טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentYigal Linkovsky
SubjectStructural and Functional Investigation of the
Mitochondrial TSPO Receptor
DepartmentDepartment of Chemistry
Supervisor Full Professor Adir Noam
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

This research was carried out on a protein called TSPO (trans-locator protein) - 18kDa in size. The protein is, located mainly in the outer membranes of mitochondria. Apparently, the main role of TSPO is to transport cholesterol through the mitochondrial outer membrane to the inner membrane. Biochemical and pharmacological studies have shown that TSPO is also involved in various pathogenic processes such as cancer and neurological and psychiatric disorders. To suggest a precise mechanism for TSPO’s activity, a detailed description of the atomic structure of the protein can be provided by X-ray crystallography

To extract the protein from the membrane of E. coli, we had to choose suitable detergents that are able to dissolve the membrane extract the protein and stabilize it. In addition, we would like to be able to preserve its structure and activity without causing denaturation. The goal was to find the most ideal detergent concentration suitable to dissolve bacterial membranes as much as possible. After separating the membranes from the rest of the contents of cell, we finally managed to extract the protein and stabilize it in aqueous solution without causing denaturation with the uncharged detergent, DDM (dodecyl β-D-maltoside), at a concentration of 4%. After the purification step, we obtained a relatively large amount of protein at a high level of purity and homogeneity that enabled us to crystallize the TSPO.

A wide screen of different detergents was carried out in order to improve the crystal lattice, to strengthen interactions within the crystal and produce a more ordered crystal. As such, the screen included a variety of detergents differing in the length of the carbon chain and the electrical charge of the polar group. We succeeded in slightly improving the crystal morphology. However, the diffraction pattern produced by these crystals was still not of high enough quality to provide structural information.

We preformed activity tests on the TSPO, using the binding of a synthetic ligand, PK11195. It was showed that the dissociation constant of the recombinant TSPO from this ligand is approximately 1nM, both when the protein is inside a lipid membrane or as proteo-liposome, and this constant is similar to that of the native protein. In addition, it appears from the activity assays that the Bmax values of the recombinant protein are similar to those of the native protein.