PhD projects


Structural Biology of Membrane Proteins

PhD project:

Structure-function of mitochondrial carriers

Crystal structures combined to in situ studies

Principal Investigator:

E. Pebay-Peyroula, Inst de Biol. Struct. UMR5075 CEA-CNRS-Univ. J.Fourier, 41, rue Jules Horowitz, F38027 Grenoble cedex 1; eva.pebay-peyroula|

In collaboration with:

H. Vogel, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL); horst.vogel|

Mitochondrial carriers are essentials membrane proteins that transport a large variety of metabolites over the inner mitochondrial membrane. They are deeply related to major metabolic cycles ongoing in mitochondrial and are also implicated in diseases. Due the presence of a specific sequence signature among all members of the mitochondrial carrier family (MCF), they most probably share a similar fold although despite the diversity of transported substrates (nucleotides, phosphates, protons, glutamate, ornithine,...). High resolution structural studies are well suited approaches to shed light on the transport mechanisms and to understand the differences that ensure specificity. Furthermore, fully understanding the transport mechanism in the native membrane, also relies on the knowledge of the protein arrangement in the membrane.

The project is focused on two members of the family, the ADP/ATP carrier (AAC) responsible for the import of ADP to the mitochondria and the export toward the cytosol of ATP after synthesis, and the uncoupling proteins (UCP) which allow the entrance of protons to the mitochondrial matrix and therefore uncouple the respiratory chain and ATPsynthase. We solved the structure of the ADP/ATP carrier in the presence of an inhibitor, carboxyatractyloside (Nature, 2003). It is the first MCF for which a structure is known and still the only one. >The structure revealed the overfold of the carrier in one of the conformations adopted during the transport, and showed a large cavity open towards the inter membrane space. At the same time, it brought new insights to the transport, and broke the dogma of the necessity of having dimers. Although this structure sheds some light of substrat specificity and transport mechanism, understanding the transport mechanism still needs additional information (structures from various conformations, structures from other MCF members, supramolecular arrangement in the native membrane,...).

For the structural work both proteins will be produced from natural sources, bovine heart mitochondria for AAC and rat brown adipous tissue for UCP1 (collaboration with B. Miroux). In parallel, overexpression systems will be set up in order to produce larger quantities of the native proteins and also mutants.  Different detergents will be tested for the solubilizing, purifying and stabilizing the proteins. Lipids, in particular cardiolipins known to influence AAC activity, will be tested as additives. Protein-detergent complexes in solution will be analyzed and characterized prior crystallization. Crystallization of both carriers will be undertaken with the classical vapor diffusion method and also by using lipidic phases. Microcrystals will be tested on a synchrotron beam-line and structures solved by X-ray crystallography. Characterization of the supramolecular arrangement of the carriers in the native membrane will be approached by FRET studies on yeast mitochondria in the group of H. Vogel.

The Institut de Biologie Structurale at Grenoble has established an ensemble of techniques for determining structures of protein and larger macromolecular assemblies. The group of E. Pebay-Peyroula has an expertise in the field of membrane proteins. The group is interested in the developments of crystallization strategies with a special focus on mitochondrial carriers. Structure determination by X-ray crystallography is favored by the proximity of the ESRF synchrotron radiation source. A first structure of the ADP/ATP structure was solved in the lab and several transport features were proposed from it. The crystallization of AAC and UCP will benefit from this experience. The group of H. Vogel has outstanding expertise in various fluorescence techniques at the cellular level. Furthermore, the group has succeeded recently to elucidate the supramolecular arrangement of GPCR receptors at the surface of eukaryotic cells. The same strategy will be applied to approach the oligomerization state of MCF carriers in the membrane of yeast mitochondria.

The main topics of this PhD project will be the high resolution structure determination of AAC complexed with an inhibitor or nucleotide analog. The analysis of at least two AAC conformations will shed led on structural modifications occurring during the transport mechanism. In addition, the interpretation of FRET signal at the surface of yeast mitochondria will bring new insights to the existence (or not) of cooperativity. In parallel, a similar approach will be undertaken on UCP1 known to transport protons. Comparison between two structurally related carriers at high resolution will allow to understand the major differences that account for such different transported entities. The project aims to study molecular processes integrating different scales spanning from the atomic details in the molecule to the scale of a molecule in a mitochondrion.

- Protein biochemistry and characterization (E. Pebay-Peyroula in coll. with B. Miroux, 12 months)

Proteins from the native tissues will be prepared based on previous established protocols. In parallel, overexpression methods in E. coli or a cell-free system will be developed to produce native proteins in larger quantities or mutants. Ligand binding will be followed by surface plasmon resonance. Protein solubilized in detergent will be characterized by combining size-exclusion chromatography with refractive index, absorbance, static and dynamic light scattering measurements in order to produce homogenous and stable protein solutions for crystallization.

- Crystallization and X-ray analysis (E. Pebay-Peyroula, 12  months)

Crystallization screens will be set up at different conditions using the classical vapor diffusion method and lipidic phases. Growth conditions of initial crystals will be refined for optimal diffraction properties. Structures will be solved by X-ray crystallography using synchrotron radiation sources.

- Yeast expression for FRET experiments (E. Pebay-Peyroula, 6 months)

Carriers will be fused to the acyl carrier protein (ACP) and expressed in yeast in order to incorporate MCF carriers fused to ACP in the inner mitochondrial membrane.

- FRET experiments (H. Vogel, 6 months)

Mitochondrial sub-particles devoid of the outer membrane will be labeled by fluorescent substrates covalently linked to ACP. FRET measurements on the single molecule level and on molecular ensembles will be performed to obtain information about the state of oligomerization of the carrier protein as well as its distribution in potential microdomains using methods developed recently (Meyer 2006; Jacquier 2006). Provided that suitable labeling is feasible, conformational changes will also be investigated by single molecule FRET on carriers reconstituted in lipid membranes or in native membranes (Guignet 2004).

Selected Publications:

  1. H. Nury, C. Dahout-Gonzalez, V. Trézéguet, G. Lauquin, G. Brandolin, E. Pebay-Peyroula (2006),
    Relations between structure and function of the mitochondrial ADP/ATP carrier. Annual Review of Biochemistry, 75, 713-741.
  2. H. Nury, C. Dahout-Gonzalez, V. Trézéguet, G. Lauquin, G. Brandolin, E. Pebay-Peyroula (2005).
    Structural basis for lipid mediated interactions between mitochondrial ADP/ATP carrier monomers. Febs Lett 579, 6031-6036.
  3. E.Pebay-Peyroula, C. Dahout-Gonzalez, R. Kahn, V. Trézéguet, G.J.-M. Lauquin and G. Brandolin (2003).
    Structure of mitochondrial ADP/AP carrier in complex with carboxyatractyloside. Nature 426, 39-44.
  4. BH Meyer, JM Segura, KL Martinez, R Hovius, N George, K Johnsson, H Vogel (206).
    FRET imaging reveals that functional neurokinin-1 receptors are  monomeric and reside in membrane microdomains of live cells. Proc Natl Acad USA 103, 2138-2143.
  5. V Jacquier, M Prummer, JMSegura, H Pick, H Vogel (2006).
    Visualizing odorant receptor trafficking in living cells down to the single-molecule level. Proc. Natl. Acad. Sci. USA, 103, 14325-14330.
  6. E.G. Guignet , R. Hovius, H. Vogel (2004).
    Reversible site-selective labeling of membrane proteins in live cells. Nat. Biotech. 22, 440-444.