La TSPO préalablement connue sous le nom de récepteur périphérique aux benzodiazépines (PBR), est une protéine membranaire principalement impliquée dans le transport du cholestérol du cytosol vers la matrice des mitochondries, étape limitante dans la synthèse des stéroïdes et des sels biliaires.La production de la forme murine de la TSPO recombinante a été réalisée par un plasmide exprimé dans la bactérie Escherichia coli. La purification a été obtenue par colonne d'affinité grâce à l'étiquette polyhistidine codée dans le gène recombinant. Différents environnements mimétiques membranaires, détergents, lysodérivés, lipides ont été testés d'un point de vue structural et fonctionnel. Parmi les détergents étudiés, la dodécyl phosphocholine (DPC) a permis le meilleur repliement de la protéine. L'ajout de lysodérivés (LMPE) ou de phospholipides (DMPC/DMPE) a permis d'accroître la stabilité. La liaison des ligands spécifiques de la TSPO (PK 11195, cholestérol, protoporphyrine IX) a été observée dans plusieurs conditions étudiées par différentes approches biophysiques et biochimiques. Les trois approches structurales (ME, RX et RMN) ont pu être réalisées après optimisation des conditions de production et de stabilisation. Les résultats obtenus sont discutés en lien avec la structure de la TSPO publiée ces derniers mois. En parallèle à l'étude structurale, des mesures fonctionnelles par mutagenèse dirigée ont été réalisées afin d'obtenir des informations sur la liaison du ligand PK 11195. Ces données ont été confrontées à la structure récemment déterminée permettant de proposer un rôle pour les résidus mutés. Le mécanisme de transport du cholestérol par la TSPO est discuté. / TSPO previously named peripheral-type benzodiazepine receptor (PBR) is a membrane protein mostly involved in cholesterol transport from the cytosol to the matrix of mitochondria, a limiting step in steroids and bile salts biosynthesis.Production of recombinant mouse TSPO has been performed by plasmid expression in Escherichia coli bacteria. Purification has been obtained by immobilized affinity chromatography (IMAC) using polyhistidine tag encoded by recombinant gene. Various membranous mimetic environments such as detergents, lysoderivates, lipids have been tested from structural and functional point of view. Among tested detergents, dodecyl phosphocholine (DPC) has permitted the best protein refolding. The presence of lysoderivate (LMPE) or phospholipids (DMPC/DMPE) increased protein stability. Binding of TSPO high affinity ligands (PK 11195, cholesterol, protoporphyrin IX) has been measured in different conditions studied by biochemical and biophysical techniques. Three structural approaches (EM, XR and NMR) have been performed after optimization of production conditions and protein stabilization. Results gained have been discussed in line with TSPO atomic structure published within these last months. In parallel with structural studies, functional measurements have been carried out by site directed mutagenesis in order to gain data on binding site of PK 11195. These data have been faced with recently published TSPO atomic structure stabilized with this ligand and enabled to propose functional implication of mutated amino acids. Transport mechanism of cholesterol by TSPO is discussed.

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The Type Three Secretion Systems (T3SSs) of Gram-negative bacteria, including Shigella, Salmonella, and Enteropathogenic/Enterohaemorrhagic Escherichia coli (EPEC/EHEC), pass virulence factors directly into the host to mediate invasion. Prior to secretion down the narrow T3SS channel, effector proteins associate with chaperone proteins. The binding enables the T3SS to keep effectors soluble and partially unfolded for secretion. In the first part of this thesis, the association of one promiscuous chaperone, Spa15 of Shigella flexneri, with three of its cognate effectors has been studied. In addition to the role this plays in secretion, the binding of one particular substrate leads to Spa15 being involved in the regulation of the T3SS. The oligomerisation and impact of substrate binding upon Spa15 has been determined by crystallography and EPR. Once secreted, T3SS effectors subvert the host cytoskeleton for the benefit of the bacteria. Soluble homologues of Spa15 effectors from EHEC and Salmonella have been purified, and their interactions with host GTPases which lead to stress fibre phenotypes observed. The Twin Arginine Translocation (Tat) pathway provides a contrasting view of bacterial secretion. Instead of preventing folding in the cytoplasm, it is a criterion of transport that the protein be folded. One of the reasons for internal folding is the necessity to insert cofactors which could not be incorporated externally. In the second part of this thesis, a protein which exemplifies this necessity is studied. This is PhoD, the model protein for Tat export from Bacillus subtilis. PhoD is an alkaline phosphodiesterase expressed to scavenge phosphate in times of phosphate deficiency. The structure of PhoD has been solved, and the protein is shown to be able to cleave a component of its own cell wall. It uses an unusual catalytic site more reminiscent of the eukaryotic purple acid phosphatases than of other currently known alkaline phosphatases. Furthermore this site appears to require metal binding before export from the bacterial cytoplasm.