CHARACTERIZATION AND CRYSTALLIZATION OF TRANSLOCATOR PROTEIN 18 kDa (TSPO) FROM RHODOBACTER SPHAEROIDES ByFei LiTranslocator protein 18 kDa (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that is a continuing research focus due to its importance in cholesterol and porphyrin transport and apoptotic signaling. Dysfunction of TSPO has been shown to relate to various diseases, including metastatic cancer, inflammation, Alzheimer, and Parkinson disease. Functions of TSPO have been investigated but the molecular details remain an enigma, due in part to two major bottlenecks: 1) the lack of atomic resolution structural information; and 2) conflicting data from both in vivo and in vitro characterization. The Rhodobacter sphaeroides homolog of TSPO (RsTSPO) was discovered as an oxygen sensor regulating photosynthesis and respiration in this bacterium and shares 34 % sequence identity with the human protein. In addition, the knock-out phenotype of R. sphaeroides can be functionally complemented by the rat homolog, confirming the use of RsTSPO as a valid model system for the mammalian protein. In this study, we have successfully established an expression, purification, and characterization methodology for the RsTSPO that allowed us to investigate RsTSPO with molecular detail. R. sphaeroides is also an highly successful system for producing crystal structures of membrane proteins. A crystallization strategy was established and optimized with RsTSPO that has resulted in the first high resolution crystal structure of a TSPO family protein. A sensitive tryptophan fluorescence quenching assay was used to characterize the binding of purified RsTSPO with various ligands. Novel ligands of TSPO previously shown to affect apoptosis were identified and support a role of TSPO in the regulation of the apoptosis pathway. Detailed characterization of ligand binding with RsTSPO and mutants in vitro combined with computational modeling of RsTSPO structure and analysis of previous mutagenesis data led to a model of TSPO-ligand interaction. A cholesterol binding enhancement motif was identified in TSPO that is highly conserved within the mammalian proteins and is able to account for the 1000 fold different binding affinity of RsTSPO compared to the mammalian homologs. This motif is also observed in other membrane proteins interacting with cholesterol, highlighting the potential general importance of this enhancement motif in the regulation of cholesterol binding in mammals. In addition, residues within the same motif were identified by evolutionary covariance analysis as playing a critical role in the stability of TSPO proteins, providing useful information for structural analysis of TSPO. Extensive screening and optimization of crystallization strategies for RsTSPO were carried out with both the vapor diffusion and the lipidic cubic phase (LCP) methods. Successful crystallization and structure determination of RsTSPO with the LCP method not only provided the long-awaited structural information to facilitate interpretation of biochemical data on TSPO, but it also provides unique and valuable knowledge to guide crystallization of other TSPO family proteins, as well as similar small α helical membrane proteins.
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