Physical Chemistry

Read the scientific activity report. (pdf 1.5Mo, last update 5th, february 2010)

Membrane proteins are involved in major cellular processes e.g. cell homeostasis, bioenergetics and communication. Nearly 25% of genes encode for a membrane protein and these include protein targets for over about 50 % of all drugs in use today.

Their knowledge at the molecular level that is needed for the conception of new pharmacological tools lacks far beyond those of the cytoplasmic proteins. This is due to their amphiphilic character that complicates their handling from the overexpression, the purification to the crystallization steps.
In this context, we are studying the function of purified membrane proteins after reconstitution into liposomes and their structure by electron microscopy (Figure 1). The membrane proteins under studies are involved in cellular multidrug resistance and detoxification and in cellular bio-energetics.

Purified membrane proteins in detergent are reconstituted into a lipid membrane as proteoliposomes to study their function in a simpler system than the native membrane. For the structural analysis we use cryo-electron microscopy to collect structural information at high resolution (1-2 nm) of purified proteins in detergent and in native like membrane environment.
We also develop new methods to decrease the amount of protein per analysis and to get access to the poorly expressed human membrane proteins.

Fig. 1: We are interested in the functional and structural analysis of membrane proteins. Functions are analyzed after purification and reconstitution into proteoliposomes. Structures are determinated by cryo-electron microscopy of purified protein in detergent, single particle analysis approach, or after reconstitution into a lipid membrane as 2D crystals that are further analyzed by electron crystallography.

Our most significant projects and findings over the past few years include:

Supramolecular organization of membrane proteins

Supercomplexes of membrane proteins are supramolecular assembly of membrane proteins able to function individually but assembled into a supercomplex to increase the field of catalytic reactions. Supercomplexes have been reported in the respiration, in plants and bacterial photosynthesis or in multidrug resistances. This concept is a recent field in membrane proteins and is the most functionally documented in the case of the bacterial photosynthesis. This makes these proteins interesting models for understanding the structural parameters involved in the assembly and stabilization of super-complexes of membranes proteins. A comparative study on photosynthetic complexes has provided new insights into the biosynthesis and the supramolecular assembly of membranes proteins involved in the energy transduction in cells (Figure 2) (1,2).

Fig. 2: Panel A : Bacterial photosynthetic apparatus. Panel B : (1) 3D reconstruction by cryo-electron microscopy of the core complex from Rb. veldkampii that contains a PufX subunit unable to form dimers. (2) Projection map of the dimeric core complex of Rb. sphaeroides with a proposed sub-units organization that includes a pair of Pufx holding the rest of the dimer. Panel C : (1, 2) Tubular membranes of Rb. sphaeroides containing dimeric core complexes. (3) Model of a dimer of PufX hold by the GXXXG motif present in Rb. sphaeroides and absent in PufX of Rb. veldkampii. (5) The angle between the two PufX leads to the formation of a convex dimeric core complex that curves the membrane in enriched areas. Panel D : Proposed mechanism of subunits assembly during the biosynthesis of the cores complexes. In Rb. sphaeroides, after dimerization of PufX (pink ellipse), α (blue circles) and β (green circles) subunits are assembled in pairs leading to antenna with opposite curvature that could not be enclosed due to steric hindrance. The opening in the antenna might be a preferred passage for the quinones to the cyt. bc1. In Rb. velkampii, Pufx is unable to dimerize and the a and b subunits are assembled in pairs up to the enclosure.

Structural analysis of Multidrugs Resistant transporters

A particular effort is devoted to the structure determination of ABC transporters that hydrolyse ATP for the transmembrane transport of various xenobiotics and the cell detoxification. Several ABC's confers a multidrug resistance phenotype (MDR) to bacteria against antibiotics and to human against drugs use in anticancer treatments. Our project includes the structural analysis of bacterial homolog and human MDR transporters involved in the transport of anticancer drugs with the final aim to contribute to the conception of new inhibitors (Figure 3). We recently described the transport of drugs at the molecular level by BmrA, a MDR transporter homolog to the human Pgp (3).

fig 3: Multidrugs Resistance Proteins hydrolyse ATP for the efflux of various xenobiotics. Three human MDR transporters have been found in tumor cells and transport several drugs used in chemiotherapy along with a mechanism of selectivity and inhibition poorly understood. Our team is working on bacterial homolog and on human MDRs. In absence of nucleotide, the conformation of the NBD's is open, leading to a conical shaped protein that upon reconstitution into lipid bilayer forms tubes (A). In presence of nucleotide, planar membrane and 2D crystals are formed (B). Projection map of ATP bound conformation or “NBD closed”. Centre, catalytic cycle of BmrA involving a large conformational change associated to the translocation of drugs. (Coll. S. Marco, INSERM U759, Institut Curie, A. Di Pietro, IBCP, Lyon, J.M.Jault, IBS, Grenoble).

New methods for the structural analysis of low expressed membrane proteins

In parallel, we develop new methods for decreasing to the picomole level the amount of proteins needed for the structural analysis and to get access to human membrane proteins that are poorly expressed. The basic concept relies on the concentration of proteins from the bulk solution to a surface, e.g. a functionalised surface of lipids or a planar supported bilayer. Both strategies are used for the analysis of eukaryotes MDR transporters (Fig 4) (4).

Fig.4: New strategies to decrease the amount of membrane proteins per structural analysis assays. A) 2D crystallization onto a lipid film that contains a lipid ligand of the proteins. The principle of the method relies on the specific recognition (1) followed by the concentration of the purified protein onto a lipid ligand (2) and the 2D crystallization (3). Example of a 2D crystal of BmrA, a bacterial multidrugs resistant transporter. B) Direct incorporation of proteins into a planar lipid bilayer for atomic force microscopy analysis. Example of a photosynthetic multicomplex inserted in the lipid bilayer (coll. P.E.Milhiet, CBS, INSERM, Montpellier) (4).

Projects

Our researches will focus on:

    * the structural analysis of prokaryotic and eukaryotic MDR transporters;
    * the development of new methods for scaling down the amount of proteins per analysis to access to poorly expressed human membrane proteins;
    * the characterization of model membranes by cryo-electron microscopy (5).