NMRによる細胞生理活性と細胞内蛋白質構造の解析

• Project Area: Novel measurement techniques for visualizing 'live' protein molecules at work
• Project/Area Number: 17H05873
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: Yokohama National University
• Principal Investigator: 児嶋 長次郎 横浜国立大学, 大学院工学研究院, 教授 (50333563)
• Project Period (FY): 2017-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥13,000,000 (Direct Cost: ¥10,000,000、Indirect Cost: ¥3,000,000); Fiscal Year 2018: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2017: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
• Keywords: NMR / 蛋白質 / 立体構造

Outline of Annual Research Achievements

本研究では生きている細胞内の蛋白質の立体構造を決定するNMR技術の開発を目的としている。具体的には、化学シフト帰属・NOE帰属・構造計算の3つのステップを一体的に自動最適化する全自動NMR構造解析プラットフォームと、細胞内で安定な還元耐性スピンラべル試薬およびキレート試薬を用いた新規NMR測定解析技術を開発し、研究代表者らが開発・整備を進めてきた独自のアミノ酸選択標識技術や本研究で開発する世界最先端のNMR技術・装置を組み合わせることで、生きている細胞内の蛋白質の立体構造を決定するIn-cell NMR技術の確立を目指した。また、様々な細胞状態下でIn-cell NMRスぺクトルを測定し、細胞の活動状態とNMRスぺクトルを相関させることでIn-cell NMR法を細胞生理活性の検出法として整備することも目的とした。
平成３０年度は、平成２９年度から継続して生きている細胞内での蛋白質の立体構造を決定するための基礎技術課題、(1)NMR統合解析プラットフォームMagROの開発と、(2)還元耐性スピンラベル試薬およびキレート試薬を用いたNMR測定解析技術の開発、を進めるとともに、独自のアミノ酸選択標識技術などと組み合わせ、(3)生きている細胞内での蛋白質の立体構造決定と、(4)In-cell NMR法による細胞生理活性の検出法の開発、に取り組んだ。特筆すべき成果として、大腸菌の系でIn-cell NMRスペクトルと増殖曲線に強い相関があることを見出し、In-cell NMR法が細胞の生理活性を鋭敏に反映する良い手法であることを示すことに成功した。

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。

Research Products

• [Int'l Joint Research] Goethe University Frankfurt(ドイツ)
• [Int'l Joint Research] Academy of Sciences(チェコ)
• [Int'l Joint Research] Marquette University(米国)
• [Int'l Joint Research] Universite de Sherbrooke(カナダ)
• [Journal Article] Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks (2018)
  • Author(s): Kobayashi, N., Hattori, Y., Nagata, T., Shinya, S., Guentert, P., Kojima, C., Fujiwara, T.
  • Journal Title: Bioinformatics Volume: 34 Issue: 24 Pages: 4300-4301
  • DOI: 10.1093/bioinformatics/bty581
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] NMR spectroscopic characterization of a model RNA duplex reflecting the core sequence of hammerhead ribozymes (2018)
  • Author(s): Hattori Yoshikazu、Yamanaka Daichi、Morioka Saori、Yamaguchi Taishi、Tomonari Honoka、Kojima Chojiro、Tanaka Yoshiyuki
  • Journal Title: Nucleosides, Nucleotides and Nucleic Acids Volume: 37 Issue: 7 Pages: 383-396
  • DOI: 10.1080/15257770.2018.1473877
  • Peer Reviewed
• [Journal Article] Phosphoinositide binding by the PH domain in ceramide transfer protein (CERT) is inhibited by hyperphosphorylation of an adjacent serine-repeat motif (2018)
  • Author(s): Sugiki Toshihiko、Egawa Daichi、Kumagai Keigo、Kojima Chojiro、Fujiwara Toshimichi、Takeuchi Koh、Shimada Ichio、Hanada Kentaro、Takahashi Hideo
  • Journal Title: Journal of Biological Chemistry Volume: 293 Issue: 28 Pages: 11206-11217
  • DOI: 10.1074/jbc.ra118.002465
  • Peer Reviewed / Open Access
• [Journal Article] Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance (2018)
  • Author(s): Sugiki Toshihiko、Furuita Kyoko、Fujiwara Toshimichi、Kojima Chojiro
  • Journal Title: Biochemistry Volume: 57 Issue: 26 Pages: 3576-3589
  • DOI: 10.1021/acs.biochem.8b00182
  • Peer Reviewed
• [Journal Article] The Helix Rearrangement in the Periplasmic Domain of the Flagellar Stator B Subunit Activates Peptidoglycan Binding and Ion Influx (2018)
  • Author(s): Kojima Seiji, Takao Masato, Almira Gaby, Kawahara Ikumi, Sakuma Mayuko, Homma Michio, Kojima Chojiro, Imada Katsumi.
  • Journal Title: Structure Volume: 26 Issue: 4 Pages: 590-598
  • DOI: 10.1016/j.str.2018.02.016
  • Peer Reviewed
• [Journal Article] NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase (2017)
  • Author(s): Shinya Shoko、Ghinet Mariana G.、Brzezinski Ryszard、Furuita Kyoko、Kojima Chojiro、Shah Sneha、Kovrigin Evgenii L.、Fukamizo Tamo
  • Journal Title: Journal of Biomolecular NMR Volume: 67 Issue: 4 Pages: 309-319
  • DOI: 10.1007/s10858-017-0109-6
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The mechanism of the glycosylase reaction with hOGG1 base-excision repair enzyme: Concerted effect of Lys249 and Asp268 during excision of 8-oxoguanine. (2017)
  • Author(s): Jakub Sebera, Yoshikazu Hattori, Daichi Sato, David Reha, Radim Nencka, Takashi Kohno, Chojiro Kojima, Yoshiyuki Tanaka, and Vladimir Sychrovsky
  • Journal Title: Nucleic Acids Research Volume: 45 Issue: 9 Pages: 5231-5242
  • DOI: 10.1093/nar/gkx157
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions (2017)
  • Author(s): Hattori Yoshikazu、Heidenreich David、Ono Yuki、Sugiki Toshihiko、Yokoyama Kei-ichi、Suzuki Ei-ichiro、Fujiwara Toshimichi、Kojima Chojiro
  • Journal Title: Journal of Biomolecular NMR Volume: 68 Issue: 4 Pages: 271-279
  • DOI: 10.1007/s10858-017-0125-6
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na + -Driven Flagella (2017)
  • Author(s): Miyanoiri Yohei、Hijikata Atsushi、Nishino Yuuki、Gohara Mizuki、Onoue Yasuhiro、Kojima Seiji、Kojima Chojiro、Shirai Tsuyoshi、Kainosho Masatsune、Homma Michio
  • Journal Title: Structure Volume: 25 Issue: 10 Pages: 1540-1548
  • DOI: 10.1016/j.str.2017.08.010
  • NAID: 120006375873
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion (2017)
  • Author(s): Fukuda N、Noi K、Weng L、Kobashigawa Y、Miyazaki H、Wakeyama Y、Takaki M、Nakahara Y、Tatsuno Y、Uchida-Kamekura M、Suwa Y、Sato T、Ichikawa-Tomikawa N、Nomizu M、Fujiwara Y、Ohsaka F、Saitoh T、Maenaka K、Kumeta H、Shinya S、Kojima C、Ogura T、Morioka H
  • Journal Title: Molecules Volume: 22 Issue: 10 Pages: 1695-1695
  • DOI: 10.3390/molecules22101695
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 創薬 NMR 技術の開発とその植物生理学分野への応用 (2018)
  • Author(s): 児嶋長次郎
  • Organizer: 日本蛋白質科学会第18回年会
  • Invited
• [Presentation] NMR tools developed for protein-drug and protein-protein interaction studies (2018)
  • Author(s): Chojiro Kojima
  • Organizer: The 6th International Symposium on Drug Discovery and Design by NMR
  • Int'l Joint Research / Invited
• [Presentation] 植物構造生物学研究と創薬 (2018)
  • Author(s): 児嶋長次郎
  • Organizer: 植物科学シンポジウム 2018
  • Invited
• [Presentation] Fully Automated NMR Structure Determination of Protein (2017)
  • Author(s): Chojiro Kojima
  • Organizer: 2017 Taiwan-Japan Biomedical Symposium on Magnetic Resonance
  • Int'l Joint Research / Invited
• [Remarks] Kojima Lab top page
  • URL: http://nmrlab.ynu.ac.jp/labotop.php