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David M. Lemaster - One of the best experts on this subject based on the ideXlab platform.
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enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures spatial propagation of differential flexibility in rubredoxin hybrids
Proteins, 2005Co-Authors: David M. Lemaster, Diana I Paredes, Jianzhong Tang, Griselda HernándezAbstract:The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in Hyperthermophile proteins below room temperature. Substitution of the Hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14–33Pf) Cp, that exhibits a 12° increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14–33Pf) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14–33Pf) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus Hyperthermophile homologs. Proteins 2005. © 2005 Wiley-Liss, Inc.
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reduced temperature dependence of collective conformational opening in a Hyperthermophile rubredoxin
Biochemistry, 2001Co-Authors: Griselda Hernández, David M. LemasterAbstract:Spatially localized differences in the conformational dynamics of the rubredoxins from the Hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the Hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX2 hydrogen exchange kinetics with conformational opening rates of >1 s-1. Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded β-sheet, while for the rest of the structure, the exchange rates at 23 °C are similar for both proteins. For the multiple-turn region comprising residues 14−32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rig...
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reduced temperature dependence of collective conformational opening in a Hyperthermophile rubredoxin
Biochemistry, 2001Co-Authors: Griselda Hernández, David M. LemasterAbstract:Spatially localized differences in the conformational dynamics of the rubredoxins from the Hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the Hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1). Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins. For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rigidity in the Hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region. The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the Hyperthermophile protein.
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millisecond time scale conformational flexibility in a Hyperthermophile protein at ambient temperature
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Griselda Hernández, Michael W W Adams, Francis E Jenney, David M. LemasterAbstract:Rubredoxin from the Hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10−6 s−1 at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the ≈millisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 104 and 106, for which EX2 exchange kinetics were directly verified, have exchange activation energy values within 2–3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of Hyperthermophile proteins is not supported by these data.
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from the cover millisecond time scale conformational flexibility in a Hyperthermophile protein at ambient temperature
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Griselda Hernández, Michael W W Adams, Francis E Jenney, David M. LemasterAbstract:Rubredoxin from the Hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10−6 s−1 at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the ≈millisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 104 and 106, for which EX2 exchange kinetics were directly verified, have exchange activation energy values within 2–3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of Hyperthermophile proteins is not supported by these data.
Katsuhide Yutani - One of the best experts on this subject based on the ideXlab platform.
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stimulated interaction between α and β subunits of tryptophan synthase from Hyperthermophile enhances its thermal stability
Journal of Biological Chemistry, 2003Co-Authors: Kyoko Ogasahara, Masami Ishida, Katsuhide YutaniAbstract:Tryptophan synthase from Hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the Hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the Hyperthermophile protein than in the mesophile one, indicating that the conformational change of the Hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the Hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.
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x ray crystalline structures of pyrrolidone carboxyl peptidase from a Hyperthermophile pyrococcus furiosus and its cys free mutant
Journal of Biochemistry, 2001Co-Authors: Hideaki Tanaka, Kyoko Ogasahara, Masanobu Chinami, Tsunehiro Mizushima, Motonori Ota, Tomitake Tsukihara, Katsuhide YutaniAbstract:In order to elucidate the mechanism of the thermostability of proteins from Hyperthermophiles, X-ray crystalline structures of pyrrolidone carboxyl peptidase from a Hyperthermophile, Pyrococcus furiosus (PfPCP), and its mutant protein with Ser substituted at Cys142 and Cys188 were determined at 2.2 and 2.7 A resolution, respectively. The obtained structures were compared with those previously reported for pyrrolidone carboxyl peptidases from a hyperthermophilie, Thermococcus litoralis (TlPCP), and from a mesophile, Bacillus amyloliquefaciens (BaPCP). The PfPCP structure is a tetramer of four identical subunits similar to that of the TlPCP and BaPCP. The largest structural changes among the three PCPs were detected in the C-terminal protrusion, which interacts with that of another subunit. A comparison of the three structures indicated that the high stability of PfPCP is caused by increases in hydrophobic interactions and hydrogen bonds, the formation of an intersubunit ion-pair network, and improvement to an ideal conformation. On the basis of the structures of the three proteins, it can be concluded that PfPCP does not have any special factors responsible for its extremely high stability and that the conformational structure of PfPCP is superior in its combination of positive and negative stabilizing factors compared with BaPCP.
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crystal structure of methionine aminopeptidase from Hyperthermophile pyrococcus furiosus
Journal of Molecular Biology, 1998Co-Authors: Tahir H Tahirov, Susumu Tsunasawa, Kyoko Ogasahara, Katsuhide Yutani, Tomitake Tsukihara, Hideyuki Oki, Kazuhiro Ogata, Yukiko Izu, Ikunoshin KatoAbstract:Abstract The structure of methionine aminopeptidase from Hyperthermophile Pyrococcus furiosus (PfMAP) with an optimal growth temperature of 100°C was determined by the multiple isomorphous replacement method and refined in three different crystal forms, one monoclinic and two hexagonal, at resolutions of 2.8, 2.9, and 3.5 A. The resolution of the monoclinic crystal form was extended to 1.75 A by water-mediated transformation to a low-humidity form, and the obtained diffraction data used for high-resolution structure refinement. This is the first description of a eukaryotic type methionine aminopeptidase structure. The PfMAP molecule is composed of two domains, a catalytic domain and an insertion domain, connected via two antiparallel β-strands. The catalytic domain, which possesses an internal 2-fold symmetry and contains two cobalt ions in the active site, resembles the structure of a prokaryotic type MAP from Escherichia coli (EcMAP), while the structure of the insertion domain containing three helices has a novel fold and accounts for a major difference between the eukaryotic and prokaryotic types of methionine aminopeptidase. Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP: (1) a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; (2) an increased number of hydrogen bonds between the positively charged side-chain and neutral oxygen; (3) a larger number of buried water molecules involved in crosslinking the backbone atoms of sequentially separate segments; (4) stabilization of two antiparallel β-strands connecting the two domains of the molecule by proline residues; (5) shortening of N and C-terminal tails and stabilization of the loop c3E by deletion of three residues.
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electrostatic stabilization in methionine aminopeptidase from Hyperthermophile pyrococcus furiosus
Biochemistry, 1998Co-Authors: Kyoko Ogasahara, Susumu Tsunasawa, Ikunoshin Kato, Yukiko Izu, Elena A Lapshina, Miyo Sakai, Katsuhide YutaniAbstract:The thermostability of methionine aminopeptidase from a Hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 °C at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (ΔH) measured at different pHs linearly correlated with Tm up to 102 °C, suggesting that the denaturation heat capacity (ΔCp) for PfMAP is constant up to 100 °C. ΔCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10−100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasin...
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The Unusually Slow Unfolding Rate Causes the High Stability of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile, Pyrococcus furiosus: Equilibrium and Kinetic Studies of Guanidine Hydrochloride-Induced Unfolding and Refolding†
Biochemistry, 1998Co-Authors: Kyoko Ogasahara, Susumu Tsunasawa, Mamoru Nakamura, Satomi Nakura, Ikunoshin Kato, Tadashi Yoshimoto, Katsuhide YutaniAbstract:To elucidate the energetic features of the anomalously high-level stabilization of a Hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, ...
Griselda Hernández - One of the best experts on this subject based on the ideXlab platform.
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enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures spatial propagation of differential flexibility in rubredoxin hybrids
Proteins, 2005Co-Authors: David M. Lemaster, Diana I Paredes, Jianzhong Tang, Griselda HernándezAbstract:The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in Hyperthermophile proteins below room temperature. Substitution of the Hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14–33Pf) Cp, that exhibits a 12° increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14–33Pf) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14–33Pf) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus Hyperthermophile homologs. Proteins 2005. © 2005 Wiley-Liss, Inc.
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reduced temperature dependence of collective conformational opening in a Hyperthermophile rubredoxin
Biochemistry, 2001Co-Authors: Griselda Hernández, David M. LemasterAbstract:Spatially localized differences in the conformational dynamics of the rubredoxins from the Hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the Hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX2 hydrogen exchange kinetics with conformational opening rates of >1 s-1. Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded β-sheet, while for the rest of the structure, the exchange rates at 23 °C are similar for both proteins. For the multiple-turn region comprising residues 14−32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rig...
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reduced temperature dependence of collective conformational opening in a Hyperthermophile rubredoxin
Biochemistry, 2001Co-Authors: Griselda Hernández, David M. LemasterAbstract:Spatially localized differences in the conformational dynamics of the rubredoxins from the Hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the Hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1). Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins. For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rigidity in the Hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region. The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the Hyperthermophile protein.
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millisecond time scale conformational flexibility in a Hyperthermophile protein at ambient temperature
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Griselda Hernández, Michael W W Adams, Francis E Jenney, David M. LemasterAbstract:Rubredoxin from the Hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10−6 s−1 at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the ≈millisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 104 and 106, for which EX2 exchange kinetics were directly verified, have exchange activation energy values within 2–3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of Hyperthermophile proteins is not supported by these data.
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from the cover millisecond time scale conformational flexibility in a Hyperthermophile protein at ambient temperature
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Griselda Hernández, Michael W W Adams, Francis E Jenney, David M. LemasterAbstract:Rubredoxin from the Hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10−6 s−1 at 100°C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the ≈millisecond time frame or faster at 28°C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 104 and 106, for which EX2 exchange kinetics were directly verified, have exchange activation energy values within 2–3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of Hyperthermophile proteins is not supported by these data.
Kyoko Ogasahara - One of the best experts on this subject based on the ideXlab platform.
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stimulated interaction between α and β subunits of tryptophan synthase from Hyperthermophile enhances its thermal stability
Journal of Biological Chemistry, 2003Co-Authors: Kyoko Ogasahara, Masami Ishida, Katsuhide YutaniAbstract:Tryptophan synthase from Hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (22) composed of and 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the and subunits in the Hyperthermophile protein were of the order of 108 M1, which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the Hyperthermophile protein than in the mesophile one, indicating that the conformational change of the Hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the subunit from the Hyperthermophile was enhanced by 17 degrees C due to the formation of the 22 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.
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x ray crystalline structures of pyrrolidone carboxyl peptidase from a Hyperthermophile pyrococcus furiosus and its cys free mutant
Journal of Biochemistry, 2001Co-Authors: Hideaki Tanaka, Kyoko Ogasahara, Masanobu Chinami, Tsunehiro Mizushima, Motonori Ota, Tomitake Tsukihara, Katsuhide YutaniAbstract:In order to elucidate the mechanism of the thermostability of proteins from Hyperthermophiles, X-ray crystalline structures of pyrrolidone carboxyl peptidase from a Hyperthermophile, Pyrococcus furiosus (PfPCP), and its mutant protein with Ser substituted at Cys142 and Cys188 were determined at 2.2 and 2.7 A resolution, respectively. The obtained structures were compared with those previously reported for pyrrolidone carboxyl peptidases from a hyperthermophilie, Thermococcus litoralis (TlPCP), and from a mesophile, Bacillus amyloliquefaciens (BaPCP). The PfPCP structure is a tetramer of four identical subunits similar to that of the TlPCP and BaPCP. The largest structural changes among the three PCPs were detected in the C-terminal protrusion, which interacts with that of another subunit. A comparison of the three structures indicated that the high stability of PfPCP is caused by increases in hydrophobic interactions and hydrogen bonds, the formation of an intersubunit ion-pair network, and improvement to an ideal conformation. On the basis of the structures of the three proteins, it can be concluded that PfPCP does not have any special factors responsible for its extremely high stability and that the conformational structure of PfPCP is superior in its combination of positive and negative stabilizing factors compared with BaPCP.
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crystal structure of methionine aminopeptidase from Hyperthermophile pyrococcus furiosus
Journal of Molecular Biology, 1998Co-Authors: Tahir H Tahirov, Susumu Tsunasawa, Kyoko Ogasahara, Katsuhide Yutani, Tomitake Tsukihara, Hideyuki Oki, Kazuhiro Ogata, Yukiko Izu, Ikunoshin KatoAbstract:Abstract The structure of methionine aminopeptidase from Hyperthermophile Pyrococcus furiosus (PfMAP) with an optimal growth temperature of 100°C was determined by the multiple isomorphous replacement method and refined in three different crystal forms, one monoclinic and two hexagonal, at resolutions of 2.8, 2.9, and 3.5 A. The resolution of the monoclinic crystal form was extended to 1.75 A by water-mediated transformation to a low-humidity form, and the obtained diffraction data used for high-resolution structure refinement. This is the first description of a eukaryotic type methionine aminopeptidase structure. The PfMAP molecule is composed of two domains, a catalytic domain and an insertion domain, connected via two antiparallel β-strands. The catalytic domain, which possesses an internal 2-fold symmetry and contains two cobalt ions in the active site, resembles the structure of a prokaryotic type MAP from Escherichia coli (EcMAP), while the structure of the insertion domain containing three helices has a novel fold and accounts for a major difference between the eukaryotic and prokaryotic types of methionine aminopeptidase. Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP: (1) a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; (2) an increased number of hydrogen bonds between the positively charged side-chain and neutral oxygen; (3) a larger number of buried water molecules involved in crosslinking the backbone atoms of sequentially separate segments; (4) stabilization of two antiparallel β-strands connecting the two domains of the molecule by proline residues; (5) shortening of N and C-terminal tails and stabilization of the loop c3E by deletion of three residues.
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electrostatic stabilization in methionine aminopeptidase from Hyperthermophile pyrococcus furiosus
Biochemistry, 1998Co-Authors: Kyoko Ogasahara, Susumu Tsunasawa, Ikunoshin Kato, Yukiko Izu, Elena A Lapshina, Miyo Sakai, Katsuhide YutaniAbstract:The thermostability of methionine aminopeptidase from a Hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 °C at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (ΔH) measured at different pHs linearly correlated with Tm up to 102 °C, suggesting that the denaturation heat capacity (ΔCp) for PfMAP is constant up to 100 °C. ΔCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10−100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasin...
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The Unusually Slow Unfolding Rate Causes the High Stability of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile, Pyrococcus furiosus: Equilibrium and Kinetic Studies of Guanidine Hydrochloride-Induced Unfolding and Refolding†
Biochemistry, 1998Co-Authors: Kyoko Ogasahara, Susumu Tsunasawa, Mamoru Nakamura, Satomi Nakura, Ikunoshin Kato, Tadashi Yoshimoto, Katsuhide YutaniAbstract:To elucidate the energetic features of the anomalously high-level stabilization of a Hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, ...
Sunghou Kim - One of the best experts on this subject based on the ideXlab platform.
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structure based experimental confirmation of biochemical function to a methyltransferase mj0882 from Hyperthermophile methanococcus jannaschii
Journal of Structural and Functional Genomics, 2002Co-Authors: Lan Huang, Rosalind Kim, Sunghou Kim, Hisao Yokota, Liwei Hung, Mark OdellAbstract:We have determined the three-dimensional (3-D) structure of protein MJ0882, which derives from a hypothetical open reading frame in the genome of the Hyperthermophile Methanococcus jannaschii. The 3-D fold of MJ0882 at 1.8 A highly resembles that of a methyltransferase, despite limited sequence similarity to any confirmed methyltransferase. The structure has an S-adenosylmethionine (AdoMet) binding pocket surrounded by motifs with similarities to those commonly found among AdoMet binding proteins. Preliminary biochemical experiments show that MJ0882 specifically binds to AdoMet, which is the essential co-factor for methyltransferases.
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crystal structure of a fibrillarin homologue from methanococcus jannaschii a Hyperthermophile at 1 6 a resolution
The EMBO Journal, 2000Co-Authors: Hongming Wang, David C Boisvert, Kyeong Kyu Kim, Rosalind Kim, Sunghou KimAbstract:Fibrillarin is a phylogenetically conserved protein essential for efficient processing of pre-rRNA through its association with a class of small nucleolar RNAs during ribosomal biogenesis. The protein is the antigen for the autoimmune disease scleroderma. Here we report the crystal structure of the fibrillarin homologue from Methanococcus jannaschii, a Hyperthermophile, at 1.6 A resolution. The structure consists of two domains, with a novel fold in the N-terminal region and a methyltransferase-like domain in the C-terminal region. Mapping temperature-sensitive mutations found in yeast fibrillarin Nop1 to the Methanococcus homologue structure reveals that many of the mutations cluster in the core of the methyltransferase-like domain.
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small heat shock protein of methanococcus jannaschii a Hyperthermophile
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Rosalind Kim, Kyeong Kyu Kim, Hisao Yokota, Sunghou KimAbstract:Small heat shock proteins (sHSPs) belong to a family of 12- to 43-kDa proteins that are ubiquitous and are conserved in amino acid sequence among all organisms. A sHSP homologue of Methanococcus jannaschii, a hyperthermophilic Archaeon, forms a homogeneous multimer comprised of 24 monomers with a molecular mass of 400 kDa in contrast to other sHSPs that show heterogeneous oligomeric complexes. Electron microscopy analysis revealed a spherically shaped oligomeric structure ≈15–20 nm in diameter. The protein confers thermal protection of other proteins in vitro as found in other sHSPs. Escherichia coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C. Similar results were obtained when purified sHSP protein was added to an E. coli cell lysate. The protein also prevented the aggregation of two purified proteins: single-chain monellin (SCM) at 80°C and citrate synthase at 40°C.
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the crystal structure of an fe superoxide dismutase from the Hyperthermophile aquifex pyrophilus at 1 9 a resolution structural basis for thermostability
Journal of Molecular Biology, 1997Co-Authors: Jae Hwan Lim, Sunghou Kim, Ye Sun Han, Seung J Cho, Byung Yoon Ahn, Yunje ChoAbstract:Superoxide dismutase (SOD) from Aquifex pyrophilus, a hyperthermophilic bacterium, is an extremely heat-stable enzyme that maintains about 70% of its activity after heat treatment for 60 minutes at 100°C. To understand the molecular basis of thermostability of this enzyme, we have determined the crystal structure of A. pyrophilus superoxide dismutase (Ap SOD), an Fe containing homotetrameric enzyme, at 1.9 A resolution, and compared it with SOD structures from a mesophile and a thermophile, and other enzyme structures from other Hyperthermophiles. The structure has been refined to a crystallographic R-factor (I > 2σ) of 17.0% and R-free (I > 2σ) of 19.9%. While the overall structure of the Ap SOD monomer is similar to the other SODs, significant conformational differences are observed in a highly variable loop region and the C-terminal helix. The conformational differences in these regions alter the subunit arrangement of this enzyme and generate a very compact tetramer. Structural comparisons of three SODs have revealed that Ap SOD has some stabilizing features at both the tertiary and the quaternary structural level: The Ap SOD monomer contains a large number of ion-pairs and the Ap SOD tetramer has a dramatically increased buried surface area per monomer. Comparisons of the Ap SOD structure with that of other known enzymes from Hyperthermophiles reveal that the increased number of intrasubunit ion-pairs is a common feature.