UMD Theses and Dissertations
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Item Probing the 3D Structure and Function of a Cation/H+ Exchanger in Plant Reproduction(2015) Czerny, Daniel; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Maintaining intracellular pH and K+ homeostasis are necessary for a cell to divide, grow, and communicate with other cell types. How a cell responds to stimuli and subsequently regulates intracellular pH and K+ content are largely unknown. Ion transporters, including cation/H+ exchangers are one potential determinant of intracellular pH and K+ content. A novel family of CHX transporters, predicted to exchange a cation for a H+, is found in all land plants, though their functions in the plant and the mode of transport are mostly unknown. What is the mode of transport of Arabidopsis thaliana CHX17? Model structures of the CHX17 transmembrane domain were built from two crystallized bacterial Na+/H+ antiporters. Based on protein architecture and homology, residues were selected for mutagenesis and CHX17 activity was tested in yeast. Thr170 and Lys383 in the discontinuous α-helices of transmembrane 4 and 11, and Asp201 and Lys355 in the middle of transmembranes 5 and 10 are necessary for CHX17 activity. Results suggest these are core residues that participate in cation binding and/or catalysis. Glu111 near the cytosolic surface of CHX17 was necessary for activity, suggesting CHX17 could be regulated by cytosolic pH. Thus the protein fold and mode of transport of Arabidopsis CHX17 resemble a K+/H+ exchanger. What roles do K+/H+ exchangers play in plant reproduction? chx17/18/19 mutant plants showed a 56%-77% reduction in seed set though the biological basis was unknown. Reciprocal crosses showed reduced seed set was primarily caused by defects in the male gametophyte. Mutant chx17/18/19 pollen grains developed normally and pollen tubes grew and reached most ovules. However, half the ovules receiving a mutant pollen tube failed to develop. Wild-type pistils that received chx17/18/19 pollen showed unfertilized ovules, ovules with single fertilizations, and some embryos that developed similarly to wild-type. Thus, some triple mutant pollen showed failure to complete fertilization. When fertilization was successful, embryos from self-fertilized chx17/18/19 pods showed delays in development. Our findings suggest maintenance of pH and K+ homeostasis in endomembrane compartments by CHX17 and its homologs could regulate membrane trafficking events necessary for pollen tube growth, male gamete fusion, and embryo development.Item MODULATION OF HIV-1 REVERSE TRANSCRIPTASE AND FAMILY A DNA POLYMERASE PRIMER-TEMPLATE BINDING(2014) Fenstermacher, Katherine Joan; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Polymerases are enzymes used by all cellular and viral organisms to replicate their genomes. The human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT), uses a single-stranded RNA template to create double-stranded DNA during the course of the viral life cycle. Successful reverse transcription relies on the speed of catalysis and the ability of the enzyme to stay bound to the template during synthesis. I demonstrate that both of these properties can be modulated by the presence of different divalent cations, fundamentally altering the behavior of HIV RT. In the presence of 2 mM Mg2+, the HIV RT primer-template complex has a half-life of 1.7±1.0 min, incorporating nucleotides at a maximum rate of 3.5 nucleotides (nt) per second (average speed 1.4±0.4 nt/sec). Substituting 2 mM Mg2+ with 400 μM Zn2+ dramatically slows the speed of catalysis (maximum 0.1 nt/sec, average 0.022±0.003 nt/sec) and promotes primer-template complexes that last hours (half-life of 220±60 min). These profound changes to the enzyme's function critically inhibit reverse transcription, even in the presence of optimal concentrations of Mg2+. In addition to the cation composition during reverse transcription, previous studies have demonstrated that the sequence of the primer-template substrate can also affect the duration of a RT-primer-template complex. In light of this discovery, I investigated the tendency of two Family A DNA polymerases, the Thermus aquaticus DNA polymerase (Taq pol) and the Klenow fragment from Escherichia coli DNA polymerase I (Klenow), to selectively and tightly bind primer-template complexes. Using Primer-Template Systematic Evolution of Ligands by Exponential Enrichment (PT SELEX), I determined that both Taq pol and Klenow tightly bind to sequences containing regions that match the initiation and melting domains of promoters for the structurally similar bacteriophage T7-like RNA polymerases. This suggests a shared sequence preference that might be present in all Family A DNA polymerases, derived from a common ancestor. I plan to exploit this primer-template binding preference to advance biotechnologies utilizing these enzymes.Item Theoretical and Experimental Investigations of High Spin Ionic Intermediates(2007-11-20) Winter, Arthur; Falvey, Daniel E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to identify high-spin organic intermediates that could potentially be used as building blocks for the construction of high-spin organic ferromagnets, density functional theory (DFT) computations were performed to assess the singlet-triplet state energy gaps for a number of substituted aryl ionic intermediates. The quantitative accuracy of these DFT computations was benchmarked by high-level multireference second order perturbation theory (CASPT2) computations for representative species. These computations led to the discovery of a novel meta pi donor effect, wherein substituting the meta positions of aryl cationic species such as arylnitrenium ions (Ar-N-H+), arylsilylenium ions (Ar-SiH2+), aryloxenium ions (Ar-O+), and benzyl cations (Ar-CH2+), with pi donors stabilizes a π,π* triplet state analogous to the electronic state of the m-xylylene diradical. Two of these benzylic cations were generated experimentally through photochemical methods and analyzed by laser flash photolysis, chemical trapping studies, and product analysis. The experimental results were consistent with the existence of low-energy triplet states. Additionally, species with an inverted connectivity (e.g. an anionic donor with two pi electron withdrawing groups) were also found to have low-energy triplet states by density functional theory computations. These anions were generated chemically and studied by NMR and EPR spectroscopy as well as quenching studies. The preliminary results of the experimental studies are consistent with the intermediacy of triplet ground state benzyl anions, in line with the theoretical predictions. Vinyl cations substituted with β pi donors were also found to have triplet ground states, as computed by DFT and CBS-QB3 methods. In many cases, the singlet vinyl cations are anticipated to have facile rearrangement pathways, but incorporating the pi donors into rings appears to discourage obvious rearrangement pathways. To permit the photogeneration of congested arylnitrenium ions, a new method for photochemically generating these species was developed through photolysis of protonated 1,1-diarylhydrazines. Additionally, the carbazolyl nitrenium ion was generated photochemically and studied by laser flash photolysis, chemical trapping studies, product analysis and computational studies. This nitrenium ion is found to be more short-lived and reactive than similar diarylnitrenium ions as a likely result of destabilizing antiaromatic character.