Chemistry & Biochemistry
Permanent URI for this collectionhttps://hdl.handle.net/11274/15812
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Browsing Chemistry & Biochemistry by Subject "Biochemistry"
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Item The dimer loop and electrostatic interactions at the dimer interface of human glutathione synthetase(2012-12) De Jesus, Margarita; Anderson, Mary; Britt, Mark; Sheardy, Richard DeanHuman glutathione synthetase (hGS) is homodimeric and negatively cooperative toward its γ-glutamyl substrate making it a good model to study protein-protein interactions. The allosteric pathway between hGS active sites is hypothesized to travel through the dimer interface. To better understand the allostery of hGS and interactions at the dimer interface, two regions, the dimer loop [35-TSQEPTSSE-43] and key amino acid residues (Serine42, Arginine221 and Aspartate24) were studied using site-directed mutagenesis and analyzed for effects on cooperativity, activity and stability. Alanine mutant enzymes of dimer loop residues did not greatly affect activity or stability of hGS, nor change the cooperativity of hGS, but did affect γ-glutamyl substrate affinity, while alanine mutant enzymes of residues Ser42, Arg221 and Asp24 did not change cooperativity, but did decrease in activity and stability, and increase in γ-glutamyl affinity, with Asp24 having the greatest loss in activity and stability, followed by Arg221, and then Ser42.Item Human glutathione synthetase residues Valine 44 and Valine 45 are required for subunit stability and negative cooperativity(2011-08) Slavens, Kerri; Anderson, Mary; Britt, Mark; Sheardy, Richard DeanValine 44 and Valine 45 serve as important residues for human glutathione synthetase (hGS) function and stability given their location at the dimer interface of this enzyme. Computational studies suggest Val45 has more impact on the structure and stability of hGS than Val44. Experimentally, enzymes with mutations at the 44 and/or 45 positions were prepared, purified and assayed for initial activity. The Val45 position mutations have a greater impact on enzyme activity than mutations at Val44. Differential scanning calorimetry experiments reveal a stability loss in all mutant enzymes. The γ-GluABA substrate affinity remains unaltered in V44A and V45A mutant enzymes, but increases with tryptophan introduction. Hill coefficients trend towards less negative cooperativity except with V45W. These results imply that residues V44 and V45 are located along the allosteric pathway of this negatively cooperative dimeric enzyme and that these residues are integral to the stability of human glutathione synthetase.Item Investigating the drug mechanism of carboplatin with DNA employing circular dichroism surface-enhanced raman scattering(12/30/2016) Khan, Sidrah; Mirsaleh-Kohan, Nasrin; Rawashdeh-Omary, Manal; Sheardy, Richard DeanItem Reaction of (Z)- and (E)-β-chlorocinnamohydroximoyl chlorides with methoxide ion(2008-12) Liu, Jin; Johnson, James; Britt, Mark; Sheardy, Richard DeanNew α,β-unsaturated hydroximoyl chlorides and hydroximates were synthesized and characterized. 13C NMR, 1H NMR, IR and elemental analysis were determined for each new compound. The kinetics of methoxide ion substitution on (Z)- and (E)-β-chlorocinnamohydroximoyl chloride were measured. Identification of the products and the measurement of the rate constants helped with an understanding of the mechanisms of these reactions.Item Understanding the conformations and stabilities of g-quadruplexes formed by human telomere sequence in ideal and less ideal solutions(1/1/2014) Sharma, Vishal Rajat; Sheardy, Richard Dean; Mirsaleh-Kohan, Nasrin; Rawashdeh-Omary, ManalThe G-rich termini of human chromosomes, called telomeres, have the ability to form unique DNA structures called G-Quadruplexes, which have been implicated in certain cancer types and, as a result, have drawn researchers' attention to study their structure inside the cell and thus consider them as potential targets for new anticancer therapies. Biophysical studies of these entities have been carried out under near ideal conditions, i.e., low phosphate concentration (e.g., 10mM), pH 7, low ionic strength (e.g., 115mM), and for optical studies, low concentration of DNA (e.g., 1 × 10-6 M). We, here, created less ideal conditions by using different osmolytes such as polyethylene glycol, acetonitrile, trifluoroethanol, and betaine. Our research focuses on the effect of these co-solutes on the structural and thermodynamic properties of telomeric DNA oligomers and report the characterization of (TTAGGG)x, where x = 1, 2, or 4. In general, increasing the percentage of any osmolytes in solutions drives the equilibrium from unimolecular hybrid to multimolecular parallel conformation. In addition, Stability of parallel conformation also increases.