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NMR approaches to investigate protein structure, folding stability and dynamics in the crowded intracellular environment

Publicly Offered Research

Project AreaNovel measurement techniques for visualizing 'live' protein molecules at work
Project/Area Number 17H05887
Research Category

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

Allocation TypeSingle-year Grants
Review Section Complex systems
Research InstitutionTokyo Metropolitan University

Principal Investigator

伊藤 隆  首都大学東京, 理学研究科, 教授 (80261147)

Project Period (FY) 2017-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2018: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Fiscal Year 2017: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Keywordsin-cell NMR / 蛋白質の立体構造 / 蛋白質のフォールディング / 蛋白質の動的平衡 / 分子クラウディング / 蛋白質
Outline of Annual Research Achievements

細胞内の分子クラウディング環境が蛋白質の様々な動態に与える影響が注目されている.In-cell NMRを用いたこれまでの国内外の研究によって,細胞内環境における蛋白質動態,フォールディング不安定化,動的平衡に対する影響等は明らかになりつつあるものの,統一的・普遍的な理解にはまだ至っていなかった.本研究では,in-cell NMRをさらに発展させることで,細胞内蛋白質の立体構造,安定性および動的平衡状態の詳細な解析を可能にする手法の確立を目指した.また,応用研究として,ヒト培養細胞を用いた薬剤スクリーニングの系の検証も試みた.
まず,ショウジョウバエDRK蛋白質やヒトGRB2蛋白質などのマルチドメイン・アダプター蛋白質群に注目し,希薄溶液状態,クラウディング状態,細胞内環境での立体構造,安定性および動的平衡状態の解析を進めた.DRK/GRB2はN末端とC末端の2つのSH3ドメインとこれらに挟まれたSH2ドメインから構成されている.具体的な結果としては,DRKのN末端SH3ドメインが,ドメイン単体では希薄溶液中でfoldとunfoldの1:1平衡になっているにもかかわらず,全長蛋白質ではほぼunfold状態になっていることを見出した.また,常磁性NMR効果を用いることで,溶液状態でのGRB2各ドメインの相対配置を決定した.さらに,少ない距離拘束条件から正しい立体構造を算出するためのベイズ推定を用いた立体構造解析手法を確立し,昆虫培養細胞を用いて,真核細胞内としては世界初となる高分解能蛋白質立体構造決定に成功した.
In-cell NMRの創薬科学への応用研究としては,ヒトHRas蛋白質とRaf-1 RBDの相互作用の系をモデルとして,細胞内の蛋白質間相互作用の詳細な解析を試みた.

Research Progress Status

平成30年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

平成30年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2018 Annual Research Report
  • 2017 Annual Research Report
  • Research Products

    (22 results)

All 2019 2018 2017 Other

All Int'l Joint Research (6 results) Journal Article (7 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 7 results,  Open Access: 5 results) Presentation (8 results) (of which Int'l Joint Research: 2 results,  Invited: 8 results) Book (1 results)

  • [Int'l Joint Research] University of Cambridge/University of Leicester(英国)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] ETH Zurich(スイス)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] Goethe University Frankfurt(ドイツ)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] University of Cambridge/University of Leicester(United Kingdom)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] Goethe University Frankfurt(Germany)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] ETH Zurich(Switzerland)

    • Related Report
      2017 Annual Research Report
  • [Journal Article] High resolution protein 3D structure determination in living eukaryotic cells2019

    • Author(s)
      Tanaka, T., Ikeya, T., Kamoshida, H., Suemoto, Y., Mishima, M., Shirakawa, M., Guentert, P., Ito, Y.
    • Journal Title

      Angewandte Chemie International Edition

      Volume: in press Issue: 22 Pages: 1-7

    • DOI

      10.1002/anie.201900840

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Structural insights into ubiquitin phosphorylation by PINK12018

    • Author(s)
      Okatsu Kei、Sato Yusuke、Yamano Koji、Matsuda Noriyuki、Negishi Lumi、Takahashi Akiko、Yamagata Atsushi、Goto-Ito Sakurako、Mishima Masaki、Ito Yutaka、Oka Toshihiko、Tanaka Keiji、Fukai Shuya
    • Journal Title

      Scientific Reports

      Volume: 8 Issue: 1 Pages: 10382-10382

    • DOI

      10.1038/s41598-018-28656-8

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Protein NMR Structure Refinement Based on Bayesian Inference for Dynamical Ordering of Biomacromolecules2018

    • Author(s)
      Teppei Ikeya and Yutaka Ito
    • Journal Title

      Journal of Computer Chemistry, Japan

      Volume: 17 Issue: 1 Pages: 65-75

    • DOI

      10.2477/jccj.2018-0009

    • NAID

      130006555815

    • ISSN
      1347-1767, 1347-3824
    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Solution NMR views of dynamical ordering of biomacromolecules2018

    • Author(s)
      Ikeya Teppei、Ban David、Lee Donghan、Ito Yutaka、Kato Koichi、Griesinger Christian
    • Journal Title

      Biochim. Biophys. Acta-General Subjects

      Volume: 1862 Issue: 2 Pages: 287-306

    • DOI

      10.1016/j.bbagen.2017.08.020

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Impact of cellular health conditions on the protein folding state in mammalian cells2017

    • Author(s)
      Inomata Kohsuke、Kamoshida Hajime、Ikari Masaomi、Ito Yutaka、Kigawa Takanori
    • Journal Title

      Chemical Communications

      Volume: 53 Issue: 81 Pages: 11245-11248

    • DOI

      10.1039/c7cc06004a

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Parkinson’s disease-related DJ-1 functions in thiol quality control against aldehyde attack in vitro2017

    • Author(s)
      Matsuda Noriyuki、Kimura Mayumi、Queliconi Bruno Barros、Kojima Waka、Mishima Masaki、Takagi Kenji、Koyano Fumika、Yamano Koji、Mizushima Tsunehiro、Ito Yutaka、Tanaka Keiji
    • Journal Title

      Scientific Reports

      Volume: 7 Issue: 1 Pages: 12816-12816

    • DOI

      10.1038/s41598-017-13146-0

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Chemical shift assignments of the first and second RRMs of Nrd1, a fission yeast MAPK-target RNA binding protein2017

    • Author(s)
      Ayaho KobayashiTeppei KanabaRyosuke SatohYutaka ItoReiko SugiuraMasaki Mishima
    • Journal Title

      Biomolecular NMR Assignments

      Volume: 印刷中 Issue: 2 Pages: 123-126

    • DOI

      10.1007/s12104-017-9731-1

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Presentation] NMRと計算科学の融合によるin site構造生物学の確立 真核細胞内蛋白質の動態研究から創薬への展開2018

    • Author(s)
      伊藤 隆
    • Organizer
      ゲノム創薬・医療フォーラム第9回談話会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] NMRを用いた細胞内蛋白質の立体構造,フォールディング安定性および動的平衡状態の解析2018

    • Author(s)
      伊藤 隆
    • Organizer
      第18回日本蛋白質科学会年会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] NMR approaches to investigating protein 3D structures in living eukaryotic cells2018

    • Author(s)
      Yutaka Ito
    • Organizer
      XXVIII International Conference on Magnetic Resonance in Biological Systems (ICMRBS 2018)
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] NMR approaches to investigating protein 3D structures in living eukaryotic cells2018

    • Author(s)
      Yutaka Ito
    • Organizer
      The 6th International Symposium on Drug Discovery and Design by NMR
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 生きた真核細胞内の蛋白質の高分解能立体構造解析2018

    • Author(s)
      伊藤 隆
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] 常磁性金属を利用したNMRによる生細胞内蛋白質の立体構造解析2018

    • Author(s)
      伊藤 隆
    • Organizer
      日本化学会第98春季年会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] in situ structural biology by NMR2017

    • Author(s)
      伊藤 隆
    • Organizer
      分子研研究会「生体金属動態」
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] NMR approaches to investigate protein folding and dynamics in the crowded intracellular environment2017

    • Author(s)
      伊藤 隆
    • Organizer
      日本生物物理学会第55回年会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Book] “Chapter 3, Advances in NMR data acquisition and processing for protein structure determination” Experimental approaches of NMR spectroscopy: Methodology and application to life science and materials science2017

    • Author(s)
      Yutaka Ito and Teppei Ikeya
    • Total Pages
      636
    • Publisher
      Springer
    • Related Report
      2017 Annual Research Report

URL: 

Published: 2017-04-28   Modified: 2019-12-27  

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