Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item COMPUTATIONAL METHODS IN PROTEIN STRUCTURE, EVOLUTION AND NETWORKS.(2013) Cao, Chen; Moult, John; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The advent of new sequencing technology has resulted in the accumulation of a large amount of information on human DNA variation. In order to make sense of these data in the context of biology and medicine, new methods are needed both for analysis and for integration with other resources. In this work: 1) I studied the distribution pattern of human DNA variants across populations using data from the 1000 genomes project and investigated several evolutionary biology questions from the perspective of population genomics. I found population level support for trends previously observed between species, including selection against deleterious variants, and lower frequency of variants in highly expressed genes and highly connected genes. I was also able to show that the correlation between synonymous and non-synonymous variant levels is a consequence of both mutation prevalence variation across the genome and shared selection pressure. 2) I performed a systematic evaluation of the effectiveness of GWAS (Genome Wide Association Studies) for finding potential drug targets and discovered the method is very ineffective for this purpose. I proposed two reasons to explain this finding, selection against variants in drug targets and the relatively short length of drug target genes. I discovered that GWAS genes and drug targets are closely associated in the biological network, and on that basis, developed a machine learning algorithm to leverage the GWAS results for the identification of potential drug targets, making use of biological network information. As a result, I identified some potential drug repurposing opportunities. 3) I developed a method to increase the number of protein structure models available for interpreting the impact of human non-synonymous variants, important for not only the understanding the mechanisms of genetic disease but also in the study of human protein evolution. The method enables the impact of approximately 40% more missense variants to be reliably modeled. In summary, these three projects demonstrate that value of computational methods in addressing a wide range of problems in protein structure, evolution, and networks.Item Energetics of Drug Interactions(2008-11-26) Todorova, Niya Ancheva; Kelman, Zvi; Schwarz, Frederick P.; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The goal of our research is to determine in terms of thermodynamic change of state functions the effects of experimental factors, such as water, mutagenesis, or the presence of a second substrate on the energetics of drug-inhibitor binding interactions. The binding of non-steroidal anti-inflammatory drugs within the rigid cavities of cyclodextrins was investigated by titration calorimetry and spectrofluorimetry. Loss of bulk water structure upon drug binding in the smaller hydrophobic β-cyclodextrin cavity results in an increase in the binding entropy, while restriction of the configurations of the drug in the cavity decreases the binding entropy. This restriction in the hydrophobic β-cyclodextrin cavity enhances the binding enthalpies so that the β-cyclodextrin binding reactions are enthalpy-driven. In the larger γ-cyclodextrin cavity, water is retained so that, not only are the interactions between the drug and the cavity reduced, there is an increase in the drug configurations resulting in increases in the binding entropies and the binding reactions become entropically-driven. These binding reactions also manifest enthalpy-entropy compensation where changes in the binding enthalpies are compensated by changes in the binding entropies. In drug binding to the more flexible p38α MAP kinase mutants, a single-point C→S mutation distal from the binding site, changes the interaction between the N- and C-terminal structural domains of the kinase as evident in differential scanning calorimetry. Calorimetric results show that drug-inhibitor binding affinities to kinase increase with size of the drugs since the binding reactions are all enthalpically-driven. Drug-inhibitors binding to trimeric human purine nucleoside phosphorylase were investigated by calorimetry in the presence of its second substrate, inorganic phosphate (Pi). Increasing concentrations of Pi modulates the driving-nature of the binding reaction, so that the acyclovir binding almost exclusively to the purine substrate binding site becomes more entropically-driven, while the binding reactions of ganciclovir and 9-benzylguanine interacting also with the adjacent Pi substrate site become more enthalpically-driven. A novel calorimetric enzyme activity assay at the low dissociation concentrations of the phosphorylase show an increase in the enzyme activity at low Pi concentrations, but also a decrease in the 9-benzylguanine binding affinity since this drug also interacts with an adjacent subunit.Item Mutational analysis of Human Immunodeficiency Virus type-1 nucleocapsid protein to evaluate its nucleic acid chaperone activity(2006-09-15) NARAYANAN, NIRUPAMA; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The highly basic 55 amino acid nucleocapsid protein (NC) that coats the HIV-1 genome has two zinc fingers that differ by five amino acids (strain pNL4-3). Previous work showed that NC's first finger (N-terminal) is primarily responsible for unwinding secondary structures (helix destabilizing activity), while the second (C-terminal) plays an accessory role. The amino acid differences between the fingers are (finger one to finger two): phenylalanine to tryptophan (F to W), asparagine to lysine (N to K), isoleucine to glutamine (I to Q), alanine to methionine (A to M), and asparagine to aspartic acid (N to D) at positions 16, 17, 24, 25, and 27 of finger one, respectively. To determine at an amino acid level the reason for the apparent distinction between the fingers, five point mutants were designed with amino acid residues in finger one incrementally replaced by those at corresponding locations in finger two. Each mutant was analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild type (wt) levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W which had substantially greater helix destabilizing activity than wt NC. All mutants retained wt levels of the condensation/aggregation activity of NC. Unlike I24Q and others, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro recombination assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a "dominant negative" effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity and hence NC's chaperone activity. Preliminary results from in vivo experiments indicated that virus with the N27D mutation is infectious at near wt NC levels. This suggests that aggregation activity may be more important than helix destabilizing for viral viability. Results from two other forms of HIV-1 NC (NCp9 and NCp15) and NC proteins from Simian Immunodeficiency Virus and Murine Leukemia Virus are also reported.