Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, December 1999. / Includes bibliographical references. Also available on the Internet.

Vector-borne diseases such as malaria, leishmaniasis, and African trypanosomiasis are a major scourge to humans and animals in some of the most impoverished nations across the globe. Enabling the transmission of these disease-causing pathogens is a highly sophisticated molecular arsenal of surface proteins. My research focuses on biophysical characterization of these proteins with the ultimate goal of deciphering the molecular crosstalk between pathogen and vector. In support of this goal, I have selected the tsetse fly-transmitted parasites of the genus Trypanosoma, the etiological agent of African sleeping sickness, as a model system. Towards elucidating the molecular mechanism of transmission, I have attempted to characterize structurally three novel proteins; TbFam50.360, TbPSSA2, and TcCISSA and get insight into their functions. Before this study, GARP (Glutamic Acid Rich Protein from T. congolense), and VSG (Variant Surface Glycoprotein from T. brucei) were the only proteins to be structurally characterized in the vector stages of the parasite. Our structural analysis revealed that while the N- terminal region of TbFam50.360 adopted a three-helical structure similar to previously characterized trypanosome surface proteins, ectodomains of both TbPSSA2 and TcCISSA adopted a previously uncharacterized bilobed architecture. The structural analysis further identified putative ligand binding regions in TbFam50.360 and TcCISSA. However, in the absence of binding partners, the exact function of these proteins could not be established. Our lab in conjunction with our collaborators is investigating the binding partners of these proteins within the tsetse. The structures of TbFam50.360, TbPSSA2, and TcCISSA can be added to the repertoire of structurally characterized surface proteins expressed by trypanosomes. The information gained from these first structures of trypanosome surface proteins offer insight into their role in the trypanosome life cycle, and may, in the future, contribute to the control of African trypanosomiasis. / Graduate

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed July 31, 2009). Includes bibliographical references (p. 179-187). Also issued in print.

Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins. This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems. A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.

This Ph.D. thesis addresses the structural characterization of the unique family of tetrameric flavoproteins WrbA, the role of which in the life of cells is still largely unknown but its enzymatic activity and expression properties implicate it in the cell protection against oxidative stress. Proteins of the WrbA family were proved to carry out two-electron reductions of quinones and in this way to prevent generation of the free radicals, similarly to other flavoproteins known as quinone oxidoreductases. Crystal structures of the liganded and unliganded forms of the prototypical WrbA from Escherichia coli were determined. Comparative analyses of these structures with the related flavoproteins were intended to identify and explain the defining structural features of the WrbA family and to clarify its structural and functional relationships to the other flavoproteins.