Browsing Chemistry & Biochemistry Theses and Dissertations by Author "Bahmanjah, Soheila"
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- ItemSTRUCTURAL STUDY OF THE EARLY SECRETORY PROTEIN B AND ATYPICAL RIO2 KINASE(2015) Bahmanjah, Soheila; LaRonde, Nicole; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mycobacterium tuberculosis, Mtb, is a successful pathogen that secretes variety of proteins to manipulate the host defense mechanisms and create a proper environment for its survival. The progression of the disease depends on the regulated secretion of essential virulence factors such as culture filtrate protein-10 (CFP-10), and early secretory antigenic target-6 (ESAT-6). Mtb regulates the secretion of the virulence factors through interactions of MycP1 protease with EspB (Early secretory protein B) protein. One of the goals of this thesis was to provide the first crystal structure of EspB protein in order to gain insight into the expression, secretion, function and transmembrane translocation of this protein. EspB292 (residue 1-292) structure has 16 monomers in the asymmetric unit that are arranged into a set of four homotetramers. To clarify the components that affect oligomerization of the EspB292 in the biologically relevant conditions, we set up experiments that mimic the phagosomal environment. In addition to work done on EspB protein, structural determination of atypical Chaetomium thermophilum Ct-Rio2 in presence of transition state analogs such as sodium orthovanadate and beryllium fluoride is reported to define the importance of individual domains in binding ATP. Rio2 is required for site D cleavage and maturation of the small subunit ribosomal RNA (rRNA). In this study, the structures of the Ct-Rio2 and its binding to ADP/BeF2 and ADP/VO42- are investigated. The structure are expected to mimic the transition state of the phosphoryl transfer from ATP to Asp257 in Rio2’s active site and the subsequent hydrolysis of the aspartyl phosphate that could power late cytoplasmic 40S subunit biogenesis. Collaborative work on the structural characterization of the G228 Ct-Rio2 mutant protein is also reported. Our goal is to investigate the evolutionary relationship between Rio kinases and the canonical eukaryotic protein kinases. Our hypothesis is that canonical eukaryotic protein kinases have evolved from Rio kinases. We have made several mutations in a key differentiating position of the kinase domain sequence of Ct-Rio2, G228, and have collected biochemical data to test the ability of the mutated Rio kinase to autophosphorylate and carry out ATP hydrolysis.