Structure et fonction en solution de la protéine transmembranaire mTSPO dans différents systèmes amphiphiles : des détergents aux environnements biomimétiques

TSPO is a small, ubiquitous, translocator membrane protein composed of five transmembrane α-helices. In mammals, it is primarily located in the outer mitochondrial membrane, where it is believed to play a role in cholesterol transport and steroid synthesis pathways. This protein has significant pharmacological interest due to its affinity for various ligands used as markers of inflammation in neuroimaging. The only known atomic structure of mammalian TSPOs is the NMR structure (2MGY.PDB) of mouse TSPO (mTSPO). However, this structure is controversial as it was obtained by refolding the protein using a high concentration of DPC and in the presence of the ligand (R)-PK11195, that stiffens it significantly. In the absence of ligand, the structure of mTSPO is too flexible to be resolved by NMR. Furthermore, to date, no amphiphilic condition has allowed the crystallization of mammalian TSPOs, with and without ligand, unlike bacterial TSPOs.The aim of the present study is to determine, using a structure/function approach, the effect of different amphiphilic environments on the structure of apo-mTSPO (i.e. without the ligand). We investigated mTSPO's structure in solution at different scales using radiation scattering and optical spectroscopy techniques. Small-angle X-ray and neutron scattering (SAXS, SANS), combined with size-exclusion chromatography (SEC), contrast variation in SANS, and ab initio modeling, allowed us to obtain the conformation of the entire mTSPO/amphiphile complex and to specifically probe those of the protein and the amphiphilic belt within the complex. The quantity of amphiphile molecules associated with mTSPO, measured by MALS, allowed the validation of the proposed models. The effect of the environment on ligand affinity was measured by microscale thermophoresis (MST).The apo-mTSPO, produced by a recombinant way in E.coli bacteria inclusion bodies, is partially unfolded following its extraction by SDS. We show that the protein refolds in DPC, both locally (significant increase of content and interactions of α-helices and in tryptophan fluorescence) and three-dimensionally, with a more extended "apo" conformation than the NMR structure 2MGY.PDB. Adding DMPC phospholipids to create a partially biomimetic environment of mixed DMPC:DPC bicelles further structures apo-mTSPO: the quantity and interactions of α-helices, as well as tryptophan fluorescence, increase significantly. This refolding is associated with a significant increase in the protein's affinity for the ligand (R)-PK11195 (0.9 μM) compared to that in DPC (70 μM) and SDS (no affinity). Thus, we demonstrate the relevance of using DMPC:DPC bicelles for studying membrane proteins in solution and confirm the crucial role of lipids in the structure and function of mTSPO. Finally, we show that this environment is favorable for the crystallization of apo-mTSPO and for reconstituting the protein in DMPC nanodiscs.To compare these results with a protein expressed under native conditions, we developed a new protocol for producing mTSPO in yeast cells. We managed to purify the protein in “apo” condition in DDM, a detergent known for solubilizing properly folded proteins, while retaining associated membrane lipids.This thesis work (i) contributes to a better understanding of the structure/function of mTSPO in different amphiphilic environments to determine optimal conditions for higher-resolution structural studies, and (ii) provides a significant methodological contribution to the study of membrane proteins in solution.