טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShahar Anat
SubjectStructural Studies on Protein Involved in Human Health: The
M. tuberculosis Cpn60.2 Chaperonin and the
Mitochondrial TSPO Receptor Protein
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir
Full Thesis textFull thesis text - English Version


Abstract

Proteins are vital components of living cells which perform a wide variety of biochemical functions. The functional properties of proteins depend on their three dimensional structure, which can be determined experimentally by X ray crystallography. Disruption of the 3D structures of proteins result in their dysfunction and as a result human diseases may arise. Understanding these structure/function relationships of proteins involved in human health can be important for treatments of diseases by using the structures as a templates for drug design. Two different and unique systems were studied, which may serve as targets for structure based drug design in the future.

Cpn60.2 from Mycobacterium tuberculosis (Mt) belongs to the Heat Shock Proteins 60 family, which is involved in protein folding and prevention of misfolding in an ATP dependent manner. In addition, Cpn60.2 is involved in the pathogenesis of the Adjuvant Arthritis disorder. The 3D structure of the Cpn60.2 was determined to a resolution of 2.75Å at our lab, and it contains a dimer in the asymmetric unit, with the two monomers interacting through their apical domains. These results are in contradiction with all known structures from the HSP60 family, which possess rings of seven or fourteen subunits in the asymmetric unit.  In addition, it was shown that the Cpn60.2 appears as a dimer in solution. These results raised the question regarding the mechanism of protein folding which is performed by the Cpn60.2.

The outer mitochondrial membrane 18KDa translocator protein (TSPO) from Rattus norvegicus. TSPO, is part of the Permeability Transition Pore (PTP), which is located at the contact site between the inner and outer mitochondrial membrane. The main function of TSPO is the regulation of cholesterol transport from cellular compartments to the inner mitochondrial membrane. TSPO binds to both cholesterol and drug ligands such as the isoquinoline carboxamide PK11195. The TSPO was found to be involved in several disorders such as various kinds of cancer, neurological diseases and psychotic diseases. After testing various cloning vectors we were able to obtain high amounts of highly purified TSPO by using the detergents sodium dodecyl sulfate (SDS) and dodecyl β-D-maltoside. Crystallization trials of TSPO were successful , however, the crystals are not yet of high enough quality for structural determination, with the maximal diffraction at 12Å. Functional analysis of TSPO revealed that the recombinant protein binds the synthetic ligand [3H] PK11195 in nanomolar affinity after it was reconstituted into proteliposomes.