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Béatrice Golinelli-pimpaneau - One of the best experts on this subject based on the ideXlab platform.
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Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase
Nucleic Acids Research, 2010Co-Authors: Amandine Guelorget, Martine Roovers, Vincent Guérineau, Carole Barbey, Béatrice Golinelli-pimpaneauAbstract:The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence.
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The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity
Nucleic Acids Research, 2008Co-Authors: Hélène Walbott, Nicolas Leulliot, Henri Grosjean, Béatrice Golinelli-pimpaneauAbstract:The 5-methyluridine is invariably found at position 54 in the TPsiC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase ((Pab)TrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of (Pab)TrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 A resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of (Pab)TrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by (Pab)TrmU54. These results were used to model yeast tRNA(Asp) in the (Pab)TrmU54 structure to get further insights into the different RNA specificities of RumA and (Pab)TrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to (Pab)TrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TPsiC loop conformational change has to occur for the flipping of the target uridine into the (Pab)TrmU54 active site during catalysis.
Rudolf Ladenstein - One of the best experts on this subject based on the ideXlab platform.
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ultrahigh resolution study on Pyrococcus abyssi rubredoxin ii introduction of an o h sγ fe hydrogen bond increased the reduction potential by 65 mv
Journal of Biological Inorganic Chemistry, 2007Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The effect of D–H···Sγ–Fe hydrogen bonding on the reduction potential of rubredoxin was investigated by the introduction of an O–H···Sγ–Fe hydrogen bond on the surface of Pyrococcus abyssi rubredoxin. The formation of a weak hydrogen bond between Ser44-Oγ and Cys42-Sγ in mutant W4L/R5S/A44S increased the reduction potential by 56 mV. When side effects of the mutation were taken into account, the contribution of the additional cluster hydrogen bond to the reduction potential was estimated to be +65 mV. The structural analysis was based on ultrahigh-resolution structures of oxidized P. abyssi rubredoxin W4L/R5S and W4L/R5S/A44S refined to 0.69 and 0.86 A, respectively.
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Ultrahigh-resolution study on Pyrococcus abyssi rubredoxin: II. Introduction of an O–H···Sγ–Fe hydrogen bond increased the reduction potential by 65 mV
Journal of Biological Inorganic Chemistry, 2007Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The effect of D–H···Sγ–Fe hydrogen bonding on the reduction potential of rubredoxin was investigated by the introduction of an O–H···Sγ–Fe hydrogen bond on the surface of Pyrococcus abyssi rubredoxin. The formation of a weak hydrogen bond between Ser44-Oγ and Cys42-Sγ in mutant W4L/R5S/A44S increased the reduction potential by 56 mV. When side effects of the mutation were taken into account, the contribution of the additional cluster hydrogen bond to the reduction potential was estimated to be +65 mV. The structural analysis was based on ultrahigh-resolution structures of oxidized P. abyssi rubredoxin W4L/R5S and W4L/R5S/A44S refined to 0.69 and 0.86 A, respectively.
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Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi.
Acta crystallographica. Section F Structural biology communications, 2007Co-Authors: Bin Ren, Hannu Myllykallio, Joëlle Kuhn, Laurence Meslet-cladiere, Rudolf LadensteinAbstract:Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 A. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 A and a complete native data set was collected to 2.65 A resolution.
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ultrahigh resolution study on Pyrococcus abyssi rubredoxin i 0 69 a x ray structure of mutant w4l r5s
Acta Crystallographica Section D-biological Crystallography, 2005Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The crystal structure of Pyrococcus abyssi rubredoxin mutant W4L/R5S was solved by direct methods. The model of the air-oxidized protein was refined by partially restrained full-matrix least-squares refinement against intensity data to 0.69 A resolution. This first ultrahigh-resolution structure of a rubredoxin provides very detailed and precise information about the Fe(SCys)4 centre and its environment, the peptide-backbone stereochemistry, H atoms and hydrogen bonds, static and dynamic disorder, the solvent structure and the electron-density distribution. P. abyssi rubredoxin W4L/R5S is the first of a series of mutants studied by atomic and ultrahigh-resolution X-ray crystallography which are expected to contribute to the understanding of structure–function relationships in iron–sulfur proteins.
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Ultrahigh-resolution study on Pyrococcus abyssi rubredoxin. I. 0.69 A X-ray structure of mutant W4L/R5S.
Acta Crystallographica Section D-biological Crystallography, 2005Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The crystal structure of Pyrococcus abyssi rubredoxin mutant W4L/R5S was solved by direct methods. The model of the air-oxidized protein was refined by partially restrained full-matrix least-squares refinement against intensity data to 0.69 A resolution. This first ultrahigh-resolution structure of a rubredoxin provides very detailed and precise information about the Fe(SCys)4 centre and its environment, the peptide-backbone stereochemistry, H atoms and hydrogen bonds, static and dynamic disorder, the solvent structure and the electron-density distribution. P. abyssi rubredoxin W4L/R5S is the first of a series of mutants studied by atomic and ultrahigh-resolution X-ray crystallography which are expected to contribute to the understanding of structure–function relationships in iron–sulfur proteins.
Didier Flament - One of the best experts on this subject based on the ideXlab platform.
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Research Article Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi
2016Co-Authors: Nicolas Leulliot, Sophie Quevillon-cheruel, Marc Graille, Claire Geslin, Didier Flament, Le M. Romancer, H. Van TilbeurghAbstract:Copyright © 2013 N. Leulliot et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiformmembers of the family Fuselloviridae or the genus Salterprovirus.The 18 kb dsDNAgenome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome.We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å.Theprotein forms dimers both in solution and in the crystal.The fold of PAV1-137 is a four
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Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi
Archaea, 2013Co-Authors: Nicolas Leulliot, Sophie Quevillon-cheruel, Marc Graille, Claire Geslin, Didier Flament, M. Le Romancer, H. Van TilbeurghAbstract:Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 A. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.
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Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels
Journal of Biological Chemistry, 2012Co-Authors: Christophe Creze, Hannu Myllykallio, Alessio Ligabue, Sébastien Laurent, Roxane Lestini, Sergey P. Laptenok, Joelle Khun, Mirjam Czjzek, Didier FlamentAbstract:Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.
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Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels
Journal of Biological Chemistry, 2012Co-Authors: Christophe Creze, Hannu Myllykallio, Alessio Ligabue, Sébastien Laurent, Roxane Lestini, Sergey P. Laptenok, Mirjam Czjzek, Joëlle Kuhn, Marten Vos, Didier FlamentAbstract:Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.
Journal of Molecular Biology, 2010Co-Authors: Benoît Castrec, Didier Flament, Ghislaine Henneke, Sébastien Laurent, Jean-paul RaffinAbstract:A glycine-rich motif described as being involved in human polymerase delta proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D.
Yannick Gueguen - One of the best experts on this subject based on the ideXlab platform.
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replication factor c from the hyperthermophilic archaeon Pyrococcus abyssi does not need atp hydrolysis for clamp loading and contains a functionally conserved rfc pcna binding domain
Journal of Molecular Biology, 2002Co-Authors: Ghislaine Henneke, Didier Flament, Joël Querellou, Jacques Dietrich, Yannick Gueguen, Philippe Azam, Ulrich Hubscher, Jean-paul RaffinAbstract:The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.
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PCR performance of the highly thermostable proof‐reading B‐type DNA polymerase from Pyrococcus abyssi
Fems Microbiology Letters, 2002Co-Authors: Jacques Dietrich, Jean-luc Rolland, Philippe Schmitt, Montserrat Zieger, Brigitte Preve, Hassan Chaabihi, Yannick GueguenAbstract:DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10−6 mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).
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pcr performance of the highly thermostable proof reading b type dna polymerase from Pyrococcus abyssi
Fems Microbiology Letters, 2002Co-Authors: Jacques Dietrich, Jean-luc Rolland, Philippe Schmitt, Montserrat Zieger, Brigitte Preve, Hassan Chaabihi, Yannick GueguenAbstract:DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10−6 mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).
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Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.
FEBS Journal, 2001Co-Authors: Yannick Gueguen, Didier Flament, Odile Lecompte, Jean-luc Rolland, Jean-paul Raffin, Philippe Azam, Gisèle Le Romancer, Jacques DietrichAbstract:The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.
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Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi.
Applied and Environmental Microbiology, 2001Co-Authors: S. Zappa, Didier Flament, Jean-luc Rolland, Joseph Boudrant, Yannick Gueguen, Jacques DietrichAbstract:This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiolreducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intraor interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.
Heiko Bonisch - One of the best experts on this subject based on the ideXlab platform.
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ultrahigh resolution study on Pyrococcus abyssi rubredoxin ii introduction of an o h sγ fe hydrogen bond increased the reduction potential by 65 mv
Journal of Biological Inorganic Chemistry, 2007Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The effect of D–H···Sγ–Fe hydrogen bonding on the reduction potential of rubredoxin was investigated by the introduction of an O–H···Sγ–Fe hydrogen bond on the surface of Pyrococcus abyssi rubredoxin. The formation of a weak hydrogen bond between Ser44-Oγ and Cys42-Sγ in mutant W4L/R5S/A44S increased the reduction potential by 56 mV. When side effects of the mutation were taken into account, the contribution of the additional cluster hydrogen bond to the reduction potential was estimated to be +65 mV. The structural analysis was based on ultrahigh-resolution structures of oxidized P. abyssi rubredoxin W4L/R5S and W4L/R5S/A44S refined to 0.69 and 0.86 A, respectively.
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Ultrahigh-resolution study on Pyrococcus abyssi rubredoxin: II. Introduction of an O–H···Sγ–Fe hydrogen bond increased the reduction potential by 65 mV
Journal of Biological Inorganic Chemistry, 2007Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The effect of D–H···Sγ–Fe hydrogen bonding on the reduction potential of rubredoxin was investigated by the introduction of an O–H···Sγ–Fe hydrogen bond on the surface of Pyrococcus abyssi rubredoxin. The formation of a weak hydrogen bond between Ser44-Oγ and Cys42-Sγ in mutant W4L/R5S/A44S increased the reduction potential by 56 mV. When side effects of the mutation were taken into account, the contribution of the additional cluster hydrogen bond to the reduction potential was estimated to be +65 mV. The structural analysis was based on ultrahigh-resolution structures of oxidized P. abyssi rubredoxin W4L/R5S and W4L/R5S/A44S refined to 0.69 and 0.86 A, respectively.
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ultrahigh resolution study on Pyrococcus abyssi rubredoxin i 0 69 a x ray structure of mutant w4l r5s
Acta Crystallographica Section D-biological Crystallography, 2005Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The crystal structure of Pyrococcus abyssi rubredoxin mutant W4L/R5S was solved by direct methods. The model of the air-oxidized protein was refined by partially restrained full-matrix least-squares refinement against intensity data to 0.69 A resolution. This first ultrahigh-resolution structure of a rubredoxin provides very detailed and precise information about the Fe(SCys)4 centre and its environment, the peptide-backbone stereochemistry, H atoms and hydrogen bonds, static and dynamic disorder, the solvent structure and the electron-density distribution. P. abyssi rubredoxin W4L/R5S is the first of a series of mutants studied by atomic and ultrahigh-resolution X-ray crystallography which are expected to contribute to the understanding of structure–function relationships in iron–sulfur proteins.
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Ultrahigh-resolution study on Pyrococcus abyssi rubredoxin. I. 0.69 A X-ray structure of mutant W4L/R5S.
Acta Crystallographica Section D-biological Crystallography, 2005Co-Authors: Heiko Bonisch, Christian L Schmidt, Pierre Bianco, Rudolf LadensteinAbstract:The crystal structure of Pyrococcus abyssi rubredoxin mutant W4L/R5S was solved by direct methods. The model of the air-oxidized protein was refined by partially restrained full-matrix least-squares refinement against intensity data to 0.69 A resolution. This first ultrahigh-resolution structure of a rubredoxin provides very detailed and precise information about the Fe(SCys)4 centre and its environment, the peptide-backbone stereochemistry, H atoms and hydrogen bonds, static and dynamic disorder, the solvent structure and the electron-density distribution. P. abyssi rubredoxin W4L/R5S is the first of a series of mutants studied by atomic and ultrahigh-resolution X-ray crystallography which are expected to contribute to the understanding of structure–function relationships in iron–sulfur proteins.
