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不均一系超分子における分子認識機構を解明するNMR戦略の開発

Research Project

Project/Area Number 20051005
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionThe University of Tokyo

Principal Investigator

大澤 匡範  The University of Tokyo, 大学院・薬学系研究科, 助教 (60361606)

Co-Investigator(Kenkyū-buntansha) 嶋田 一夫  東京大学, 大学院・薬学系研究科, 教授 (70196476)
Project Period (FY) 2008 – 2009
Project Status Completed (Fiscal Year 2009)
Budget Amount *help
¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 2009: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2008: ¥3,700,000 (Direct Cost: ¥3,700,000)
Keywords構造生物学 / NMR / 安定同位体標識 / 相互作用解析 / 複合体構造
Research Abstract

これまでに我々はビーズに固定化した膜蛋白質を脂質二重膜中に再構成するBPL法を開発し、相互作用残基を同定する転移交差飽和法(TCS法)を開発してきた。TCS法は、交差飽和を受け取るアクセプター分子上の結合界面を明らかとすることが可能であるが、ドナーであるBPL上のアクセプター結合部位に関する情報は得られない。そこで、ドナーに対して特定のアミノ酸以外を^2Hとする、アミノ酸選択的^1Hラベルを行い、交差飽和源を1種類のアミノ酸に限定する、アミノ酸選択的交差飽和(ASCS)法を開発した。本手法を、yeast ubiquitin (Ub)と、その加水分解酵素であるUb hydrolase 1(C90S変異体,以下YUH1)に対して、Ubをアクセプターに、YUH1をドナーとして適用したところ、結晶構造に一致するYUH1上の結合部位の同定に成功したものの、同一結合部位の中での複数の異なる組み合わせがASCS実験結果を満たすことが判明した。そこで、ASCS法のみでは近接残基対の同定および複合体モデルの構築が困難な巨大タンパク質複合体の構造解析を目指し、特定の一残基を^1H、他を^2H標識して交差飽和源を一残基のみに限定する「残基選択的TCS法(RSTCS法)」を開発した。Leu165選択[^1H,^<13>C]標識YUHをCSドナー、均一[^2H,^<15>N]標識UbをCSアクセプターとしたRSTCS実験の結果、UbのR74およびG75において、L165からの交差飽和によるNMRシグナル強度減少が観測された。YUH上のL165とUb上のR74は、結晶構造において近接する残基対である。したがって、RSTCS法を用いることにUb-YUH複合体の正確な近接残基対を同定できたと結論した。

Report

(2 results)
  • 2009 Annual Research Report
  • 2008 Annual Research Report
  • Research Products

    (17 results)

All 2010 2009 2008 Other

All Journal Article (10 results) (of which Peer Reviewed: 10 results) Presentation (6 results) Remarks (1 results)

  • [Journal Article] Structural basis underlying the dual gate properties of KcsA2010

    • Author(s)
      Imai, S.
    • Journal Title

      Proc.Natl.Acad.Sci.USA. 107

      Pages: 6216-6221

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Evidence for the direct interaction of spermine with the inwardly rectifying potassium channel2009

    • Author(s)
      Osawa M
    • Journal Title

      J.Biol.Chem. 284

      Pages: 26117-26126

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Backbone resonance assignments for the ligand binding subunit of the histidine permease complex(HisJ)from Escherichia coli, under histidine-bound and unbound states2009

    • Author(s)
      Igarashi S
    • Journal Title

      Biomol.NMR Assign (In press)

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Backbone resonance assignments for the cytoplasmic regions of G protein-activated inwardly rectifying potassium channel 1(GIRK1)2009

    • Author(s)
      Yokogawa M
    • Journal Title

      Biomol.NMR Assign. 3

      Pages: 125-128

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cross-saturation and transferred cross-saturation experiments2009

    • Author(s)
      Shimada I
    • Journal Title

      Prog.Nuc.Magn.Reson.Spect. 54

      Pages: 123-140

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Backbone resonance assignments for the cytoplasmic regions o f G protein-activated inwardly rectifying potassium channel 1 (GIRK1)2009

    • Author(s)
      Yokogawa M.
    • Journal Title

      Biomolecular NMR Assignments (In press)

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Stable isotope labeling of protein by Kluyveromyces lactis for NMR study2008

    • Author(s)
      Sugiki T.
    • Journal Title

      J Biomol NMR 42

      Pages: 159-162

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Carbohydrate binding mechanism of the macrophage galactose-type C-type lecti n 1 revealed by saturation transfer experiments2008

    • Author(s)
      Sakakura M.
    • Journal Title

      J Biol Chem 283

      Pages: 33665-33673

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Chondroitin sulfate E fragments enhance CD44 cleavage and CD44-dependent motility in tumor cells2008

    • Author(s)
      Sugahara K.N.
    • Journal Title

      Cancer Res 68

      Pages: 7191-7199

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Amino acid selective cross-saturation method for identification of proximal resi due pairs in a protein-protein complex2008

    • Author(s)
      Igarashi S.
    • Journal Title

      J. Am. Chem. Soc 130

      Pages: 12168-12176

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Presentation] Structural basis on the specificity and efficiency of the G-protein signaling for the GIRK-gating2009

    • Author(s)
      Mase Y.
    • Organizer
      日本分子生物学会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2009-12-09
    • Related Report
      2009 Annual Research Report
  • [Presentation] Structural basis of the inhibitory mechanism of collagen-binding to vWF and α2β1integlin mediated by leech product LAPP2009

    • Author(s)
      Izawa N.
    • Organizer
      日本分子生物学会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2009-12-09
    • Related Report
      2009 Annual Research Report
  • [Presentation] 生体超分子における分子認識機構解明に向けたHR-MASの適用2008

    • Author(s)
      豊永翔
    • Organizer
      第47回NMR討論会
    • Place of Presentation
      茨城県つくば市
    • Year and Date
      2008-11-13
    • Related Report
      2008 Annual Research Report
  • [Presentation] 巨大タンパク質複合体のモデル構築を目的とした残基選択的交差飽和法の開発2008

    • Author(s)
      小澤新一郎
    • Organizer
      第47回NMR討論会
    • Place of Presentation
      茨城県つくば市
    • Year and Date
      2008-11-13
    • Related Report
      2008 Annual Research Report
  • [Presentation] Structural studies on the interaction of spermine with inwar dly rectifying potassium channel2008

    • Author(s)
      Osawa M.
    • Organizer
      XXIIIrd International Conference on Magnet ic Resonance in Biological Systems
    • Place of Presentation
      San Diego, CA, USA
    • Year and Date
      2008-08-24
    • Related Report
      2008 Annual Research Report
  • [Presentation] Structural basis of the gating of G protein-activated inward ly rectifying potassium channel by Gbg2008

    • Author(s)
      Yokogawa M.
    • Organizer
      XXIIIrd International Conference on Magnet ic Resonance in Biological Systems
    • Place of Presentation
      San Diego, CA, USA
    • Year and Date
      2008-08-24
    • Related Report
      2008 Annual Research Report
  • [Remarks]

    • URL

      http://ishimada.f.u-tokyo.ac.jp/publichtml/index_j.html

    • Related Report
      2009 Annual Research Report

URL: 

Published: 2008-04-01   Modified: 2018-03-28  

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