Investigating protein behaviours in cultured human cells by paramagnetic in-cell NMR

• Project Area: Science on Function of Soft Molecular Systems by Cooperation of Theory and Experiment
• Project/Area Number: 16H00847
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Tokyo Metropolitan University
• Principal Investigator: 伊藤 隆 首都大学東京, 理工学研究科, 教授 (80261147)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000); Fiscal Year 2017: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2016: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
• Keywords: in-cell NMR / 常磁性NMR / 蛋白質

Outline of Annual Research Achievements

ヒト培養細胞を用いたin-cell NMR実験は，外部から導入した安定同位体標識蛋白質の細胞内濃度やNMR試料管中の細胞寿命の問題などから，測定感度が低いという問題が避けられない．このような状況では通常のNOEを用いた蛋白質の立体構造情報の取得は極めて困難であるため，ランタノイド（Ln3+）結合タグ（LBT）で化学修飾を行い，感度の良い2D 異種核相関NMRスペクトルで観測される常磁性擬コンタクトシフト（PCS）情報の有用性が注目されている．
2017年度は，まず，ヨードアセトアミド型LBT（M8-CAM-I）の改良を行った．私たちはM8-CAM-Iを用いてヒト培養細胞中でのPCS観測に成功したが，リンカー部のフレキシビリティーのために，得られたPCS値は同種のLBTに比べてやや小さかった．そこでリンカーにピペラジン構造を導入しフレキシビリティーを抑制することを狙った．新LBTを用いて実験を行った結果，M8-CAM-Iより大きなPCS値を得ることに成功した．
次に，非天然型アミノ酸とアジド型LBTのクリック反応を用いてLn3+を導入する方法を確立した．多くのLBTは蛋白質のシステイン残基側鎖SH基に導入される．しかし，蛋白質の物性を変えずに，任意の場所にシステイン残基を導入することは困難な場合も多く．異なる戦略でLBTを導入する方法が模索されている．今回はリンカー末端にアジド基を持つM8誘導体を合成し，一方で蛋白質には末端アルキンを持つチロシン誘導体を組み込み，クリック反応でLBTを導入する方法を試した．実験の結果PCSの観測に成功した．
これに加えて，培養昆虫細胞内の蛋白質に適用可能な新しいNMR解析，立体構造計算法を確立し，真核細胞の系では初となる蛋白質の高分解能立体構造決定に成功した．

Research Progress Status

29年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

29年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] University of Cambridge/University of Leicester(United Kingdom)
• [Int'l Joint Research] Goethe University Frankfurt(Germany)
• [Int'l Joint Research] ETH Zurich(Switzerland)
• [Int'l Joint Research] University of Leicester(United Kingdom)
• [Journal Article] Solution NMR views of dynamical ordering of biomacromolecules (2018)
  • 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
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Impact of cellular health conditions on the protein folding state in mammalian cells (2017)
  • 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
  • Peer Reviewed / Open Access
• [Journal Article] Parkinson’s disease-related DJ-1 functions in thiol quality control against aldehyde attack in vitro (2017)
  • 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
  • Peer Reviewed / Open Access
• [Journal Article] Chemical shift assignments of the first and second RRMs of Nrd1, a fission yeast MAPK-target RNA binding protein (2017)
  • 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
  • Peer Reviewed
• [Journal Article] In-cell structure determination of proteins at near-physiological concentration (2016)
  • Author(s): Teppei Ikeya, Tomomi Hanashima, Saori Hosoya, Manato Shimazaki, Shiro Ikeda, Masaki Mishima, Peter Guentert and Yutaka Ito
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 38312-38312
  • DOI: 10.1038/srep38312
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] A new carbamidemethyl-linked lanthanide chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells. (2016)
  • Author(s): Yuya Hikone, Go Hirai, Masaki Mishima, Kohsuke Inomata, Teppei Ikeya, Souichiro Arai, Masahiro Shirakawa, Mikiko Sodeoka, and Yutaka Ito
  • Journal Title: Journal of Biomolecular NMR Volume: 66 Issue: 2 Pages: 99-110
  • DOI: 10.1007/s10858-016-0059-4
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [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
  • Invited
• [Presentation] NMRによる生細胞内蛋白質の立体構造・ダイナミクスの解析 (2017)
  • Author(s): 伊藤 隆
  • Organizer: 日本薬学会第137年会
  • Place of Presentation: 仙台国際センター，仙台
  • Year and Date: 2017-03-25
  • Invited
• [Presentation] in situ structural biology by NMR (2017)
  • Author(s): 伊藤 隆
  • Organizer: 分子研研究会「生体金属動態」
  • Invited
• [Presentation] NMR approaches to investigate protein folding and dynamics in the crowded intracellular environment (2017)
  • Author(s): 伊藤 隆
  • Organizer: 日本生物物理学会第55回年会
  • Invited
• [Presentation] Protein structure determination in living eukaryotic cells by in-cell NMR spectroscopy (2016)
  • Author(s): Yutaka Ito
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜，横浜
  • Year and Date: 2016-12-01
  • Invited
• [Presentation] In situ structural biology by NMR (2016)
  • Author(s): Yutaka Ito
  • Organizer: The 42nd Naito Conference “In the Vanguard of Structural Biology: Revolutionizing Life Sciences”
  • Place of Presentation: シャトレーゼガトーキングダムサッポロ，札幌
  • Year and Date: 2016-10-05
  • Int'l Joint Research / Invited
• [Presentation] In situ structural biology by NMR (2016)
  • Author(s): Yutaka Ito
  • Organizer: International Conference on Magnetic Resonance in Biological Systems 2016
  • Place of Presentation: 京都国際会館，京都
  • Year and Date: 2016-08-22
  • Int'l Joint Research / Invited
• [Presentation] In situ structural biology by NMR (2016)
  • Author(s): Yutaka Ito
  • Organizer: UK-Japan Symposium “From single molecules to cells and tissues
  • Place of Presentation: University of Leiceister, UK
  • Year and Date: 2016-07-05
  • Int'l Joint Research / 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 Science (2017)
  • Author(s): Yutaka Ito and Teppei Ikeya
  • Total Pages: 636
  • Publisher: Springer