Cell Biology & Molecular Genetics Theses and Dissertations
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Item STRUCTURE FUNCTION DIVERSITY WITHIN THE PHOSPHOENOLPYRUVATE MUTASE / ISOCITRATE LYASE SUPERFAMILY AS REVEALED BY THE ENZYMES OXALOACETATE DECARBOXYLASE AND 2,3-DIMETHYLMALATE LYASE(2008) Narayanan, Buvaneswari Coimbatore; Herzberg, Osnat; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Two members of the phosphoenolpyruvate mutase (PEPM) / isocitrate lyase (ICL) superfamily were investigated to study their structure-function relationships and to identify sequence signatures that define a particular function. The first enzyme (PA4872) was a protein of unknown function from Pseudomonas aeruginosa. The second enzyme from Aspergillus niger (An07g08390) was thought to be an oxaloacetate acetyl hydrolase (OAH) because of its high sequence identity (~60%) to an enzyme with confirmed OAH activity. The X-ray crystal structure determination of PA4872 revealed unique features that guided the design of biochemical experiments, which ultimately led to the discovery that the enzyme is an oxaloacetate decarboxylase (OAD). Two structures of An07g08390, one with bound Mg2+ and the second with bound Mn2+ and the inhibitor 3,3-difluorooxaloacetate, were determined. The functional studies demonstrated that although the enzyme has OAH activity, it has a far better activity as a 2R,3S-dimethylmalate lyase (DMML). The active site structure of DMML indicated a proline residue (Pro240) as a marker of DMML function along with confirming the conserved locations of previously established signature residues for lyase activity. OAD is the founding member of a family within the PEPM / ICL superfamily and thus defines the function of the remaining family members. However, the biological context in which OAD functions remains unknown. DMML is known to function in the nicotinate catabolism pathway but not all the members of the pathway are present in A. niger. Transcriptome analysis suggests that the DMML encoding gene is under carbon catabolite repression but the pathway in which the enzyme functions has not yet been identified.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 The Structural Basis for Function of the Escherichia coli Mechanosensitive Channel of Small Conductance, MscS(2007-04-26) Akitake, Bradley Chun-Yee; Sukharev, Sergei; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The 'small' mechanosensitive channel, MscS, resides in cytoplasmic membranes of most free-living bacteria. MscS is gated directly by membrane tension and functions as an osmolyte release valve in bacterial turgor regulation. In contrast to previously studied MscL, which is a strictly prokaryotic molecule, MscS homologs are found in eukaryotes increasing the value of this channel as a general model for gating by membrane stretch. Presented here are the results of three studies aimed at characterizing the structural basis for function of Escherichia coli MscS. In study one, we provide the first electrophysiological characterization of the wild-type channel in its native membrane free of other mechanosensitive channels. It is, to date, the most complete description the gating cycle specifying the kinetic scheme and dependencies of major rates on tension and voltage. Study two represents a collaborative effort to probe the strength of intersubunit contacts in the homo-heptameric MscS channel. In patch-clamp experiments we show that the dissociating effects of TFE alter MscS gating in a manner that provides significant insight into the mechanics of channel inactivation. In the final study our research group utilized a novel extrapolated motion technique to explore the conformational pathways of the MscS functional cycle. Guided by these new models, channel mutants were generated to alter helical propensity along the pore lining TM3 helix. Patch-clamp analysis revealed a vivid picture of the functioning MscS in which these TM3 domains provide a structural frame for the open channel. Dynamic collapse of these 'struts' at flexible points along TM3 modulates transitions from the open state to the inactivated and closed states. My contributions to these studies have allowed for (1) refinement of the MscS functional cycle including identification of a new desensitized state; (2) determination of the physical parameters and spatial scales of channel opening, closing and inactivation; and (3) identification of key hinge elements, residing in TM3, that along with membrane tension serve to modulate the functional cycle of MscS. These findings have led to a better understanding of the biophysical principles that underlie mechanotransduction and provide insights into the larger family of mechanically activated phenomena.