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Dynamics and loop function analyses of dihydrofolate reductase

Research Project

Project/Area Number 10480159
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionHiroshima University

Principal Investigator

GEKKO Kunihiko  Hiroshima Univ., Graduate School of Science, Professor, 大学院・理学研究科, 教授 (10023467)

Co-Investigator(Kenkyū-buntansha) IWAKURA Masahiro  National Institute of Bioscience and Human-Technology, Research Director, 生命工学工業技術研究所, 室長
OHMAE Eiji  Hiroshima Univ., Graduate School of Science, Research Associate, 大学院・理学研究科, 助手 (30284152)
KATAYANAGI Katsuo  Hiroshima Univ., Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (20291479)
巖倉 正寛  工業技術院, 生命工学工業技術研究所, 室長
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥10,300,000 (Direct Cost: ¥10,300,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥6,100,000 (Direct Cost: ¥6,100,000)
Keywordsdihydrofolate reductase / dynamics / loop function / mutagenesis / アミノ酸置換
Research Abstract

In order to elucidate the structure-dynamics-function relationships of protein, we investigated the effects of mutation and ligand binding of E.Coli dihydrofolate reduactase (DHFR) on the enzymatic function, adiabatic compressibility, and X-ray structure. Single amino acid substitutions at Gly67, Gly121, and Ala145 in three different flexible loops induced large changes in stability and adiabatic compressibility, indicating the modified flexibility of the structure. The B-factor and cavity distribution at sites far from the mutation sites were largely modified by these mutations, indicating that the local changes in structure due to mutation extend to overall of the protein molecule. The compressibility of the mutants increased with increasing the total cavity volume, indicating the importance of atomic packing in generating the protein dynamics. Although the mutation sites are largely separated from the active site, the mutants having a large compressibility showed high enzymatic activity dominantly due to the enhanced catalytic rate. High pressure NMR experiments revealed that the flexible loops and hinge-motion of active-sites including cofactor-binding domain are strongly influenced by pressure and the equilibrium of two conformers is shifted to open form under high pressure. The compressibility of DHFR changed on ligand binding with the change in the cavity volume, reflecting the characteristic flexibility of the intermediates in the reaction coordinate. These results demonstrate that the local structural changes due to mutation and ligand binding affect the dynamics and function of protein molecule through the modified atomic packing (cavity) and that the flexible loops play important role in protein dynamics and function.

Report

(4 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (43 results)

All Other

All Publications (43 results)

  • [Publications] Ohmae,E.: "Nonadditive Effects of Double Mutations at the Flexible Loops, Glycine-67 and Glycine-121, of E.coli Dihydrofolate Reductase on Its Stability and Function."J.Biochem.. 123. 33-41 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ohmae,E.: "Effects of Point Mutation at a Flexible Loop Alanine-145 of Escherichia coli Dihydrofolate Reductase on the Stability and Function."J.Biochem.. 123. 839-846 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwakura,M.: "Topological Mutation of Eschrichia coli Dihydrofolate Reductase."J.Biochem.. 124. 769-777 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kamiyama,T.: "Large Flexibility of Dihydrofolate Reductase as Revealed by Temperature Effects on the Volume and Compressibility."Chem.Lett.. 507-508 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakamura,T.: "Circular Permutation Analysis as a Methods for Distinction of Functional Elements in the M20 Loop of Eschrichia coli Dihydrofolate Reductase."J.Biol.Chem.. 274. 19041-19047 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Katayanagi,K.: "The Flexibility of Protein Molecule in terms of the Crystallography of DHFR Mutants."Spring-8 User Experimental Report. 5A. 304 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kamiyama,T.: "Effects of Ligand Binding on the flexibility of Dihydrofolate Reductase as revealed by Compressibility."Biochim.Biophys.Acta. 1478. 257-266 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko,K.: "Single Amino Acid Substitutions in Flexible Loops can induce Large Compressibility Changes in Dihydrofolate Reductase."J.Biochem.. 128. 21-27 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kitahara,R.: "High Pressure NMR reveals Active-Site Hinge Motion of Folate-Bound Eschrichia coli Dihydrofolate Reductase."Biochemistry. 39. 12789-12795 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwakura,M.: "Systematic Circular Permutation of an Entire Protein Reveals Essential Folding Elements."Nat.Struct.Biol.. 7. 580-585 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] 月向邦彦: "タンパク質の圧縮率と立体構造"高圧力の科学と技術. 10. 101-107 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko,K.: "The Encychlopedia of Molecular Biology"(ed., Creighton, T.E.), Wiley, New York. 8(16) (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko,K.: "Trends in High Pressure Bioscience and Biotechnology"(ed., Hayashi,R.), Elsevier Science B.V.(印刷中). 8(8) (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ohmae, E.: "Nonadditive Effects of Double Mutations at the Flexible Loops, Glycine-67 and Glycine-121, of E.coli Dihydrofolate Reductase on Its Stability and Function."J.Biochem.. 123. 33-41 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ohmae, E.: "Effects of Point Mutation at a Flexible Loop Alanine-145 of Escherichia coli Dihydrofolate Reductase on the Stability and Function."J.Biochem.. 123. 839-846 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwakura, M.: "Topological Mutation of Eschrichia coli Dihydrofolate Reductase."J.Biochem.. 124. 769-777 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kamiyama, T.: "Large Flexibility of Dihydrofolate Reductase as Revealed by Temperature Effects on the Volume and Compressibility."Chem.Lett.. 507-508 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakamura, T.: "Circular Permutation Analysis as a Methods for Distinction of Functional Elements in the M20 Loop Of Eschrichia coli Dihydrofolate Reductase."J.Biol.Chem.. 274. 19041-19047 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Katayanagi, K.: "The Structural Effects of the Flexible Loops on Protein to its Function."Spring-8 User Experimental Report. 5A. 304 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kamiyama, T.: "Effects of Ligand Binding on the flexibility of Dihydrofolate Reductase as revealed by Compressibility."Biochim.Biophys.Acta. 1478. 257-266 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko, K.: "Single Amino Acid Substitutions in Flexible Loops can induce Large Compressibility Changes in Dihydrofolate Reductase."J.Biochem.. 128. 21-27 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kitahara, R.: "High Pressure NMR reveals Active-Site Hinge Motion Of Folate-Bound Eschrichia coli Dihydrofolate Reductase."Biochemistry. 39. 12789-12795 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwakura, M.: "Systematic Circular Permutation of an Entire Protein Reveals Essential Folding Elements."Nat.Struct.Biol.. 7. 580-585 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko, K.: "Compressibility-Structure Relationship of Proteins."Science and Technology of High Pressure. 10 (in Japanese). 101-107 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko, K.: "Accessible Surface, Compressibility, Excluded Volume, Expansibility, Molecular Surface, Volume, Partial Specific (or Molar) Volume, van der Waals Surface. The Encyclopedia of Molecular Biology (ed., Creighton, T.E.)"Wiley, New York. 10-11, 553-555, 873-874, 879, 1530-1531, 1765-1768, 2736-2737 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Gekko, K.: "Effects of Mutation and Ligand Binding on the Compressibility of a Protein. Trends in High Pressure Bioscience and Biotechnology (ed., Hayashi, R.)"Elsevier Science B.V.(in press). 8

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tadashi Kamiyama: "Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility."Biochim.Biophys,Acta. 1478. 257-266 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kunihiko Gekko: "Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase."J.Biochem.. 128. 21-27 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ryo Kitahara: "High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase."Biochemistry. 39. 12789-12795 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Masahiro Iwakura: "Systematic circular permutation of an entire protein reveals essential folding elements,"Nat.Struct.Biol.. 7. 580-585 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tadashi Kamiyama: "Large flexibility of dihydrofocate reductase as revealed by temperature effects on the volume and compressibility"Chem. Lett.. 507-508 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsutomu Nakamura: "Circular permutation analysis as a method for distinction of functional elements in the M20 loop of Escherichia coli dihydrofolate reductase"J. Biol. Chem.. 274. 19041-19047 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tadashi Kamiyama: "Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility"Biochim. Biophys. Acta. (印刷中).

    • Related Report
      1999 Annual Research Report
  • [Publications] Kunihiko Gekko: "The Encyclopedia of Molecular Biology"Wiley. 16 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 月向 邦彦: "水と生命-熱力学から生理学へ-"共立出版. 23 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Eiji Ohmae: "Effects of point mutation at a flexible loop Alanine-145 of Escherichia coli dihydrofolate reductase on the stability and function" J.Biochem.123. 839-846 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Eiji Ohmae: "Nonadditive effects of double mutations at the flexible loops, glycine-67 and glycine-121 of Escherichia coli dihydrofolate reductase on its stability and function" J.Biochem.123. 33-41 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kunihiko Gekko: "Compressibility and volume changes of lysozyme due to inhibitor binding" Chem.Lett.839-840 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Masahiro Iwakura: "Topological mutation of Escherichia coli dihydrofolate reductase" J.Biochem.124. 769-777 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Masahiro Iwakura: "Effects of the length of a glycine linker connecting the N- and C-termini of a circularly permuted dihydrofolate reductase" Protein Eng.11. 707-713 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Masahiro Iwakura: "In search of circular permuted variants of Escherichia coli dihydrofolate reductase" Biosci.Biotechnol.Biochem.62. 778-781 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 月向邦彦: "熱分析ハンドブック" 丸善, 1 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kunihiko Gekko: "The Encyclopedia of Molecular Biology" Wiley(印刷中), (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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