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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BREYTON Cécile / EBEL Christine / SCHAACK Beatrice

Structure and Stability of Integral Membrane Protein and Phage Assemblies

Team Leader: Cécile Breyton (CNRS)


Team


- Cécile Breyton (CR, CNRS)
- Christine Ebel (DR, CNRS)
- Eric Forest (DR, CEA)
- Béatrice Schaack (DR, CEA)
- Aline Le Roy (AI, CEA)
- Charles Arnaud (Th)

Main Research themes


Structural study of membrane proteins related to virulence and pathogenicity

New approaches for in vitro studies of membrane proteins: novel surfactants; droplet interface bilayer; H/D exchange in mass spectrometry

Expertise


Biochemistry of membrane proteins
Biophysics
Cristallography
X-rays and neutron scattering
Nanotechnology

Responsibility of platforms


Analytical Ultracentrifugation
Protein analysis on line

Research theme: Structural study of proteins related to virulence and pathogenicity

- Infection of E. coli by phage T5

C. Breyton, in collaboration with P. Boulanger, Orsay, M. Chami, Basel, P. Schanda and G. Schoehn, IBS, investigates the early steps of the infection process at the molecular and structural level, notably the structure of the phage tail and channel formation for DNA entrance, using SANS, X-ray crystallography, NMR, and electron microscopy.

- Flayhan, Vellieux, Lurz, Maury, Contreras-Martel, Girard, Boulanger, Breyton. Crystal structure of pb9, the distal tail protein of bacteriophage T5: a conserved structural motif among all siphophages. (2014) J Virol 88 220-8.
- Zivanovic, Confalonieri, Ponchon, Lurz, Chami, Flayhan, Renouard, Huet, Decottignies, Davidson, Breyton, Boulanger. Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components. (2014) J Virol 88 1162-74.
- Breyton, Flayhan, Gabel, Lethier, Durand, Boulanger, Chami, Ebel. Assessing the conformation changes of pb5, the Receptor Binding Protein of phage T5, upon binding to its E. coli receptor FhuA. (2013) J Biol Chem 288 30763-72.

- Membrane proteins related to pathogenicity

We are studying the association state, structure and conformation changes of the membrane transporter FhaC from Bordetella pertussis (coll. V. Villeret, Lille and F. Gabel, IBS), of the neutrophilic NADPH oxidase complex and C-type lectin receptors of dendritic cells (coll. F. Fieschi, IBS) and of multidrug ABC transporters (coll. J.-M. Jault, Lyon and F. Gabel, IBS).

- Gabel, Lensink, Clantin, Jacob-Dubuisson, Villeret, Ebel. Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling. (2014) Biophys J 107 185-96.
- Sutkeviciute, Thepaut, Sattin, Berzi, McGeagh, Grudinin, Weiser, Le Roy, Reina, Rojo, Clerici, Bernardi, Ebel, Fieschi. Unique DC-SIGN Clustering Activity of a Small Glycomimetic: A Lesson for Ligand Design. (2014) ACS Chem Biol 9 1377-85.
- Boncoeur, Durmort, Bernay, Ebel, Di Guilmi, Vernet, Jault. PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of /Streptococcus pneumoniae /to//fluoroquinolones. (2012) Biochemistry 51 7755-65.

Research theme: New approaches for in vitro studies of membrane proteins

- Novel surfactants

We are studying new fluorinated surfactants and new amphiphilic polymers for the structural study of membrane proteins (coll. G. Durand and A. Polidori, Avignon, S. Keller, Kaiserslautern DE, F. Gabel, IBS).

- Breyton, Gabel, Lethier, Flayhan, Durand, Jault, Juillan-Binard, Imbert, Moulin, Ravaud, Hartlein, Ebel. Small angle neutron scattering for the study of solubilised membrane proteins. (2013) Eur Phys J E Soft Matter 36 71.
- Abla, Unger, Keller, Bonnete, Ebel, Pucci, Breyton, Durand. Micellar and biochemical properties of a propyl-ended fluorinated surfactant designed for membrane-protein study. (2015) J Colloid Interface Sci 445 127-36.
- Popot, Althoff, Bagnard, Banères ... Ebel ... Zoonens. Amphipols from A to Z. (2011) Ann Rev Biophys 40 379-408.
- Lethier, Moulin, Härtlein, Ebel. Report on expression and purification of a deuterated model membrane protein: OmpX

- Droplet Interface Bilayer (DIB)

The aim of the VenomPicoScreen project (ANR RPIB 2012) is the insertion of potassium channels and GPCRs as single protein in an artificial bilayer, to identify potential drug candidates from peptide venoms modulating membrane proteins activity. We are using biochips, combining microfluidics and electrophysiology. The partnership between IBS (B. Schaack, C. Moreau), the CNRS (B. Cross), the CEA (N. Gilles) and the startup Smartox (R. Beroud) aims to develop a novel drug screening dedicated to small volumes. Targets are inserted in an artificial bilayer obtained between two nanodroplets and their electric activity is measured.

-Hydrogen/deuterium exchange mass spectrometry HDXMS

It enables a rather fine identification (5-10 amino acids) of regions of proteins implied in conformational changes or interactions with partners.

- Lennartz, Bengtsson, Olsen, Joergensen, Brown, Remy, Man, Forest, Barfod, Adams, Higgins, Jensent. Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding. (2015) J Immunol 195 3273-83.

Others themes

- Biochemistry and structural biology of other membrane complexes
- Allorent, Tokutsu, Roach, Peers, Cardol, Girard-Bascou, Seigneurin-Berny, Petroutsos, Kuntz, Breyton, Franck, Wollman, Niyogi, Krieger-Liszkay, Minagawa, Finazzi. A dual strategy to cope with high light in Chlamydomonas reinhardtii. (2013) Plant Cell 25 545-57.
- Dach, Olesen, Signor, Nissen, le Maire, Møller, Ebel. Active detergent solubilized H+,K+-ATPase is a monomer. (2012) J Biol Chem 287 41963-78.
- Breyton, Haase, Rapoport, Kuhlbrandt, Collinson. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. (2002) Nature 418 662-5.

- Thermodynamics and experimental approaches

We propose protocols about membrane proteins and surfactants, intrinsically disordered proteins, polysaccharides and their complexes with proteins, analysis of weak interactions and hydration. We developped an expertise in solvent mediated protein-protein interactions. We have also expertise in biochips.
- Le Roy, Wang, Schaack, Schuck, Breyton, Ebel. AUC and Small-Angle Scattering for Membrane Proteins. (2015) Method Enzymol 562 257-86.
- Publication list related to AUC and PAOL
- Schaack, Wei, Thiery, Auger, Hochepied, Castellan, Ebel, Chaneac, Achouak. Design of a live biochip for in situ nanotoxicology studies: a proof of concept. (2015) RSC Adv 5 82169–78.

- Molecular adaptation to extreme conditions

We proposed that the very acidic surface of halophilic proteins allows specific interactions with solvent salt ions, in order to preserve high solubility needed for in vivo protein function using multimolar salt concentrations. Our data also allows to understand the complex protocol for the protein crystallization. We are also investigating the interactions and structure in solution of an antifreeze protein AFPIII (collaboration with A. Salvay and E. Howard, Argentina).
- Salvay, Gabel, Pucci, Santos, Howard, Ebel. Structure and interactions of fish type III antifreeze protein in solution. (2010) Biophys J 99 609-18.
- Ebel, Zaccai. Crowding in extremophiles: Linkage between solvation and weak protein – protein interactions, stability and dynamics, provides insight into molecular adaptation. (2004) J Mol Recognit 17 382-89.

Key words

• Structure-function relationships of receptors, channels and transporters
• Tools and methods: from expression to crystallization
• Biophysics and biochemistry
• Molecular and structural virology
• Bacteriophages
• Surfactants