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Development of time-resolved spectroscopic observation systems for the fast dynamics of protein folding

Research Project

Project/Area Number 12558081
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Biophysics
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

TAKAHASHI Satoshi  kyoto University, Graduate School of Engineering, Instructor, 工学研究科, 助手 (30283641)

Co-Investigator(Kenkyū-buntansha) ISHIMORI Koichiro  kyoto University, Graduate School of Engineering, Assistant Professor, 工学研究科, 助教授 (20192487)
HARADA Yoshiyuki  Shimadzu Corporation, 分析機器事業部, 副主任研究員
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 2001: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥7,600,000 (Direct Cost: ¥7,600,000)
Keywordsprotein folding / time-resoloved observation / rapid mixing device / CD spectrometer / cytochrome c / poly-L-glutamic acid / apomyoglobin / 折れ畳み過程 / 高速分光システム / CD分光法
Research Abstract

We developed the following original instruments to observe the early dynamics of protein folding. 1) The rapid mixing device that can initiate protein folding within 50 μs. The previous mixing devices has the mixing time of 300〜400 μs. Thus, we achieved to reduce the mixing times to nearly an order shorter. 2) We constructed to the observation system for the CD spectroscopy based on the prism polychrometer and a CCD detector. We resolved several problems associated with the new ideas of spectroscopic observation system, such as the effect of optical retardation. The developed system enabled us to observe the circular dichroism spectra with a high S/N ratio based on the multichannel detection. 3) We tried to combine the developped flow cell system and the spectroscopic observation system ; however, faild to observe reliable data for the repid-mixing cell. We are currently working to reduce the optical retardations of the cells.
Using the developed flow cell systems, we obtained the follo … More wing results that were already reported in scientific journals. 1) We observed time-resolved small-angle X-ray scattering profiles for the protein folding dynamics of cytochrome c. The developed cell enabled us to obtain information on the early submillisecond phase of cytohrome c folding that were inaccessible by the conventional devices. We conclude that the protein compaction and the secondary structure formations proceed simultaneously in the folding process of cytochrome c. 2) We observed the helix formation processes of polyglutamic acid using the developed rapid-mixing cell. We demonstrated that a short helix formation precedes the longer helix formation process in the PGA helix formation. This is a new dynamical event observed in the helix formation of polypeptides. 3) The helix-formation and the collapse process of apomyoglobin were observed using the developed rapid mixing systems in the submillisecond time domain. We clarified that the apomyoglobin folding proceed as a stepeise process that is similar to the observation for cytochrome c. Less

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (17 results)

All Other

All Publications (17 results)

  • [Publications] 高橋 聡: "蛋白質の折り畳み研究の展開"生化学. 73. 54-59 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kimura, T. et al.: "Direct observation of the multi-step helix formation of poly-L-glutamic acids"J. Am. Chem. Soc.. 124. 11596-11597 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Akiyama, S. et al.: "Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering"Proc. Natl. Acad. Sci. USA. 99. 1329-1334 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 高橋 聡 他一名: "蛋白質の折り畳み運動を捉える"蛋白質・核酸・酵素 増刊「新世紀における蛋白質科学の進展」. 46. 1545-1552 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Akiyama, S. et al.: "Stepwise formation of α-helices during cytochrome c folding"Nat. Struct. Biol.. 7. 514-520 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 高橋 聡 他一名: "「顕微赤外分光法と高速溶液混合装置を用いた時分割赤外分光システムの開発」"(株)アイピーシー 「顕微赤外分光法」(共著). 10 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Akiyama, S. et. al.: "Stepwise formation of α-helices during cytochrome c folding"Nat. Struct. Biol.. 7. 514-520 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takahashi, S. et. al.: "Experimental studies on the folding dynamics of proteins (Japanese)"Protein, Nucleic Acid, and Enzyme. 46. 1545-1552 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Akiyama, S. et. al.: "Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle X-ray scattering"Proc. Natl. Acad. Sci. U.S.A.. 99. 1329-1334 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kimura, T. et. al.: "Direct Observation of the Multi-step Helix Formation of Poly-L-glutamic acids"J. Am. Chem. Soc.. 124. 11596-11597 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takahashi S.: "Recent progress in protein folding research (Japanese)"Seikagaku. 75. 54-59 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takahashi S. et. al.: "Studies on proten folding dynamics using IR microscope and fast mixing device (Japanese)"IR microscope. (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Akiyama, S., et al.: "Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering"Proc. Natl. Acad. Sci. USA. 99. 1329-1334 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 高橋・ほか一名: "蛋白質の折り畳み運動を捉える"蛋白質・核酸・酵素 増刊号「新世紀における蛋白質科学の進展」. 46. 1545-1552 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Akiyama, S., et al.: "Stepwise formation of α-helices during cytochrome c folding"Nat. Struct. Biol.. 7. 514-520 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Akiyama,S., et al.: "Stepwise formation of α-helices during cytochrme c folding"Nature Structural Biology. 7. 514-520 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 高橋秋山: "蛋白質の折り畳み運動を捉える"蛋白質・核酸・酵素. (予定). (2001)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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