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Research Interests: Biochemistry, Physiology, Chemistry, Kinetics, Biology, and 15 moreMedicine, Biocatalysis, Enzyme Kinetics, Phylogeny, Molecular Phylogenetics and Evolution, Enzyme, Phytophthora, Kinase, Biochemistry and molecular biology, Glycine, Amino Acid Sequence, Phytophthora sojae, Oomycota, Biochemistry and cell biology, and Molecular Sequence Data
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The uncE114 mutation (Gln42----Glu) in subunit c of the Escherichia coli H+ ATP synthetase causes uncoupling of proton translocation from ATP hydrolysis (Mosher, M. E., White, L. K., Hermolin, J., and Fillingame, R. H. (1985) J. Biol.... more
The uncE114 mutation (Gln42----Glu) in subunit c of the Escherichia coli H+ ATP synthetase causes uncoupling of proton translocation from ATP hydrolysis (Mosher, M. E., White, L. K., Hermolin, J., and Fillingame, R. H. (1985) J. Biol. Chem. 260, 4807-4814). In the background of strain ER, the mutation led to dissociation of F1 from the membrane. Ten revertants to the uncE114 mutation were isolated, and the uncE gene was cloned and sequenced. Six of the revertants were intragenic and had substitutions of glycine, alanine, or valine for the mutant glutamate residue at position 42. The intragenic, revertant uncE genes were incorporated into an otherwise wild type chromosome of strain ER. Membrane vesicles prepared from each of the revertants showed a restoration of F1 binding to F0. The Val42 revertant differed from the other two revertants in that the ATPase activity of F1 was inhibited when membrane bound. This was shown by the stimulation of ATPase activity when F1 was released from the membrane. The Gly42 and Ala42 revertants demonstrated membrane ATPase activity that was resistant to dicyclohexylcarbodiimide treatment. Resistance was shown to be due to the increased dissociation of F1 from the membrane under ATPase assay conditions. The Ala42 revertant showed a significant reduction in ATP-dependent quenching of quinacrine fluorescence that was attributed to less efficient coupling of ATP hydrolysis to H+ translocation, whereas the other revertants showed responses very near to that of wild type. Minor changes in the F1-F0 interaction in all three revertants were indicated by an increase in H+ leakiness, as judged by reduced NADH-dependent quenching of quinacrine fluorescence. The minor defects in the revertants support the idea that residue 42 is involved in the binding and coupling of F1 to F0 but also show that the conserved glutamine (or asparagine) is not absolutely necessary in this function.
Research Interests: Chemistry, Biology, Membrane Proteins, Biological Chemistry, Medicine, and 15 moreBiological Sciences, ATPase, Mutation, Escherichia coli, Bacteria, Phenotype, CHEMICAL SCIENCES, Glutamine, Oxidative phosphorylation, Coupling, Base Sequence, Protein Binding, Adenosine Triphosphate, DNA mutational analysis, and Medical and Health Sciences
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Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that... more
Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.
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H+ translocating, FIFo type ATP synthases catalyze the synthesis of ATP during oxidative phosphorylation. These enzymes are composed of two functionally and structurally distinct sectors, termed FI and Fo. The Fl sector lies extrinsic to... more
H+ translocating, FIFo type ATP synthases catalyze the synthesis of ATP during oxidative phosphorylation. These enzymes are composed of two functionally and structurally distinct sectors, termed FI and Fo. The Fl sector lies extrinsic to the membrane, and in soluble form it catalyzes the hydrolysis of ATP. The Fo sector traverses the membrane, and on removal of F1 from the membrane, it catalyzes the reversible translocation of protons across the membrane. When the two sectors are coupled, the enzyme functions as a reversible, H+ translocating ATPase. In Eschen'chia coli the Fl sector is composed of five subunits in a stoichiometry of a$3y181~l and the Fo sector is composed of three types of subunits in an alb2clo+1 stoichiometry.' A counterpart of each subunit is found in the ATP synthases of other bacteria, mitochondria, and chloroplasts.* Proton translocation through the enzyme appears to be coupled to ATP synthesis by an indirect mechanism. The active site for ATP synthesis lies within subunit f3, perhaps at an interface with subunit a, and this site may lie more than 50 A from the surface of the membrane.3.4 Proton translocation through Fo is thought to cause a conformational change in Fl that leads to release of ATP product, and the tight binding of substrates at a second, alternating catalytic site.5 The key residue catalyzing proton translocation is thought to be Asp61 of subunit c, and this residue appears to lie towards the center of the lipid bilayer at least 10 A below the surface.6 In summary, the protonation-deprotonation of Asp61 at the center of the membrane is proposed to be linked to conformational changes that are ultimately transmitted over a distance of more than 50 A to the active site for ATP synthesis in F1. This essay reviews work from our own laboratory relating to the role of subunit c in proton translocation and to the coupling of proton translocation to ATP synthesis. We discuss what is known about the structure of subunit c in the membrane and relate this to a recently determined structural model obtained with purified subunit c by nuclear magnetic resonance (NMR) methods. We also discuss genetic experiments that suggest a site of functional interaction between subunit c and subunit a during proton translocation.
Research Interests: Chemistry, Library Science, Medicine, Multidisciplinary, Protein Structure and Function, and 9 moreEscherichia coli, Structural equation models, ATP synthase, Protein Secondary Structure Prediction, Medical School, Amino Acid Sequence, Structure Function, Site-directed Mutagenesis, and Molecular Sequence Data
Research Interests: Kinetics, Biology, Biological Chemistry, Medicine, Biological Sciences, and 12 moreEscherichia coli, Structural equation models, ATP synthase, D-Aspartic Acid, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, Genetic variation, Glycine, Alanine, Amino Acid Sequence, Cell Membrane, and Medical and Health Sciences
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Research Interests: Biochemistry, Chemistry, Organic Chemistry, Medicine, Electroporation, and 15 moreAnimals, Temperature, Peptide, RNAse P, Bioconjugate Chemistry, High Pressure Liquid Chromatography, Cell nucleus, Calmodulin, Cysteine, Amino Acid Sequence, Base Sequence, Structure activity Relationship, Protein Denaturation, Biochemistry and cell biology, and Molecular Sequence Data
Research Interests: Biology, Biological Chemistry, Medicine, Biological Sciences, Escherichia coli, and 13 moreBiological, ATP synthase, CHEMICAL SCIENCES, Arginine, Base Sequence, Protons, Site-directed Mutagenesis, Adenosine Triphosphate, Molecular Sequence Data, Cell Membrane, Cell membrane permeability, binding sites, and Medical and Health Sciences
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Research Interests: Biochemistry, Chemistry, Biology, Biological Chemistry, Medicine, and 13 moreBiological Sciences, Sequence alignment, Escherichia coli, Microbial genetic and drug resistance, ATP synthase, CHEMICAL SCIENCES, Alanine, Amino Acid Sequence, Base Sequence, Structure activity Relationship, Molecular Sequence Data, DNA mutational analysis, and Medical and Health Sciences
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Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome... more
Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp. PE36 have revealed what appears to be a phosphagen kinase homolog present in their genomes. Based on sequence comparisons these putative homologs bear a strong resemblance to arginine kinases found in many invertebrates and some protozoa. We describe here a biochemical characterization of one of these homologs from D. psychrophila expressed in E. coli that confirms its ability to reversibly catalyze phosphoryl transfer from ATP to arginine. A phylogenetic analysis suggests that these bacteria homologs are not widely distributed in proteobacteria species. They appear more related to protozoan arginine kinases than to similar proteins seen in some Gram-positive bacteria that share key catalytic residues but encode protein tyrosine kinases. This raises the possibility of horizontal gene transfer as a likely origin of the bacterial arginine kinases.
Research Interests: Biochemistry, Physiology, Kinetics, Biology, Horizontal Gene Transfer, and 15 moreMedicine, Phylogeny, Sequence alignment, Animals, Protein Tyrosine Kinase, Phylogenetic analysis, Creatine Kinase, Gram Positive Bacteria, Kinase, Arginine, Genome sequence, Amino Acid Sequence, High energy, Biochemistry and cell biology, and Molecular Sequence Data
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Research Interests: Kinase and Cooperativity
Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an... more
Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.
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Research Interests: Biological Chemistry, Biological Sciences, Sequence alignment, Escherichia coli, Microbial genetic and drug resistance, and 8 moreCHEMICAL SCIENCES, Alanine, Amino Acid Sequence, Base Sequence, Structure activity Relationship, Molecular Sequence Data, DNA mutational analysis, and Medical and Health Sciences
Arginine kinases (AK) are members of the phosphagen kinase family and like other phosphagen kinases, they help in regulating energy metabolism in the cell. They catalyze the transfer of a high-ener...
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Real‐time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished... more
Real‐time PCR is a recent modification to the polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. Detection can be accomplished using specific as well as nonspecific fluorescent probes. Real‐time PCR is often used in the quantification of gene expression levels. Prior to using real‐time PCR to quantify a target message, care must be taken to optimize the RNA isolation, primer design, and PCR reaction conditions so that accurate and reliable measurements can be made. This short overview of real‐time PCR discusses basic principles behind real‐time PCR, some optimization and experimental design considerations, and how to quantify the data generated using both relative and absolute quantification approaches. Useful Web sites and texts that expand upon topics discussed are also listed. Curr. Protoc. Essential Lab. Tech. 8:10.3.1‐10.3.40. © 2014 by John Wiley & Sons, Inc.