Browsing by Subject "Mutagenesis"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item A mutagenesis assay for the carcinogen 1,2-dimethylhydrazine(Texas Tech University, 1979-08) Wells, Barbara C.Not availableItem Crystallization and mutational studies of carbon monoxide dehydrogenase from moorella thermoacetica(Texas A&M University, 2004-09-30) Kim, Eun JinCarbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the ? subunit of heterotetrameric ?2?2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an ?-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the ? subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.Item DNA transformation and mutagenesis studies on dithane with Bacillus subtilis repair-deficient strains(Texas Tech University, 1980-08) Lee, Ming-chouNot availableItem Identification of Substrates and Pathways Regulated by WNK1(2004-12-15) Lee, Byung-Hoon; Cobb, Melanie H.WNK (With No lysine (K)), a serine/threonine protein kinase, is a unique molecule not belonging to any other canonical protein kinase family including mitogen-activated protein (MAP) kinases. The name of the WNK protein kinase family reflects the fact that a catalytic lysine lies in a position different in WNKs from that in all other protein kinases. The urgency of a mechanistic examination of the WNK family protein kinase was heightened by the discovery that mutations in at least two of the four human WNKs, WNK1 and 4, caused a heritable form of hypertension. My study focused on unveiling WNK1 substrates and interactors for a better understanding of the molecular pathways served by WNK kinases. Yeast two-hybrid screening was performed to identify the binding partners of WNK1 and yielded genuine interactors including synaptotagmin (Syt) isoforms, Smad2, and dynein light chain (LC8/PIN). WNK1, not WNK4, selectively binds to and phosphorylates Syt2 within its calcium binding C2 domains. Calcium strongly enhanced their binding in vitro. Essential Ca2+-binding residues in the Syt2 C2 domains were critical for formation of a WNK1-Syt2 complex and for Syt2 phosphorylation. WNK1 displayed specificity among Syt isoforms and mutational analysis implicated a hydrophobic residue on the WNK1 kinase domain surface as essential for the high affinity WNK1-Syt2 interaction and phosphorylation. Endogenous WNK1 and Syt2 coimmunoprecipitated and colocalized on a subset of secretory granules in the INS-1 cell line, a pancreatic beta cell model system. Importantly, phosphorylation by WNK1 increased the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of Thr202, a WNK1 phosphorylation site identified from mass spectrometric analysis, partially prevented this change. These findings provide a biochemical scenario that could lead to the retention or insertion of proteins in the plasma membrane. WNK1 may serve as a molecular switch for vesicle trafficking and other membrane events that regulate ion balance. The interaction with and phosphorylation of other molecules by WNK1 were also investigated here.Item The molecular basis of nucleotide recognition for T7 DNA polymerase(2008-08) Jin, Zhinan, 1972-; Johnson, Kenneth AllenDNA replication demands extraordinary specificity and efficiency of catalysis from a DNA polymerase. Previous studies on several DNA polymerases suggested that a rate-limiting conformational change preceding chemistry accounts for the high specificity following the induced fit mechanism. However, the identity of this rate-limiting conformational change and how it contributes to the fidelity is still under debate. An important study of T7 DNA polymerase performed by Tsai and Johnson using a conformationally sensitive fluorophore (CSF) characterized a conformational change directly and presented a new paradigm for nucleotide selectivity. This thesis describes work to further characterize the underlying molecular basis regulating the conformational change by a combination of site-directed mutagenesis, transient kinetics and crystallography. One flexible segment (gly-ala-gly) within the fingers domain was mutated to (ala-alaala). The kinetic analysis on this mutant showed that the mutations decreased the forward rate of the conformational change reported by the fluorophore about 1200-fold but there was no significant change on the reverse rate. The data suggested that the movement of the fingers domain is not a rigid body motion but may be complex due to the movements of various helices within the fingers domain. Quantification of the kinetics of incorporation of correct and incorrect base pairs showed the decrease of fidelity mainly was from the decreased forward rate during correct nucleotide incorporation. The roles of three active site residues, K522, H506, and R518, which form polar interactions with [alpha]-,[beat]- and [gamma]-phosphates of the incoming nucleotide respectively, in conformational change and catalysis were also characterized. All the mutants showed a slower conformational change than the wild type enzyme. After this conformational change, there was a rate limiting step with a rate comparable to kpol measured by quench-flow experiments. Correct nucleotide binding caused an increase in fluorescence, suggesting that the conformational change of the fingers domain delivers incoming nucleotide to a misaligned status even for a correct nucleotide with each of the mutants. The data suggested that active site residues play important roles in maintaining a fast conformational change and an accurate alignment of the active site during correct nucleotide incorporation. Yellow crystals of CSF-labeled T7 DNA polymerase with DNA and correct nucleotide (closed complex), incorrect nucleotide (misaligned complex) or no nucleotide (open complex) were grown to good size and diffracted to 3 Å during X-ray data collection. The structures of these complexes are still under refinement.Item Probing the structure and reactivity of the Bacillus cereus 5/B/6 metallo-Beta-lactamase through site-directed mutagenesis and mechanism-based inactivation(Texas Tech University, 1997-12) Yi, XiaomingThe discovery of peniciUin by Sir Alexander Fleming in 1929 was accidental but revolutionary. In 1938, Chain, Foley and their associates purifíed the active agent from a culture filtrate of Penicilium notatum. The subsequent introduction of penicillin into clinical use revolutionized the treatment of many infectious diseases and started a new era of chemotherapy in the history of medicine (Abraham, 1981).Item Single scaffold antibody libraries created with high rates of mutagenesis or diversity focused for peptide recognition(2007-12) Cobaugh, Christian Wessel, 1971-; Iverson, Brent L.; Georgiou, GeorgeThis dissertation describes several strategies used to create diversity in non-immune antibody libraries. Two of the strategies were used to create two separate peptide focused libraries. Both of these strategies used to create these antigen-class focused libraries used a single scaffold antibody gene that contained diversity only in the variable heavy region. The scaffold antibody gene one of the libraries, the M:anti-pep library, was chosen based on hypervariable loop canonical structures that are characteristic of other anti-peptide antibodies. Additionally, all of the contact residues of this antibody are commonly used contact residues in other anti-peptide antibodies. These positions and others were varied to incorporate the natural diversity of other anti-peptide antibodies. The second library, the Hu:anti-pep, is based on a widely used, unique combination of human germline antibody segments that express well in bacterial expression. Positions were chosen for variation based on their usage as contact residues in both anti-peptide and anti-protein antibodies. The diversity was less focused than with the M:anti-pep library, incorporating all 20 amino acids at "high usage" positions and only four amino acids at "low usage" positions. Both libraries were validated by phage display selections against the peptide angiotensin (AT) and neuropeptide Y (NPY). The M:anti-pep library yielded specific antibodies to both peptides with dissociation constants as low as 14 nM against AT and 18 nM against NPY. The Hu:anti-pep library yielded specific clones with higher dissociation constants: 49 nM against NPY and 11 [mu]M against AT. The final strategy used to introduce diversity is widely used for affinity maturation of low affinity, previously selected antibodies. Extremely high rates of mutagenesis (2.2% of the gene to 2.7%) were used to create two libraries of the anti-digoxin antibody 26-10. The libraries had been screened by others in an attempt to examine the effects of highrates of mutagenesis on the directed evolution of an antibody. A total of 91 isolated clones from both libraries were sequenced. Several consensus mutations were identified near the CDRH3 in the isolated clones, indicating that they had a positive, selectable effect. This study confirmed that high-error rate antibody libraries contain more active clones than expected. Combinations of the selected consensus mutations from these libraries provide moderate enhancements to the kinetics and expression of the wild-type antibody in a non-synergistic manner.Item Structural analysis and discovery of lead compounds for the fungal methionine synthase enzyme(2013-12) Ubhi, Devinder Kaur; Robertus, Jon D.Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine.Item Studies on Protein Engineered Soybean Sterol Methyltransferase to Probe the Structure-Function relationship of enzyme activities(Texas Tech University, 2009-08) Nguyen, Thi Thuy Minh; Nes, William David; Shaw, Robert W.; Pare, PaulSterol C24-methyltransferase (SMT) enzymes convert Δ24-sterol acceptor molecules to one or more methyl products and set the pattern of 24-alkyl sterols used as membrane inserts in plants. For this study, a series of cycloartenol-based acceptors that differed in the nature of the side chain construction were evaluated as substrates of the cloned soybean (Glycine max) SMT. Three natural substrates were tested with the soybean SMT1, cycloartenol (CA), cyclobranol (24-methyl cycloartenol, CB) and 24(28)-methylene cycloartanol (MCA); CA converted to a single product MCA, CB converted to a novel Δ24(28)-C25-methyl sterol and MCA converted to a triplet of 24-ethyl(idene) sterols. When these substrates were tested against a panel of soybean mutants designed using the results of the yeast SMT recently found to generate plant-like C1- or C2-transfer activity distinct differences were observed in product distributions between the plant and fungal SMTs. On the one hand, new transalkylation activities catalyzed by the fungal SMT assayed with zymosterol were not carried out by the plant SMT mutants assayed with CA. On the other hand, Tyr to Phe soybean SMT mutants at positions 81 and 223 (ERG6 nomenclature) generated novel products from assay with CB. A new substrate analog 26-difluorocycloartenol (diFCA) was studied to understand further the reaction mechanism catalyzed by the soybean SMT1. By use of fluorine as an isosteric replacement for a hydrogen atom in the sterol side chain, it was expected that the inductive electron-withdrawing effect of the fluoro substituent would impair the critical proton transfer of H24 to C25 affording covalent attachment of the enzyme-generated intermediate to the recombinant protein. Steady-state kinetic experiments with CA and diFCA showed that the overall catalytic efficiencies (Vmax/KM) differ markedly with the diFCA substrate much less efficient than the CA. The diFCA was found to be a competitive inhibitor of CA to MCA conversion catalyzed by the soybean SMT1 exhibiting a KI of 71 µM. In addition, conversion of the diFCA was evident in the GC-MS analysis of the enzyme-generated products which showed C24-methylated isomeric compounds of molecular weight 458 and 476 amu. These sterol derivatives were assigned the tentative structures of the monol derivatives (turnover products that possess 1 or 2 fluorine atoms at C26) and diol derivatives (kill products bound to the enzyme and released by saponification of the enzyme extract). This work affords compelling evidence in support of a cationic mechanism for C24-methylation of the cycloartenol side chain and that variant side chain features recognized by wild-type and mutant soybean SMT1 can affect catalytic competence, product diversity and/or enzyme inhibition.Item The Tuning of DNA Mutability Via Condon Context and Usage Bias : Identifying Predispositions to Nonneutral Evolution Within Human Genes(2005-05-03) Horvath, Monica Marie; Garner, Harold R.Nonrandom human point mutation trends have been identified across numerous SNP databases to show that CpG dinucleotides in particular display hierarchal mutabilities depending upon the surrounding DNA sequence microenvironment. This information can be harnessed to create a scoring system to contrast the relative mutability of gene sequences, which as a result highlights a gene's rigidity or malleability towards point mutation throughout its evolution. Nonsynonymous mutation probabilities for human genes are calculated and contrasted using four mutation models derived from distinct sources: Diseasecausing variants, single nucleotide polymorphisms, intronic mutations, and interspecific substitutions from aligned orthologs. The most mutable human genes are those that mediate reaction to environmental stimuli, including those involved in immune response, pathogen response, and olfaction. As expected, genes using context inclining low point mutation are those involved in essential processes such as cell proliferation and DNA repair. Coupled with observations from studies indicating these classes have experienced positive selection in humans, such results imply that codon usage may shape the size and diversity of the mutation pool on which selection acts. A preinclination towards either radical or safe mutation can be encoded by a gene through using a set of codons with innate tendencies that enhance variation in the required evolutionary direction. The importance of such 'internal forces' in shaping genome evolution signals a need for adjustment of a key principle underlying Neo-Darwinism, which holds that natural selection is the single driving force of genome evolution because underlying point mutation is thought to be random and ubiquitous.