Browsing by Subject "Structural biology"
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Item Development of top-down methods for evaluating protein structure and protein unfolding utilizing 193 nm ultraviolet photodissociation mass spectrometry(2014-12) Cammarata, Michael B.; Brodbelt, Jennifer S.; Webb, Lauren J.Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed another ion activation method, electron transfer dissociation (ETD), in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation supported that PARP-C exists as a monomer in solution with a close-packed C-terminal alpha helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins. UVPD mass spectrometry was also employed to investigate the structure of holo-myoglobin as well as its apo form transferred to the gas phase by native electrospray. The fragmentation yields from UVPD showed the greatest overall correlation with B-factors generated from the crystal structure of apo-myoglobin, particularly for the more disordered loop regions. Comparison of UVPD of holo- and apo- myoglobin revealed similarities in fragmentation yields, particularly for the lower charge states (8 and 9+), but those regions involved in harboring the heme group (for the holo form) exhibited significantly lower fragmentation than the apo-myoglobin state. Both holo- and apo-myoglobin exhibited low fragmentation yields for the AGH helical core (reflecting its highest stability).Item Structural Studies of Bacteriophage Lysins and their Implication in Human Diseases(2011-08-08) Sun, QinganStructural biology lays the molecular foundation for the modern field of life sciences. In this thesis, X-ray crystallography is the primary resource for atomic detail structural information and is the major technology employed in our research. Three examples show how structural biology addresses the basic processes of life. Firstly, two crystal structures of R21, corresponding to two biological states, reveal a new activation mechanism of SAR-endolysin, which not only complements the previous model, but is also more generally applicable to the endolysin family. The structural information was further corroborated by NMR data in solution. The second example is the crystal structure of mycobacteriophage lysin B, which identified the function of the protein, and tackles the unique problem of how mycobacteriophage circumvent the mycolic acid-rich outer membrane of mycobacterium. The last example is the homology modeling of the Plasmodium ribosomal L4 protein. The action mode for the drug in Plasmodium was proposed based on that, which accounts for the anti-malaria effect of azithromycin.Item Understanding and Targeting Lipid Metabolism of Mycobacterium tuberculosis(2013-12-09) Liu, ZhenMycobacterium tuberculosis (M. tuberculosis) contains a wide array of genes responsible for the synthesis and secretion of a variety of bioactive lipids. The genes represent attractive drug-targets due to their involvement in essential cell cycles, the implication in pathogenesis, and the interference with therapeutics. In this thesis, I report our efforts to understand the biological functions of, and to develop inhibitors against, multiple genes related to M. tuberculosis lipid metabolism. Firstly, dioctylamine, a substrate mimic of the mycolic acid cyclopropane synthases, is shown to inhibit CmaA2 in vitro. Its inhibition action is explained by the structural characterization. Together with our collaborators, we have found dioctylamine able to intervene multiple mycolic acid cyclopropane synthases in vivo, and hence established the first model study for the single-drug-multiple-target strategy to inhibit the mycolic acid biosynthesis of M. tuberculosis. In addition, dioctylamine can serve as the platform for the design of more potent and selective drugs in the future. Secondly, the action mechanism of isoniazid and ethionamide, both of which are pro-drugs targeting the mycolic acid biosynthesis, is explored via biochemical, X-ray crystallographic or modeling studies. We have determined that the intracellular target of isoniazid is the enoyl reductase InhA; and we have discovered the correlation between mycothiol and ethionamide susceptibility. Thirdly, I have investigated the function and mechanism of FadD10, an enzyme involved in the synthesis of a virulence-related lipopeptide. The results reveal that FadD10 was mis-annotated as a fatty acyl-CoA ligase, but it indeed transfers fatty acids to an acyl carrier protein (Rv0100). Further crystallographic characterization provides the molecular basis for the mechanism of FadD10, leading to the discovery of a new type of adenylate-forming enzyme.Item Unveiling the architectures of five bacterial biomolecular machines(2014-08) Fage, Christopher Dane; Keatinge-Clay, Adrian Tristan; Hoffman, David W; Whitman, Christian P; Appling, Dean R; Iverson, Brent L; Hackert, Marvin LNatural products represent an incredibly diverse set of chemical structures and activities. Given this fathomless, ever-evolving diversity, a reasonable approach to designing new molecules entails taking a closer look at the biochemistry that Nature has crafted over billions of years on Earth. In particular, much can be learned by unveiling the architectures of proteins, life’s molecular machines, through methods like X-ray crystallography. Acquiring the blueprints of an enzyme brings us closer to understanding the mechanism by which the enzyme transforms a simple substrate it into a complex product with biological function, and inspires us to engineer such systems to our own ends. With a focus on macromolecular structural characterization, this document elaborates on five Gram-negative bacterial biosynthetic enzymes from two categories: Cell-surface modifiers and polyketide synthases. Among the first category are the glycyl carrier protein AlmF and its ligase AlmE of Vibrio cholerae and the phosphoethanolamine transferase EptC of Campylobacter jejuni. These proteins are responsible for decorating cell-surface molecules (e.g., lipid A) of pathogenic bacteria with small functional groups to promote antibiotic resistance, motility, and host colonization. AlmE and EptC represent potential drug targets and their structures lay the groundwork for the design of therapeutics against food-borne illnesses. Included in the second category are the [4+2]-cyclase SpnF and two ketoreductase-linked dimerization elements, each from the spinosyn biosynthetic pathway in Saccharopolyspora spinosa. The former catalyzes a putative Diels-Alder reaction to form a tricyclic precursor of the insecticide spinosad, while the latter two organize ketoreductase domains within modules of a polyketide synthase. The second category also includes Ralstonia eutropha β-ketoacyl thiolase B, a substrate-permissive enzyme that can make or break carbon-carbon bonds with assistance from Coenzyme A or an analogous thiol. Each of these proteins exhibit intriguing structural features or catalyze reactions that show promise for biochemical engineering.