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Development of practical strategies to support protein crystallization for X-ray crystallography

Research Project

Project/Area Number 10558098
Research Category

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

Allocation TypeSingle-year Grants
Section展開研究
Research Field Structural biochemistry
Research InstitutionRIKEN (1999-2000)
Kyoto University (1998)

Principal Investigator

KATO Hiroaki  RIKEN, Kinetic Crystallography Research Team, Team Leader, 速度論的結晶学研究チーム, チームリーダー(研究職) (90204487)

Co-Investigator(Kenkyū-buntansha) YAMANO Akihito  RIGAKU Corporation, X-Ray Research laboratory, Scientist, X線研究所, 研究員
HIRATAKE Jun  Kyoto University, Institute for Chemical Research, Associate Professor, 化学研究所, 助教授 (80199075)
MAEDA Yuichiro  RIKEN, Structural Biochemistry Laboratory, Chief Scientist, 構造生物化学研究室, 主任研究員 (10321811)
中津 享  京都大学, 化学研究所, 教務職員 (50293949)
Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥9,200,000 (Direct Cost: ¥9,200,000)
Keywordsprotein crystallization / dynamic light scattering / crystallizability / X-ray crystallography / transition-state analogue / protein chemistry / γ-glutamylcysteine synthetase / asparagine synthetase / γグルタミルシステイン合成酵素 / 抗菌性タンパク質
Research Abstract

In this study, we have applied dynamic light scattering analysis to evaluate crystallizability of proteins. We also tried to explore practical strategy to alter the crystallizability when a protein preparation showed poor crystallizability. Finaly, we applied the strategy to some proteins that have not been crystallized yet. We did to crystallize all proteins that we tried.
The following results were archived.
1. We altered the crystallizability of pathogen related protein 5d (PR-5d) and determined its crystal structure at 1.8Å resolution.
2. We crystallized two tropinone reductases and solved their three-dimensional structures by multiple isomorphous replacement methods independently. The results implicated the structural basis for their stereospecific reaction. We also investigated their stereospecificity by site-directed mutagenesis.
3. We also succeed to synthesize its transition-state analogue inhibitor and the crystal structure of the enzyme complexed with the inhibitor was determined.
4. We succeed to crystallize endopolygalacturonase from a pathogenic fungus, Stereum purpureum and pyruvate phosphate dikinase from maiz. The crystals of the endopolygalacturonase diffracted X-ray to 0.95Å resolution.
5. We succeed to crystallize g-glutamylcysteine synthetase by alteration of its surface cysteine residues into serine residues. We also synthesized its transition state analogue inhibitors. Kinetic analysis using the inhibitors suggested some structural motives of the active site architecture of this enzyme.

Report

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

    (21 results)

All Other

All Publications (21 results)

  • [Publications] Tokutake,Nobuya: "Design,Synthesis and Evaluation of Transition-state Analogue Inhibitors of Escherichia colig-Glutamylcysteine Synthetase"Bioorg.Med.Chem.. 6. 1935-1953 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima,Keiji: "Crystal Structures of Two Tropinone Reductases : Different Reaction Stereospecificities in the same protein fold"Proc.Natl.Acad.Soc.USA. 95. 4876-4881 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Koiwa,H.: "Crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein at 1.8Å resolution."J.Mol.Biol.. 286. 1137-1145 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamashit,a,Atsuko: "Structure of tropinone reductase-II complexed with NADP+ and y-tropine at 1.9Å resolution : Imprication for Stereospecific catalysis."Biochemistry. 38. 7630-7637 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Koizumi,M.: "A Potent Transition-State Analogue Inhibitor of Escherichia coli Asparagine Synthetase A."J.Am.Chem.Soc.. 121. 5799-5800 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima,Keiji: "Site-directed Mutagenesis of Putative Substrate-binding Residues Reveals a Mechanism Controlling the Different Stereospecificities of Two Tropinone Reductases."J.Biol.Chem.. 274. 16563-16568 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tokutake, Nobuya: "Design, Synthesis and Evaluation of Transition-state Analogue Inhibitors of Escherichia coli g-Glutamylcysteine Synthetase"Bioorg.Med.Chem.. 6. 1935-1953 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima, Keiji: "Crystal Structures of Two Tropinone Reductases : Different Reaction Stereospecificities in the same protein fold"Proc.Natl.Acad.Soc.USA. 95. 4876-4881 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Koiwa, H.: "Crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein at 1.8Å resolution."J.Mol.Biol.. 286. 1137-1145 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamashit, a, Atsuko: "Structure of tropinone reductase-II complexed with NADP+ and y-tropine at 1.9Å resolution : Imprication for Stereospecific catalysis."Biochemistry. 38. 7630-7637 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Koizumi, M.: "A Potent Transition-State Analogue Inhibitor of Escherichia coli Asparagine Synthetase A."J.Am.Chem.Soc.. 121. 5799-5800 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakajima, Keiji: "Site-directed Mutagenesis of Putative Substrate-binding Residues Reveals a Mechanism Controlling the Different Stereospecificities of Two Tropinone Reductases."J.Biol.Chem.. 274. 16563-16568 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hisashi Koiwa: "Crystal structure of tobacco PR-5d, an antifungal thaumatin like protein at 1.8 Å resolution"Journal of Molecular Biology. 286. 1137-1145 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Atsuko Yamashita: "Structure of tropinone reductase-II complexed with NADP^+ and Ψ-tropine at 1.9 Å resolution : Imprication for Stereospecific catalysis"Biochemistry. 38. 7630-7637 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Mitsuteru Koizumi: "A Potent Transition-State Analogue Inhibitor of Escherichia coli Asparagine Synthetase A"Journal of American Chemical Society. 121. 5799-5800 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Keiji Nakajima: "Site-directed Mutagenesis of Putative Substrate-binding Residues Reveals a Mechanism Controlling the Different Stereospecificities of Two Tropinone Reductases"Journal of Biological Chemistry. 274. 16563-16568 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tokutake, Nobuya: "Design, Synthesis and Evaluation of Transition-state Analogue Inhibitors of Escherichia coli γ-Glutamylcysteine Synthetase" Bioorg.& Med.Chem.6. 1935-1953 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Katoh, Makoto: "ATP-Dependent Inactivation of Escherichia coli γ-Glutamylcysteine Synthetase by l-Glutamic Acid γ-Monohydroxamate" Biosci.Biotech.Biochem.62. 1455-1457 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Tokutake, Nobuya: "Absolute Configuration of an Intermediate Cyclic Sulfoximine in the Asymmetiric Synthesis of Transition-state Analogue Inhibitors of γ-Glutamylcysteine Synthetase" Acta Crystallogr.C. in press. (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Nakajima, Keiji: "Crystal structures of two tropinone reductases:different reaction stereospecificities in the same protein fold" Proc.Nat.Acad.Soc.USA. 95. 4876-4881 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Yamashita, Aatsuko: "Crystallization and preliminary X-ray study of tropinone reductase II." Acta Crystallgr.D54. 1405-1407 (1998)

    • Related Report
      1998 Annual Research Report

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

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