title: Mouse TSPO in a lipid environment interacting with a functionalized monolayer.
abstract: Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR). Electron microscopy and two-dimensional crystallization under functionalized monolayers have been successfully developed for recombinant tagged proteins. The difficulty comes from the detergent carried by membrane proteins that disrupt the lipid monolayer. We identified the best conditions for injecting the histidine tagged recombinant TSPO in detergent in the subphase and to keep the protein stable. Reconstituted recombinant protein into a lipid bilayer favors its adsorption to functionalized monolayers and limits the disruption of the monolayer by reducing the amount of detergent. Finally, we obtained the first transmission electron microscopy images of recombinant mouse TSPO negatively stained bound to the lipid monolayer after injection into the subphase of pre-reconstituted TSPO in lipids. Image analysis reveals that circular objects could correspond to an association of at least four monomers of mouse TSPO. The different amino acid compositions and the location of the polyhistidine tag between bacterial and mouse TSPO could account for the formation of dimer versus tetramer, respectively. The difference in the loop between the first and second putative transmembrane domain may contribute to distinct monomer interaction, this is supported by differences in ligand binding parameters and biological functions of both proteins.
authors: Université Paris, Denis Diderot, Paris, France.
authors: David
authors: D
authors: Teboul
authors: Sylvie
authors: S
authors: Beaufils
authors: Jean-Christophe
authors: JC
authors: Taveau
authors: Soria
authors: S
authors: Iatmanen-Harbi
authors: Anne
authors: A
authors: Renault
authors: Catherine
authors: C
authors: Venien-Bryan
authors: Véronique
authors: V
authors: Vie
authors: Jean-Jacques
authors: JJ
authors: Lacapere
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D011510:Proteolipids / Q000737:chemistry; D018079:Receptors, GABA / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: C463819:Bzrp protein, mouse; D008055:Lipids; D011510:Proteolipids; D018079:Receptors, GABA