed or enzymatically generated ceramides

Peñalva DA 1 , Wilke N 2 , Maggio B 2 , Aveldaño MI 1 , Fanani ML 2 1 INIBIBB, CONICET-UNS, Bahía Blanca, 2 CIQUIBIC, UNC-CONICET, Córdoba, 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. Peñalva, et al, J. Lipid Res. (2013)

54:2225-35.

2-

D.A. Peñalva, 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, Frédéric 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 Caillé 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.

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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 Fisicoquímica Dpto. de Química. INQUISUR Universidad Nacional del Sur 2 -Laboratorio de Biointerfases. Centro de Investigación 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 50ºC and 60ºC 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.

BLM15_Surface

properties

of

liposomes

mixtures

of

phosphatidylcholines

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

monolayers

Pinzón Barrantes, J. a ,Hoyos de Rossi, R. a , Maggio, B b ., Vico, R. a

of

amphiphilic

cyclodextrins

with

phospholipids

in

Langmuir

a INFIQC-CONICET,

Departamento

de

Química

Orgánica,

b CIQUIBIC-CONICET,

Departamento

de

Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba.

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 πA isotherms and the excess of free energy of mixing (G exc ). The results indicate non ideal mixing. Additionally, the more stable composition is π dependent, al low π

the more stable film is enriched in POPC, while at high π 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., Djedaíni-Pilard, F. Langmuir 2013. 3677.

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BLM17_Folic acid: pro-oxidant action and characterization of a system based in SPC liposomes

Marsanasco M. a , Piotrkowski B. b , Calabró 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 Fisicoquímica, Facultad de Farmacia y Bioquímica, UBA e Instituto de Bioquímica 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. Álvarez a a INSIBIO (CONICET-UNT), Instituto Superior de Investigaciones Biológicas, Chacabuco 461, San M. de Tucumán, Tucumán, T4000ILI, Argentina b Instituto de Química Física, Universidad Nacional de Tucumán, San Lorenzo 456, San. M. de Tucumán, Tucumán, 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.Álvarez, Spectrochim Acta Part A, (2013) 112, 403

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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. Álvarez a a INSIBIO (CONICET-UNT), Instituto Superior de Investigaciones Biológicas, Chacabuco 461, San M. de Tucumán, Tucumán, T4000ILI, Argentina b Instituto de Química Física, Universidad Nacional de Tucumán, San Lorenzo 456, San. M. de Tucumán, Tucumán, 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, André, International Journal of Peptides, (2011), Volume 2011, Article ID 175145

BLM20_Interactions

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 .

and

Elastic

Properties

Of

Lipid

Membranes:

The

Role

of

A

1 Complex Fluids Group, Experimental Physics Department, Institute of Physics, University of São Paulo,

São 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 Caillé 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.

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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 Física, 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 ; Peñalva, D.A. a ; Vitale, A.J. b , Aveldaño, M.I. a ; Barrantes F.J c ; Antollini S.S. a a Instituto de Investigaciones Bioquímicas de Bahía Blanca / Universidad Nacional del Sur, Argentina. b Instituto Argentino de Oceanografía / 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, 4°C). 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.

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BLM23_Changes in biophysical properties of membranes containing sphingomyelin with very long chain PUFA induced by its hydrolysis

Peñalva DA 1 , Fanani ML 2 , Maggio B 2 , Aveldaño MI 1 , Antollini SS 1 1 INIBIBB, CONICET-UNS, Bahía Blanca, and 2 CIQUIBIC, UNC-CONICET, Córdoba, 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 Química Orgánica, 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.

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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 Biológicas (INSIBIO) CONICET-UNT. Chacabuco 461, San Miguel de Tucumán. 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 Química Inorgánica, CEQUINOR (UNLP-CONICET). 2 Departamento de Ciencias Biológicas, 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.

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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 , González Flecha FL 1 1 IQUIFIB-CONICET, Dpto. de Química Biológica, FFyB, Universidad de Buenos Aires 2 IQUIBICEN-CONICET, Dpto. de Química Biológica, 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. Simão 1 , Kiffer-Moreira, T. 2 , M.F. Hoylaerts 3 , J.L. Millán 2 and P. Ciancaglini 1

1 Departamento de Química, FFCLRP-USP, Ribeirão 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.

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BLM29_Structural

simulations.

properties

of

CHAPS

micelles,

studied

by

molecular

dynamics

Herrera, Fernando E. a ; Garay, A. Sergio a ; Rodrigues, Daniel E. a,b

a Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral (UNL), Ciudad Universitaria, 3000 Santa Fe, Argentina. b INTEC (CONICET-UNL), Güemes 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.; Marić, I.; Benedini, L.; Dodero, V. I Depto de Química- INQUISUR, Universidad Nacional del Sur-CONICET, Bahía 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.

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BLM31_Compressibility modulus and states of water in lipid monolayers.

Frías, M.A.,Salcedo, C.L.,Cutro, A.C. and Disalvo, E.A Laboratorio de Biointerfases y Sistemas Centro de Investigación 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.

Cutró, A.C. 1 , Salcedo, C.L. 1,2 , Frías, M.A., Nazareno, M.A. 2 , Disalvo, E.A. 1 Laboratorio de Biointerfases y Sistemas Biomiméticos 1 y Laboratorio de Antioxidantes y Procesos Oxidativos 2 , Centro de Investigación 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., López de Mishima B.A., Nazareno M.A. Food Res. Int., 2010. 43, 1187.

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BLM33_FTIR interaction between glycine and DPPC in hydrated state.

Norma M. Ale 1 , Aida Ben Altabef 1,2 , Andrea Gómez Zavaglia 3 and Sonia B. Díaz 1 1 Instituto de Química Física. Facultad de Bioquímica, Química y Farmacia. U.N.T. San Lorenzo 456. T4000CAN Tucumán, Argentina. e-mail:sobedi63@yahoo.com.ar. 2 Instituto de Química del Noroeste (INQUINOA)-CONICET-Tucumán, 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. Gómez Zavaglia, A.; Tymczyszyn, E.; De Antoni, G.; A. Disalvo J. Appl. Microbiol., 95(6): 1315-1320

(2003).

2. Díaz, S. B.; Biondi de López, 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. Díaz 1 and A. Ben Altabef 1,2 1 Instituto de Química Física. Facultad de Bioquímica, Química y Farmacia. Universidad Nacional de Tucumán San Lorenzo 456. T4000CAN Tucumán. 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. Goñi, Chem. Phys. Lipids 96 (1998) 5368.

2-

C. B. Fox, R. H. Uibel, J. M. Harris, J. Phys Chem. 2007, 111, 1142811436.

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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.QuímicaBiológica, Fac. Cs.Químicas, 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 α-helix structure. Melittin form homogeneous monolayers at BAM, but when mixed with a lipid that forms Liquid Expanded (LE) monolayers (T c,dmPC 21ºC) 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 -2ºC), the domains were not observed. On the other hand, the β-sheet Aβ1-42 peptide exhibits a different behaviour when mixed with either DMPC or POPC. In Aβ1-42/DMPC mixtures, although phase segregation did not occur at a defined lateral pressure, a fibrillar domain pattern became apparent upon compression. In Aβ1-42/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), Frías, M.A.(2), Disalvo, E. A.(2) (1) Laboratorio de Sistemas Nanoestructurados y Electroquímica., (2) Laboratorio de Biointerfases y Sistemas Biomiméticos. Centro de Investigación 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. Frías, 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

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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 Bioquímicas de Bahía Blanca, Universidad Nacional del Sur, Camino “La Carrindanga” Km 7, B8000FWB Bahía 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 (0´s and 1´s). 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.

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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 Biomiméticos 1 y Laboratorio de Antioxidantes y Procesos Oxidativos 2 , Centro de Investigación 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., FríasM.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 , Calabró 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 Fisicoquímica, Facultad de Farmacia y Bioquímica, UBA e Instituto de Bioquímica 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 thermolábil 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

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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 Biofísica, Instituto de Física, Universidade de São 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-

Berberián M.V. 1,2 , Mayorga L.S. 2 , Ciocco Aloia F. 1 and Del Popolo M.G 1 Instituto de Ciencias Básicas (ICB) 1 e Instituto de Histología y Embriología 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

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 Física, Facultad de Bioquímica y Ciencias Biológicas, 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

Dep.

gborioli@mail.fcq.unc.edu.ar

CIQUIBICCONICET,

de

Química

Biológica,

Fac.

de

Ciencias

Químicas,

UN

Córdoba.

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 Químicas, Univ. Nac. de Córdoba

SAB 2013

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BLM45_Thermotropic and structural study of sphingolipid mixtures

Dupuy, F; Oliveira, R; Maggio, B.

Centro de Investigaciones en Química Biológica de Córdoba CIQUIBIC-CONICET/UNC, Haya de la Torre S/N, Ciudad Universitaria, Córdoba, 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 Biotecnología,CEPROCOR- Min.CyT Pcia. de Córdoba; b INSIBIO-CONICET/UNT; c CIQUIBIC-CONICET/UNC, Depto. Qca. Biológica, Facultad de Ciencias Químicas, UNC

Paclitaxel (Ptx) is an antitumoral drug that inhibits microtubule depolymerization. However, its low water solubility (less than 1 μg.ml 1 ) is a drawback in treatment of cancer patients. Recently, we demonstrated that paclitaxel (Ptx) can be loaded in