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変性構造転移による膜蛋白質シグナル伝達機構の固体NMRによる解明

Publicly Offered Research

Project AreaTarget recognition and expression mechanism of intrinsically disordered protein
Project/Area Number 22113512
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionOsaka University

Principal Investigator

藤原 敏道  大阪大学, たんぱく質研究所, 教授 (20242381)

Project Period (FY) 2010 – 2011
Project Status Completed (Fiscal Year 2011)
Budget Amount *help
¥12,480,000 (Direct Cost: ¥9,600,000、Indirect Cost: ¥2,880,000)
Fiscal Year 2011: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Fiscal Year 2010: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Keywords核磁気共鳴 / 膜蛋白質 / 膜タンパク質 / 変性構造
Research Abstract

ppR二量体および ppR・pHtrII複合体など膜蛋白質の変性領域について固体NMR法で解析を行った。その複合体の試料調製で、必要な数mgの安定同位体標識試料を作り、機能のある状態で脂質二重膜へ再構成した。対象の分子量が約10万と大きく、NMR感度が十分でないため、固体NMRの新しい非線形サンプリングなどの手法で分解能・感度を向上させる。構造解析では、NMRプロテオミクス研究や計算機科学の最先端の技術として、データベースに基づくNMRスペクトルシミュレーション法と脂質膜を考慮したレプリカ交換動力学法を組み合わせて利用した。この方法論を確立した。複合体を活性状態に転移させるために、超伝導磁石内のNMR試料に光照射できるよう装置を改造すした。特にpHtrIIの水溶性領域の天然変性構造の変化に注目してNMR測定するために、J結合相関に基づく高速試料回転多次元NMR法や電子スピンとの距離測定法も開発して応用した。

Current Status of Research Progress
Reason

24年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

24年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • Research Products

    (12 results)

All 2013 2012 2011 2010 Other

All Journal Article (6 results) (of which Peer Reviewed: 6 results) Presentation (5 results) (of which Invited: 2 results) Remarks (1 results)

  • [Journal Article] Secondary Structural Analysis of Proteins Based on 13C Chemical Shift Assignments in Unresolved Solid-State NMR Spectra Enhanced by Fragmented Structure Database2013

    • Author(s)
      Keisuke Ikeda, Ayako Egawa, and Toshimichi Fujiwara
    • Journal Title

      J. Biomol. NMR,

      Volume: 55 Pages: 189-200

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Helium-Cooling and -Spinning Dynamic Nuclear Polarization for Sensitivity-Enhanced Solid-State NMR at 14 T and 30 K2012

    • Author(s)
      Yoh Matsuki, Keisuke Ueda, Toshitaka Idehara, Ryosuke Ikeda, Isamu Ogawa, Shinji Nakamura, Mitsuru Toda, Takahiro Anai, Toshimichi Fujiwara
    • Journal Title

      J. Magn. Reson.

      Volume: 225 Pages: 1-9

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Combined use of replica-exchange molecular dynamics and magic-angle-spinning solid-state NMR spectral simulations for determining the structure and orientation of membrane-bound peptide2011

    • Author(s)
      Keisuke Ikeda, Tomoshi Kameda, Erisa Harada, Hideo Akutsu, and Toshimichi Fujiwara
    • Journal Title

      J. Phys. Chem. B

      Volume: 115 Pages: 9327-9336

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Combined use of replica-exchange molecular dynamics and magic-angle-spinning solid-state NMR spectral simulations for determining the structure and orientation of membrane-bound peptide2011

    • Author(s)
      Keisuke Ikeda, Tomoshi Kameda, Erisa Harada, Hideo Akutsu, Toshimichi Fujiwara
    • Journal Title

      J.Phys.Chem.B

      Volume: 115 Pages: 9327-9336

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Structure analysis of membrane-reconstituted subunit c-ring of E.coli H^+-ATP synthase by solid-state NMR under MAS2010

    • Author(s)
      Yasuto Todokoro, Masatoshi Kobayashi, Takeshi Sato, Toru Kawakami, Ikuko Yumen, Saburo Aimoto, Toshimichi Fujiwara, Hideo Akutsu
    • Journal Title

      J.Biomol.NMR

      Volume: 48 Pages: 1-11

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Continuously Frequency Tunable High Power Sub-THz Radiation Source-Gyrotron FU CW VI for 600 MHz DNP-NMR spectroscopy2010

    • Author(s)
      Yoh Matsuki, Hiroki Takahashi, Keisuke Ueda, Toshitaka Idehara, Isamu Ogawa, Mitsuru Toda, Hideo Akutsu, Toshimichi Fujiwara
    • Journal Title

      Phys.Chem.Chem.Phys.

      Volume: 12 Pages: 5799-5803

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Presentation] 固体NMR法を用いた生理的環境での膜蛋白質pHtrIIの構造解析2012

    • Author(s)
      江川文子,池田恵介,Lili Mao,林こころ,児嶋長次郎,井上正順,藤原敏道
    • Organizer
      NMR討論会
    • Place of Presentation
      名古屋ウインク愛知
    • Related Report
      2011 Annual Research Report
    • Invited
  • [Presentation] Automated Signal Assignment and Secondary Structural Analysis of Non-Crystalline Proteins by Fragment-Based Solid-State NMR Spectral Reconstruction2012

    • Author(s)
      Keisuke Ikeda, Ayako Egawa, Kokoro Hayashi, Chojiro Kojima, Toshimichi Fujiwara
    • Organizer
      NMR討論会
    • Place of Presentation
      名古屋ウインク愛知
    • Related Report
      2011 Annual Research Report
    • Invited
  • [Presentation] Solid-state NMR structural analysis of membrane proteins and sensitivity enhancement2010

    • Author(s)
      Toshimichi Fujiwara
    • Organizer
      Pachifi Chem 2010
    • Place of Presentation
      Convention Center, Honolulu(米国)(招待講演)
    • Year and Date
      2010-12-15
    • Related Report
      2010 Annual Research Report
  • [Presentation] 固体NMRスペクトルシミュレーションによる高度好塩菌由来トランスデューサー膜蛋白質pHtrIIの動的構造解析2010

    • Author(s)
      池田恵介、江川文子、亀田倫史、林こころ、児嶋長次郎、阿久津秀雄、藤原敏道
    • Organizer
      第49回NMR討論会
    • Place of Presentation
      タワーホール船堀、東京
    • Year and Date
      2010-11-16
    • Related Report
      2010 Annual Research Report
  • [Presentation] 多次元固体NMRによる膜蛋白質pHtrIIの主鎖構造解析2010

    • Author(s)
      江川文子、林こころ、児嶋長次郎、阿久津秀雄、藤原敏道
    • Organizer
      第48回日本生物物理学会
    • Place of Presentation
      東北大学川内北キャンパス
    • Year and Date
      2010-09-21
    • Related Report
      2010 Annual Research Report
  • [Remarks] 蛋白質研究所機能構造計測学研究室

    • URL

      http://www.protein.osaka-u.ac.jp/biophys/

    • Related Report
      2011 Annual Research Report

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Published: 2010-08-23   Modified: 2018-03-28  

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