Protein 19F Labeling Strategy for In-Cell NMR and Functional Analyses

• Project/Area Number: 26440018
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Tohoku University
• Principal Investigator: Koshiba Seizo 東北大学, 東北メディカル・メガバンク機構, 教授 (70332301)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: フッ素 / タンパク質 / NMR / 細胞内環境 / 相互作用 / 動的構造変化 / 19F-NMR / In-Cell NMR / 動的構造

Outline of Final Research Achievements

Fluorine 19 (19F) is a widely used nucleus for NMR because of its high sensitivity and large chemical shift dispersion. In particular, 19F-labeling is useful for In-Cell NMR, because most of biological systems contain no fluorine, resulting that 19F-labeled molecules in living cells can be selectively analyzed. In this study, I developed several 19F-labeling methods for proteins and effectively incorporated trifluoromethyl-labeled amino acids into proteins. I expressed several kinds of trifluoromethyl-labeled proteins and could observe highly sensitive 19F NMR signals both in vitro and in vivo. I also investigated the interactions of ligands with large molecular weight proteins by 19F-NMR and could identify binding sites of the ligands in a short time. These results show that my methods are particularly useful for investigating behavior of intracellular proteins or ligand-interactions. I expect that this labeling strategy is useful for wide variety of biological sciences.

Research Products

• [Journal Article] JMorp: Japanese Multi Omics Reference Panel (2018)
  • Author(s): Tadaka S, Saigusa D, Motoike, I.N, Aoki Y, Shirota M, Koshiba S, Yamamoto M, and Kinoshita K.
  • Journal Title: Nucleic Acids Res. Volume: 46 Issue: D1 Pages: D551-D557
  • DOI: 10.1093/nar/gkx978
  • URL: https://pure.teikyo.jp/en/publications/d91494df-d7be-4ad7-8407-c4aa3c4b66c9
  • Peer Reviewed / Open Access
• [Journal Article] Evaluation of reported pathogenic variants and their frequencies in a Japanese population based on a whole-genome reference panel of 2049 individuals article (2018)
  • Author(s): Y Yamaguchi-Kabata, J Yasuda, O Tanabe, Y Suzuki, H Kawame, N Fuse, M Nagasaki, Y Kawai, K Kojima, F Katsuoka, S Saito, I Danjoh, I Motoike, R Yamashita, S Koshiba, D Saigusa, G Tamiya, S Kure, N Yaegashi, Y Kawaguchi, F Nagami, S Kuriyama, J Sugawara, N Minegishi, A Hozawa, T Takai-Igarashi, K Kinoshita, M Yamamoto.
  • Journal Title: Journal of Human Genetics. Volume: 63 Issue: 2 Pages: 213
  • DOI: 10.1038/s10038-017-0347-1
  • URL: https://pure.teikyo.jp/en/publications/68f9baf0-ba55-4278-8354-a10148b91802
  • Peer Reviewed / Open Access
• [Journal Article] The structural origin of metabolic quantitative diversity (2016)
  • Author(s): Koshiba S, Motoike I, Kojima K, Hasegawa T, Shirota M, Saito T, Saigusa D, Danjoh I, Katsuoka F, Ogishima S, ... Yamamoto M.
  • Journal Title: Sci Rep Volume: 6 Issue: 1 Pages: 31463-31463
  • DOI: 10.1038/srep31463
  • URL: https://pure.teikyo.jp/en/publications/f23f66dd-859e-4088-8e53-de40d99cc561
  • Peer Reviewed / Open Access
• [Journal Article] Establishment of protocols for global metabolomics by LC-MS for biomarker discovery (2016)
  • Author(s): Saigusa D, Okamura Y, Motoike IN, Katoh Y, Kurosawa Y, Saijyo R, Koshiba S, Yasuda J, Motohashi H, Sugawara J, Tanabe O, Kinoshita K, Yamamoto M
  • Journal Title: PLoS One Volume: 11 Issue: 8 Pages: e0160555-e0160555
  • DOI: 10.1371/journal.pone.0160555
  • URL: https://pure.teikyo.jp/en/publications/789e31b9-dbb0-4124-986b-c259a28c9328
  • Peer Reviewed / Open Access
• [Journal Article] The Tohoku Medical Megabank Project: Design and Mission (2016)
  • Author(s): Kuriyama S, Yaegashi N, et al. (Tamiya G; 11th in 58 authors)
  • Journal Title: Journal of Epidemiology Volume: 26 Issue: 9 Pages: 493-511
  • DOI: 10.2188/jea.JE20150268
  • NAID: 130005409018
  • ISSN: 0917-5040, 1349-9092
  • Peer Reviewed / Open Access
• [Journal Article] Stable isotope labeling strategy based on coding theory. (2015)
  • Author(s): Kasai T, Koshiba S, Yokoyama J, Kigawa T
  • Journal Title: J. Biomol. NMR Volume: 63 Issue: 2 Pages: 213-221
  • DOI: 10.1007/s10858-015-9978-8
  • Peer Reviewed / Open Access
• [Presentation] コホート研究におけるオミックス解析 (2017)
  • Author(s): 小柴生造
  • Organizer: 日本人類遺伝学会（招待講演）
  • Invited
• [Presentation] Metabolomics Approach in Tohoku Medical Megabank Project Prospective Cohort Studies (2017)
  • Author(s): Seizo Koshiba, Daisuke Saigusa, Ikuko Motoike, Natsuyuki Shirota, Jun Yasuda, Kenga Kinoshita, Masayuki Yamamoto
  • Organizer: Metabolomics 2017
  • Int'l Joint Research
• [Presentation] 東北メディカル・メガバンク機構のオミックス解析 (2017)
  • Author(s): 小柴生造
  • Organizer: 第11回メタボロームシンポジウム
• [Presentation] 酸化ストレス応答におけるKeap1の構造・相互作用解析 (2017)
  • Author(s): 井上仁, 村松亜紀, 鈴木隆史, 磯達朗, 小柴生造, 山本雅之
  • Organizer: 第56回NMR討論会
• [Presentation] 高精度日本人多層オミックス参照パネルの提供 (2017)
  • Author(s): 小柴生造
  • Organizer: 第90回日本生化学会大会
• [Presentation] メタボローム解析による血液検体保管条件の検討 (2017)
  • Author(s): 井上仁, 三枝大輔, 工藤久智, 峯岸直子, 小柴生造, 山本雅之.
  • Organizer: 第90回日本生化学会大会
• [Presentation] 19F標識技術を利用したKeap1-Nrf2タンパク質の構造機能解析 (2016)
  • Author(s): 小柴生造，渡部暁，碇正臣，松田夏子，磯達郎，鈴木隆史，木川隆則，山本雅之
  • Organizer: 第39回日本分子生物学会大会
  • Place of Presentation: 横浜
  • Year and Date: 2016-11-30
• [Presentation] 日本人多層オミックス参照パネルの拡張 (2016)
  • Author(s): 小柴生造, 三枝大輔，元池育子，小島要，城田松之，齊藤智，勝岡史城，河合洋介，山口由美，田邉修，長﨑正郎，安田純，木下賢吾，山本雅之
  • Organizer: 第８９回日本生化学会大会
  • Place of Presentation: 仙台
  • Year and Date: 2016-09-25
• [Presentation] Protein 19F Labeling Strategy for In-Cell NMR and Functional Analyses. (2016)
  • Author(s): Koshiba S., Watanabe S., Ikari M., Matsuda N., Iso T., Suzuki, T. Yamamoto M., and Kigawa T.
  • Organizer: International Conference on Magnetic Resonance in Biological Systems (ICMRBS 2016)
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2016-08-21
  • Int'l Joint Research
• [Book] 遺伝子医学MOOK(32) 難病研究up-to-date (2017)
  • Author(s): 小柴生造、山本雅之
  • Total Pages: 280
  • Publisher: メディカルドゥ
  • ISBN: 9784944157624
• [Remarks] Japanese Multi Omics Reference Panel
  • URL: https://jmorp.megabank.tohoku.ac.jp/