Browsing by Author "Long, Eric C. (Eric Charles)"
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Item Characterization of a fatty acid elongase condensing enzyme by site-directed mutagenesis and biochemical analysis(2014) Hernandez-Buquer, Selene; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Li, LeiFatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.Item A Computational Study of the Mechanism for F1-ATPase Inhibition by the Epsilon Subunit(2013) Thomson, Karen J.; Pu, Jingzhi; Ge, Haibo; Sardar, Rajesh; Long, Eric C. (Eric Charles)The multi-protein complex of F0F1 ATP synthase has been of great interest in the fields of microbiology and biochemistry, due to the ubiquitous use of ATP as a biological energy source. Efforts to better understand this complex have been made through structural determination of segments based on NMR and crystallographic data. Some experiments have provided useful data, while others have brought up more questions, especially when structures and functions are compared between bacteria and species with chloroplasts or mitochondria. The epsilon subunit is thought to play a signi cant role in the regulation of ATP synthesis and hydrolysis, yet the exact pathway is unknown due to the experimental difficulty in obtaining data along the transition pathway. Given starting and end point protein crystal structures, the transition pathway of the epsilon subunit was examined through computer simulation.The purpose of this investigation is to determine the likelihood of one such proposed mechanism for the involvement of the epsilon subunit in ATP regulation in bacterial species such as E. coli.Item Design and Synthesis of Small-Molecule Protein-Protein Interaction Antagonists(2014) Han, Xu; Meroueh, Samy; Long, Eric C. (Eric Charles); McLeish, Michael J.Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.Item Design of Biomembrane-Mimicking Substrates of Tunable Viscosity to Regulate Cellular Mechanoresponse(2012-03-20) Minner, Daniel Eugene; Naumann, Christoph A.; Long, Eric C. (Eric Charles); Suter, Daniel; Shah, KavitaTissue cells display mechanosensitivity in their ability to discern and respond to changes in the viscoelastic properties of their surroundings. By anchoring and pulling, cells are capable of translating mechanical stimuli into a biological response through a process known as mechanotransduction, a pathway believed to critically impact cell adhesion, morphology and multiple cellular processes from migration to differentiation. While previous studies on polymeric gels have revealed the influence of substrate elasticity on cellular shape and function, a lack of suitable substrates (i.e. with mobile cell-substrate linkers) has hindered research on the role of substrate viscosity. This work presents the successful design and characterization of lipid-bilayer based cell substrates of tunable viscosity affecting cell-substrate linker mobility through changes in viscous drag. Here, two complementary membrane systems were employed to span a wide range of viscosity. Single polymer-tethered lipid bilayers were used to generate subtle changes in substrate viscosity while multiple, polymer-interconnected lipid bilayer stacks were capable of producing dramatic changes in substrate viscosity. The homogeneity and integrity of these novel multibilayer systems in the presence of adherent cells was confirmed using optical microscopy techniques. Profound changes in cellular growth, phenotype and cytoskeletal organization confirm the ability of cells to sense changes in viscosity. Moreover, increased migration speeds coupled with rapid area fluctuations suggest a transition to a different migration mode in response to the dramatic changes in substrate viscosity.Item Design, Synthesis and Study of DNA-Targeted Benzimidazole-Amino Acid Conjugates(2013-07-12) Garner, Matthew L.; Long, Eric C. (Eric Charles); Minto, Robert; O'Donnell, Martin J.The DNA minor groove continues to be an important biological target in the development of anticancer, antiviral, and antimicrobial compounds. Among agents that target the minor groove, studies of well-established benzimidazole-based DNA binders such as Hoechst 33258 have made it clear that the benzimidazole-amidine portion of these molecules promotes an efficient, site-selective DNA association. Building on the beneficial attributes of existing benzimidazole-based DNA binding agents, a series of benzimidazole-amino acid conjugates was synthesized to investigate their DNA recognition and binding properties. In this series of compounds, the benzimidazole-amidine moiety was utilized as a core DNA “anchoring” element accompanied by different amino acids to provide structural diversity that may influence DNA binding affinity and site-selectivity. Single amino acid conjugates of benzimidazole-amidines were synthesized, as well as a series of conjugates containing 20 dipeptides with the general structure Xaa-Gly. These conjugates were synthesized through a solid-phase synthetic route building from a resin-bound amino acid (or dipeptide). The synthetic steps involved: (1) the coupling of 4-formylbenzoic acid to the resin-bound amino acid (via diisopropylcarbodiimide and hydroxybenzotriazole); followed by (2) introduction of a 3,4-diaminobenzamidoxime in the presence of 1,4-benzoquinone to construct the benzimidazole ring; and, finally, (3) reduction of the resin-bound amidoxime functionality to an amidine via treatment with 1M SnCl2•2H2O in DMF before cleavage of final product from the resin. The synthetic route developed and employed was simple and straightforward except for the final reduction that proved to be very arduous. All target compounds were obtained in good yield (based upon weight), averaging 73% mono-amino acid and 78% di-amino acid final compound upon cleavage from resin. Ultimately, the DNA binding activities of the amino acid-benzimidazole-amidine conjugates were analyzed using a fluorescent intercalator displacement (FID) assay and calf thymus DNA as a substrate. The relative DNA binding affinities of both the mono- and di-amino acid-benzimidazole-amidine conjugates were generally weaker than that of netropsin and distamycin with the dipeptide conjugates showing stronger binding affinities than the mono-amino acid conjugates. The dipeptide conjugates containing amino acids with positively charged side chains, Lys-Gly-BI-(+) and Arg-Gly-BI-(+), showed the strongest DNA binding affinities amongst all our synthesized conjugates.Item DNA Recognition and Cleavage by Phenyl-Benzimidazole Modified Gly-Gly-His-Derived Metallopeptides(2010-04-08T16:20:15Z) Wang, Tianxiu; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Naumann, Christoph A.Metallopeptides of the general form M(II)∙Gly1-Gly2-His induce DNA strand scission via minor groove interactions. This peptide system can serve as a nucleic acid-targeted cleavage agent – either as an appendage to other DNA binding agents, or as a stand alone complex. In an effort to further our knowledge of DNA recognition and cleavage, a novel series of phenyl-benzimidazole modified Gly-Gly-His-derived metallopeptides was synthesized via solid phase methods and investigated. The new systems allow the formation of additional contacts to the DNA minor groove through the incorporation of a DNA binding phenyl-benzimidazole moiety, thus strengthening the overall binding interaction and further stabilizing the metal complex-DNA association. In addition, how Lys side chains and an amidinium group influence the efficiency of DNA cleavage was also studied. DNA cleavage studies suggested that the phenyl-benzimidazole-modified Gly-Gly-His metallopeptides possess enhanced DNA cleavage abilities. In particular, when amidines are placed on the benzimidazole moieties, these moieties appeared to play an important role in increasing the DNA cleavage activity of the metal complex, most likely through an enhanced electrostatic attraction to the DNA.Item Exploring the mechanism of action of spore photoproduct lyase(2014-08-27) Nelson, Renae; Li, Lei; Long, Eric C. (Eric Charles); McLeish, Michael J.Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.Item Identification of Tobacco-Related Compounds in Tobacco Products and Human Hair(2014-09-04) Rainey, Christina; Goodpaster, John V. (John Vincent); Minto, Robert; Shepson, Paul; Kissinger, Peter T., 1944-; Long, Eric C. (Eric Charles)Analyses of tobacco products and their usage are well-researched and have implications in analytical chemistry, forensic science, toxicology, and medicine. As such, analytical methods must be developed to extract compounds of interest from tobacco products and biological specimens in order to determine tobacco exposure. In 2009, R.J. Reynolds Tobacco Co. released a line of dissolvable tobacco products that are marketed as a smoking alternative. The dissolvables were extracted and prepared by ultrasonic extractions, derivatization, and headspace solid phase microextraction (SPME) with analysis by gas chromatography-mass spectrometry (GC-MS). The results show that the compounds present are nicotine, flavoring compounds, humectants and binders. Humectant concentrations vary among different tobacco types depending on the intended use. Humectants were quantified in various tobacco types by GC and “splitting” the column flow between a flame ionization detector (FID) and an MS using a microfluidic splitter in order to gain advantage from the MS’s selectivity. The results demonstrated excellent correlation between FID and MS and show that MS provides a higher level of selectivity and ensures peak purity. Chemometrics was also used to distinguish products by tobacco type. Hair is a common type of evidence in forensic investigations, and it is often subjected to mitochondrial DNA (mtDNA) analysis. Preliminary data was gathered on potential “lifestyle” markers for smoking status as well as any indications of subject age, gender, or race by investigating the organic “waste” produced during a mtDNA extraction procedure. The normally discarded organic fractions were analyzed by GC-MS and various lipids and fatty acids were detected. At this point, a total vaporization-SPME (TV-SPME) method was theorized, developed, and optimized for the specific determination of nicotine and its metabolite, cotinine. The theory of TV-SPME is to completely vaporize an organic extract which will eliminate the partitioning between the sample and the headspace, thereby simplifying the thermodynamic equilibrium. Parameters such as sample volume, incubation temperature, and extraction time were optimized to achieve the maximum analyte signal. Response surface methodology (RSM) is a statistical model that is very useful in predicting and determining optimum values for variables to ensure the ideal response. RSM was used to optimize the technique of TV-SPME for the analysis of nicotine and cotinine. Lastly, quantitation of nicotine and cotinine in human hair typically requires large sample sizes and extensive extraction procedures. Hence, a method using small sample sizes and a simple alkaline digestion followed by TV-SPME-GC-MS has been developed. Hair samples were collected from anonymous volunteers and nicotine and cotinine were identified and quantitated in the hair of tobacco users.Item Investigating the Biosynthetic Pathways to Polyacetylenic Natural Products in Fistulina hepatica and Echinacea purpurea(2013-08-20) Ransdell, Anthony S.; Minto, Robert; Long, Eric C. (Eric Charles); Li, LeiPolyacetylenic natural products, compounds containing multiple carbon-carbon triple bonds, have been found in a large collection of organisms. Radiochemical tracer studies have indicated that these bioactive metabolites are synthesized from fatty acid precursors through a series of uncharacterized desaturation and acetylenation steps. To date, there are three main pathways believed to be involved in acetylenic natural product biosynthesis. However, it is apparent that the crepenynic acid pathway is the origin of a vast majority of the known plant and fungal acetylenic products. This investigation provides concrete evidence that the polyacetylenic natural products found in the fungus Fistulina hepatica and the medicinal plant species Echinacea purpurea are biosynthesized from crepenynic acid. Through heterologous expression in Yarrowia lipolytica, two acetylenases capable of producing crepenynic acid were identified from E. purpurea. Furthermore, heterologous expression of two diverged desaturases isolated from F. hepatica, uncovered a ∆12-acetylenase and the first multifunctional enzyme capable of ∆14-/∆16- desaturation and ∆14-acetylenation.Item Investigations of lipid metabolism in Yarrowia lipolytica(2014-07-31) Blocher-Smith, Ethan Charles; Minto, Robert; Long, Eric C. (Eric Charles); Jones, LisaAn investigation of the lipid metabolism pathway in the yeast Yarrowia lipolytica was conducted. Yarrowia is an oleaginous ascomycete that is capable of growing on many different substrates, which derives its name from its high efficiency of growth on lipids. Once the exogenous lipids are converted into free fatty acids and internalized by the yeast, the primary mode of degradation is through β-oxidation mediated by the peroxisomal oxidases, or POX genes. These enzymes catalyze the formation of a trans double bond, producing the trans-2-enoyl product. Our study looked at the comparison of the Y. lipolytica prototrophic strain against a knockout of the Pox2 gene on the uptake, incorporation, and degradation of relevant fatty acids. To construct this gene knockout, a novel gene deletion method using a combination of Cre recombinase and the AHAS* gene was synthesized, developed, and tested successfully. This knockout system allows for serial deletion of genes with the use of only one resistance marker, with excision of the marker after selection.