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Folding mechanism of a protein from a hyperthermophile with unusually slow folding rates

Research Project

Project/Area Number 17570102
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionRIKEN

Principal Investigator

YUTANI Katsuhide  RIKEN, Protein Crystallographic Methodology Team, Senior Scientist, 構造解析高度化研究チーム, 上級研究員 (90089889)

Co-Investigator(Kenkyū-buntansha) SAWANO Masahide  RIKEN, Protein Crystallographic Methodology Team, Research Associate, 構造解析高度化研究チーム, リサーチアソシエイト (10415177)
SEGAWA Shin-ichi  Kansei Gakuin University, School of Science and Technology, Professor, 理工学部, 教授 (70103132)
Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
KeywordsProtein Folding / Denatured Structure of a Protein / Hyperthermophile / NMR / Protein Stability / α-Helix / Pyrrolidone Carboxyl Peptidase / Mutant Protein / 蛋白質の変性 / CD / 蛋白質の変異型
Research Abstract

We have hound that the refolding reaction of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus, is unusually slow at acidic pH and can be controlled by regulating the incubation temperature ; the refolding reaction significantly stops at pH 2.3 and 4 ℃. In this period, in order to elucidate the folding mechanism of a protein from a hyperthermophile with unusually slow folding rates, we have completed two papers using cysteine-free PCP (PCP-OSH) with unusually slow refolding rates. PCP-OSH was used in place of PCP to avoid troubles due to the formation of disulfide bonds. (1) PCPs from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core ; in contrast, the corresponding residue in mesophile protein is a hydrophobic residue, He. To elucidate the role of the buried Glu in stability and folding rates of PCP from hyperthermophiles, we examined changes in stability and structure due to mutations at Glu192. The results indicated that completely buried Glu192 contributes to stabilization of PCP-OSH due to the formation of strong intramolecular hydrogen bonds, and the hydrogen bonds by the non-ionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins (Kausahik et al., 2006). (2) The above denatured state (D_1 state) of PCP corresponds to the denatured structure that exists in equilibrium with the native state under physiological conditions. To elucidate the structural basis of the D_1 state, H/D exchange experiments with PCP-OSH were performed at pD 3.4 and 4 ℃. The results indicated that amide protons in the C-terminal a6-helix region hardly exchanged in the D_1 state with deuterium even after 7 days, suggesting that the a6-helix (from Ser188 to Glu205) of PCP-OSH was stably formed in the D_1 state (Iimura et al., 2007).

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (24 results)

All 2007 2006 2005 Other

All Journal Article (22 results) Book (2 results)

  • [Journal Article] Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile under Non-denaturing Conditions : Role of the C-terminal a-helix of the Protein in Folding and Stability.2007

    • Author(s)
      Iimura, S. et al.
    • Journal Title

      Biochemistry 46(In press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile under Non-denaturing Conditions : Role of the C-terminal a-helix of the Protein in Folding and Stability.2007

    • Author(s)
      Satoshi Iimura, Taro Umezaki, Makoto Takeuchi, Mineyuki Mizuguchi, Hiromasa Yagi, Kyoko Ogasahara, Hideo Akutsu, Yasuo Noda, Shin-ichi Segawa, Katsuhide Yutani
    • Journal Title

      Biochemistry (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Characterization of the Denatured Structure of Pyrrolidone Carboxyl Peptidase from a Hyperthermophile under Non-denatureing Conditions : Role of the C-terminal α-helix of the protein in Folding and Stability.2007

    • Author(s)
      Iimura, S. et al.
    • Journal Title

      Biochemistry 46(In press)

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Completely-buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles.2006

    • Author(s)
      Kaushik, J., et al.
    • Journal Title

      Biochemistry 45

      Pages: 7100-7112

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 ℃2006

    • Author(s)
      Tanaka, T. et al.
    • Journal Title

      FEBS Letters 580

      Pages: 4224-4230

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Completely-buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles.2006

    • Author(s)
      Jai K.Kaushik, Satoshi Iimura, Kyoko Ogasahara, Yuriko Yamagata, Shin-ichi Segawa, Katsuhide Yutani
    • Journal Title

      Biochemistry 45

      Pages: 7100-7112

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 °C2006

    • Author(s)
      Tomoyuki Tanaka, Masahide Sawano, Kyoko Ogasahara, Yasushi Sakaguchi, Bagautdin Bagautdinov, Etsuko Katoh, Chizu Kuroishi, Akeo Shinkai, Shigeyuki Yokoyama, Katsuhide Yutani
    • Journal Title

      FEBS Letters 580

      Pages: 4224-4230

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Crystal structure of alanyl-tRNA synthetase editing domain homolog (PH0574) from a hyperthermophile, Pyrococcus horikoshi OT3 at 1.45 Å resolution.2006

    • Author(s)
      Ishijima, J. et al.
    • Journal Title

      Proteins 62

      Pages: 1133-1137

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Completely-vuried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles2006

    • Author(s)
      Kaushik, J., et al.
    • Journal Title

      Biochemistry 45

      Pages: 7100-7112

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Hyper-thermostability of CutAl protein, with a denaturation temperature of nearly 150℃2006

    • Author(s)
      Tanaka, T. et al.
    • Journal Title

      FEBS Letters, 580

      Pages: 4224-4230

    • Related Report
      2006 Annual Research Report
  • [Journal Article] 約150℃まで熱安定な超好熱菌由来のタンパク質2006

    • Author(s)
      澤野雅英, 油谷克英
    • Journal Title

      Bionics[バイオニクス] No.24

      Pages: 68-69

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Conformational changes in the α-subunit coupled to binding of the β_2-subunit of tryptophan synthase from Escherichia coli : Crystal structure of tryptophan synthase α-subunit alone2005

    • Author(s)
      Nishio, K., et al.
    • Journal Title

      Biochemistry 44

      Pages: 1184-1192

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Stabilization Mechanism of the Tryptophan Synthase α-Subunit from Thermus thermophilus HB8 : X-Ray Crystallographic Analysis and Calorimetry.2005

    • Author(s)
      Asada, Y. et al.
    • Journal Title

      J. Biochem. 138

      Pages: 343-353

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Conformational Changes in the Tryptophan Synthase from a Hyperthermophile upon α_2β_2 Complex Formation : Crystal Structure of the Complex.2005

    • Author(s)
      Lee, S.J.et al.
    • Journal Title

      Biochemistry 44

      Pages: 11727-11427

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary 2005 Annual Research Report
  • [Journal Article] Conformational changes in the α-subunit coupled to binding of the (32-subunit of tryptophan synthase from Escherichia coli : Crystal structure of tryptophan synthase α-subunit alone.2005

    • Author(s)
      Kazuya Nishio, Yukio Morimoto, Manabu Ishizuka, Kyoko Ogasahara, Tomitake Tsukihara, Katsuhide Yutani
    • Journal Title

      Biochemistry 44

      Pages: 1184-1192

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Stabilization Mechanism of the Tryptophan Synthase α-Subunit from Thermus thermophilus HB8 : X-Ray Crystallographic Analysis and Calorimetry.2005

    • Author(s)
      Yukuhiko Asada, Masahide Sawano, Kyoko Ogasahara, Junji Nakamura, Motonori Ota, Chizu Kuroishi, Mitsuaki Sugahara, Katsuhide Yutani, Naoki Kunishima
    • Journal Title

      J. Biochem. 138

      Pages: 343-353

    • NAID

      10017347311

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Conformational changes in the α-subunit coupled to binding of the β_2-subnit of tryptophan synthase from Escherichia coli : Crystal structure of tryptophan synthase α-subunit alone2005

    • Author(s)
      Nishio, K., et al.
    • Journal Title

      Biochemistry 44

      Pages: 1184-1192

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Stabilization due to Dimer Formation of Phosphoribosyl Anthranilate Isomerase from Thermus thermophilus HB8 : X-Ray Analysis and DSC Experiments2005

    • Author(s)
      Taka, J.et al.
    • Journal Title

      J.Biochem. 137

      Pages: 569-578

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Stabilization Mechanism of the Tryptophan Synthase α-Subunit from Thermus thermophilus HB8 : X-Ray Crystallographic Analysis and Calorimetry.2005

    • Author(s)
      Asada, Y., et al.
    • Journal Title

      J.Biochem. 138

      Pages: 343-353

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Purification, crystallization and preliminary X-ray crystallographic study of the L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB82005

    • Author(s)
      Jeyakantahn, J.et al.
    • Journal Title

      Acta Cryst.F 61

      Pages: 1075-1077

    • Related Report
      2005 Annual Research Report
  • [Journal Article] 蛋白質立体構造から安定化のメカニズムを定量的に理解する方法2005

    • Author(s)
      舩橋順, 油谷克英
    • Journal Title

      日本結晶学会誌 47

      Pages: 253-260

    • NAID

      130000800519

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Conformational Changes in the Tryptophan Synthase from a Hyperthermophile upon α_2β_2 Complex Formation : Crystal Structure of the Complex.

    • Author(s)
      Soo Jae Lee, Kyoko Ogasahara, Jichun Ma, Kazuya Nishio, Masami Ishida, Yuriko Yamagata, Tomitake Tsukihara, Katsuhide Yutani
    • Journal Title

      Biochemistry 44

      Pages: 11727-11427

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Book] 生物工学ハンドブック(日本生物工学会編)(コロナ社)2005

    • Author(s)
      黒木良太など
    • Publisher
      蛋白質の立体構造、蛋白質の安定性、蛋白質の構造解析・構造予測技術
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Book] 生物工学ハンドブック(日本生物工学会編)(コロナ社)2005

    • Author(s)
      黒木良太, 玉田太郎, 油谷克英
    • Publisher
      蛋白質の立体構造、蛋白質の安定性、蛋白質の構造解析・構造予測技術
    • Related Report
      2005 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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