Download or read book Binding Studies of the Translocator Protein (TSPO) written by Nora Susanti and published by . This book was released on 2014 with total page 66 pages. Available in PDF, EPUB and Kindle. Book excerpt: The 18 kDa translocator protein (TSPO) is an integral membrane protein known to be involved in porphyrin transport and heme biosynthesis. TSPO also plays a role in modulating immune response, regulation of cell proliferation and programmed cell death. TSPO is associated with several health conditions such as ischemic heart reperfusion, cancers and neurodegenerative diseases. While the potential of TSPO as a major therapeutic target has been well recognized, the lack of detailed understanding of its function and ligand binding sites have hindered drug design. In this thesis we will describe the expression, extraction and purification of Rhodobacter sphaeroides TSPO (RsTSPO). Rhodobacter is one of the closest ancestors of mitochondria and RsTSPO can be used as a model for mammalian TSPO. RsTSPO was overexpressed in E. coli, and it was extracted from the membranes with detergent (i.e. n-dodecylphosphocoline (DPC) and n-dodecyl-ß-D-maltoside (DDM)) then purified by Ni-affinity and size exclusion chromatographies. The RsTSPO could be expressed and purified in high yield (mg quantitites), high purity, and in its functional state. Binding studies of RsTSPO were performed utilizing fluorescence quenching of intrinsic tryptophans with a number of porphyrins ligands such as protoporphyrin IX (PPIX), hemin, meso-Tetra(4-sulfonatophenyl)porphine dihydrochloride (2HTPPS), Zn (II) meso-Terta(4-sulfonatophenyl)porphine dihydrochloride (ZnTPPS) and with the diagnostic ligand, PK11195. Binding studies of RsTSPO were performed in both detergents (DPC and DDM) to assess the effect of the membrane mimic environment on binding. We will also describe the first evidence of the catalytic activity of the purified RsTSPO under ambient light in the presence of molecular oxygen on its natural ligand, protoporphyrin IX. We will demonstrate the photodegradation of protoporphyrin IX by RsTSPO in the presence of oxygen and light. In addition, we have studied the effect of molecular oxygen and reactive oxygen scavengers (sodium azide and glutathione) on the decomposition of protoporphyrin IX. Also, we will describe preliminary NMR studies in an effort to characterize the ligand binding site(s) of TSPO. We have performed both solution and solid state NMR studies on pure RsTSPO and RsTSPO-ligand complexes.