The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Zvi Kelman - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of conserved aspartic acid residues in the methanothermobacter thermautotrophicus mcm helicase
Extremophiles, 2011Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi KelmanAbstract:Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the Protein is divided into three domains, A, B, and C, and was shown to be involved in Protein Multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM Proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM Protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant Proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant Proteins. Possible roles of these conserved residues in MCM function are discussed.
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different residues on the surface of the methanothermobacter thermautotrophicus mcm helicase interact with single and double stranded dna
Archaea, 2010Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi KelmanAbstract:The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM Protein, while the N-terminal portion plays an important role in DNA binding and Protein Multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.
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Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM Protein
Nucleic Acids Research, 2008Co-Authors: Nozomi Sakakibara, Eugene Melamud, Rajesh Kasiviswanathan, Frederick P. Schwarz, Zvi KelmanAbstract:Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM Protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the Protein was shown to be involved in Protein Multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM Proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.
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biochemical characterization of the methanothermobacter thermautotrophicus minichromosome maintenance mcm helicase n terminal domains
Journal of Biological Chemistry, 2004Co-Authors: Rajesh Kasiviswanathan, Jaeho Shin, Eugene Melamud, Zvi KelmanAbstract:Abstract Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal Protein it was shown that the N-terminal portion of the Protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for Protein Multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.
Rajesh Kasiviswanathan - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of conserved aspartic acid residues in the methanothermobacter thermautotrophicus mcm helicase
Extremophiles, 2011Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi KelmanAbstract:Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the Protein is divided into three domains, A, B, and C, and was shown to be involved in Protein Multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM Proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM Protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant Proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant Proteins. Possible roles of these conserved residues in MCM function are discussed.
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different residues on the surface of the methanothermobacter thermautotrophicus mcm helicase interact with single and double stranded dna
Archaea, 2010Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi KelmanAbstract:The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM Protein, while the N-terminal portion plays an important role in DNA binding and Protein Multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.
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Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM Protein
Nucleic Acids Research, 2008Co-Authors: Nozomi Sakakibara, Eugene Melamud, Rajesh Kasiviswanathan, Frederick P. Schwarz, Zvi KelmanAbstract:Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM Protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the Protein was shown to be involved in Protein Multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM Proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.
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biochemical characterization of the methanothermobacter thermautotrophicus minichromosome maintenance mcm helicase n terminal domains
Journal of Biological Chemistry, 2004Co-Authors: Rajesh Kasiviswanathan, Jaeho Shin, Eugene Melamud, Zvi KelmanAbstract:Abstract Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal Protein it was shown that the N-terminal portion of the Protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for Protein Multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.
Nozomi Sakakibara - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of conserved aspartic acid residues in the methanothermobacter thermautotrophicus mcm helicase
Extremophiles, 2011Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi KelmanAbstract:Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the Protein is divided into three domains, A, B, and C, and was shown to be involved in Protein Multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM Proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM Protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant Proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant Proteins. Possible roles of these conserved residues in MCM function are discussed.
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different residues on the surface of the methanothermobacter thermautotrophicus mcm helicase interact with single and double stranded dna
Archaea, 2010Co-Authors: Nozomi Sakakibara, Rajesh Kasiviswanathan, Zvi KelmanAbstract:The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM Protein, while the N-terminal portion plays an important role in DNA binding and Protein Multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.
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Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM Protein
Nucleic Acids Research, 2008Co-Authors: Nozomi Sakakibara, Eugene Melamud, Rajesh Kasiviswanathan, Frederick P. Schwarz, Zvi KelmanAbstract:Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM Protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the Protein was shown to be involved in Protein Multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between β7 and β8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM Proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.
Raymond J Turner - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance Protein, EmrE.
Biochemistry and biophysics reports, 2015Co-Authors: S. Junaid S. Qazi, Raymond H. Chew, Raymond J TurnerAbstract:Abstract EmrE is a member of the small multidrug resistance (SMR) Protein family in Escherichia coli . EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane Proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His 6 ) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All Proteins were solubilized in the same buffered n-dodecyl-β- d -maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of Protein Multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both Proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both Proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants ( K d ) and maximum specific one-site binding ( B max ) values for particular QCCs. In vitro analyses of both Proteins demonstrated subtle but significant differences in Multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.
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a survey for small multidrug resistance Protein Multimerization in the presence of ligand using sds page analysis
Biophysical Journal, 2010Co-Authors: Raymond J TurnerAbstract:EmrE and SugE are members of the small multidrug resistance Protein family that can efflux quaternary cation compounds (QCC) via proton motive force within the Escherichia coli plasma membrane. Members of this integral membrane Protein family are characterized by their short (∼100-140 amino acids) four transmembrane (TM) alpha- helix conformation and highly conserved glutamate residue within the active site. EmrE Protein can confer broad multidrug resistance to the host strain unlike SugE Protein, which demonstrates limited multidrug resistance. The exact multimeric state or states of both Proteins during transport and ligand binding is not well understood and often yield conflicting results that are specific to the conditions of study. To explore SMR Multimerization as influenced by QCC ligands, organic solvent extracted EmrE and SugE Protein from E. coli membranes were characterized in the detergents, sodium dodecyl sulfate (SDS) and dodecyl maltoside (DDM) at varying Protein concentrations. SMR Proteins solubilized in both detergents demonstrated a predominately monomeric state but upon increasing particular QCC ligand concentrations resulted in multimer formation or enhancement using SDS- tricine polyacrylamide gel electrophoresis (PAGE). The results from this PAGE based assay demonstrate that: i) SMR multimers are induced by particular ligands that may relate to ligand shape and ii) only EmrE Multimerization is induced by particular ligands, whereas SugE appears to be insensitive to drug enhanced oligomerization. Therefore, SMR multimer variability may be dependent upon the nature of the transported substrate and SMR subclass; only EmrE can alter its subunit composition in response to particular QCC substrates. This PAGE based assay provides the framework to explore the influence of diverse QCC substrates for its affects on SMR Multimerization.
Regina Kuliawat - One of the best experts on this subject based on the ideXlab platform.
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lumenal Protein Multimerization in the distal secretory pathway secretory granules
Current Opinion in Cell Biology, 2002Co-Authors: Peter Arvan, Bao Yan Zhang, Lijun Feng, Regina KuliawatAbstract:Differences in Protein solubility appear to play an important role in lumenal Protein trafficking through Golgi/post-Golgi compartments. Recent advances indicate that multimeric Protein assembly is one of the factors regulating the efficiency of Protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.
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Lumenal Protein Multimerization in the distal secretory pathway/secretory granules.
Current Opinion in Cell Biology, 2002Co-Authors: Peter Arvan, Bao Yan Zhang, Lijun Feng, Regina KuliawatAbstract:Differences in Protein solubility appear to play an important role in lumenal Protein trafficking through Golgi/post-Golgi compartments. Recent advances indicate that multimeric Protein assembly is one of the factors regulating the efficiency of Protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.
