• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to project page

2000 Fiscal Year Final Research Report Summary

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)
Project Period (FY) 1999 – 2000
Keywordsprotein crystallization / dynamic light scattering / crystallizability / X-ray crystallography / transition-state analogue / protein chemistry / γ-glutamylcysteine synthetase / asparagine synthetase
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.

  • Research Products

    (12 results)

All Other

All Publications (12 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

URL: 

Published: 2002-03-26  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi