title: Production and initial structural characterization of the TM4TM5 helix-loop-helix domain of the translocator protein.
abstract: Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix-helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C-terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far-UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n-dodecyl-β-d-maltoside, n-octyl-β-d-glucoside, n-dodecylphosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5-DHPC complex.
authors: Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Institute of Biology and Technology (iBiTecS), Gif-sur-Yvette, France. cg393@cam.ac.uk
authors: C
authors: C
authors: Galvagnion
authors: P
authors: P
authors: Montaville
authors: Y-M
authors: YM
authors: Coïc
authors: N
authors: N
authors: Jamin
mesh_terms: D002942:Circular Dichroism; D018257:Helix-Loop-Helix Motifs; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D018079:Receptors, GABA / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D018079:Receptors, GABA; C499229:TSPO protein, human