Investigating protein behaviours in mitochondria by in-organelle NMR

• Project/Area Number: 15K14494
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Tokyo Metropolitan University
• Principal Investigator: ITO Yutaka 首都大学東京, 理工学研究科, 教授 (80261147)
• Co-Investigator(Kenkyū-buntansha): 美川 務 国立研究開発法人理化学研究所, その他, 研究員 (20321820)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: In-cell NMR / In-organelle NMR / ミトコンドリア / 蛋白質 / in-cell NMR / オルガネラ

Outline of Final Research Achievements

Protein behaviours in eukaryotic organelles were investigated by extending the in-cell NMR approaches to be available for the analysis of proteins localised in organelles, particularly mitochondria.
A model protein with a mitochondrial targeting signal was incorporated into cultured human HeLa.S3 cells by electroporation, and then the mitochondria faction was isolated for NMR experiments. Well-resolved in-mitochondria NMR spectra showed that the proper 3D structure was formed, while different effects of the mitochondria matrix environment to the conformation and dynamics of proteins was suggested from the observed spectral differences when compared with the samples in cytosol or in diluted solution suggested.

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] 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
• [Journal Article] In-cell NMRとnonlinear sampling - there is no other way (2015)
  • Author(s): 伊藤 隆
  • Journal Title: 分光研究 Volume: 64 Pages: 413-426
  • NAID: 40020544418
  • Peer Reviewed
• [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 Leicester, UK
  • Year and Date: 2016-07-05
  • Int'l Joint Research / Invited
• [Presentation] NMRを用いた生細胞中の蛋白質の立体構造解析 (2016)
  • Author(s): 伊藤 隆
  • Organizer: シンポジウム「細胞環境における蛋白質の動態解析のためのNMRおよび計算科学的アプローチ」
  • Place of Presentation: 京都大学 吉田キャンパス（京都府京都市）
  • Year and Date: 2016-03-28
  • Invited
• [Presentation] In situ structural biology by NMR (2015)
  • Author(s): Yutaka Ito
  • Organizer: Pacifichem 2015
  • Place of Presentation: Honolulu, Hawaii, USA
  • Year and Date: 2015-12-15
  • Int'l Joint Research / Invited
• [Presentation] In situ structural biology by NMR (2015)
  • Author(s): 伊藤 隆
  • Organizer: BMB2015
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市）
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] 細胞内クラウディング環境における蛋白質のフォールディングとダイナミクスをNMRで観察する (2015)
  • Author(s): 伊藤 隆
  • Organizer: 第53回日本生物物理学会年会
  • Place of Presentation: 金沢大学 角間キャンパス（石川県金沢市）
  • Year and Date: 2015-09-14
  • 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