Site selective cellular analysis by solid-state NMR

• Project/Area Number: 26291029
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Biophysics
• Research Institution: Osaka University
• Principal Investigator: Fujiwara Toshimichi 大阪大学, たんぱく質研究所, 教授 (20242381)
• Co-Investigator(Renkei-kenkyūsha): KOJIMA Chojiro 大阪大学, 蛋白質研究所, 准教授 (50333563) ; MATSUKI Yoh 大阪大学, 蛋白質研究所, 助教 (70551498)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥16,640,000 (Direct Cost: ¥12,800,000、Indirect Cost: ¥3,840,000); Fiscal Year 2016: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2015: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2014: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
• Keywords: 核磁気共鳴 / 細胞生物学 / 構造生物学 / 細胞内分子構造 / 常磁性造影剤 / 細胞内局在イメージング / 生物物理学 / 生物物理

Outline of Final Research Achievements

The thermal equilibrium NMR signal intensities give the quantitative information for all the cellular molecules at low temperatures where molecular motions are suppressed. The molecules for a single cell were counted by combined use of optical and electron microscope analyses, which enabled us to trace the biosynthetic processes by using the 13C labeled media without the purification. Applications of our magic-angle-spinning DNP NMR spectrometer with a closed-cycle He-gas system at low temperature 30 K and 16.4 T are also shown.

Research Products

• [Journal Article] Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure (2017)
  • Author(s): Kang Su-Jin、Todokoro Yasuto、Bak Suyeon、Suzuki Toshiharu、Yoshida Masasuke、Fujiwara Toshimichi、Akutsu Hideo
  • Journal Title: Journal of Biomolecular NMR Volume: 70 Issue: 1 Pages: 53-65
  • DOI: 10.1007/s10858-017-0158-x
  • Peer Reviewed / Int'l Joint Research
• [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] Mechanistic and structual basis of bioengineered bovine Cathelicidin-5 with optimized therapeutic activity (2017)
  • Author(s): Bikash R. Sahoo, Kenta Maruyama, Jyotheeswara R. Edula, Takahiro Tougan, Yuxi Lin, Young-Ho Lee, Toshihiro Horii, and Toshimichi Fujiwara
  • Journal Title: Scientific Reports Volume: 7:44781 Issue: 1 Pages: 44781-44796
  • DOI: 10.1038/srep44781
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Nanodisc-to-Nanofiber Transition of Noncovalent Peptide?Phospholipid Assemblies (2017)
  • Author(s): Ikeda Keisuke、Horiuchi Ayame、Egawa Ayako、Tamaki Hajime、Fujiwara Toshimichi、Nakano Minoru
  • Journal Title: ACS Omega Volume: 2 Issue: 6 Pages: 2935-2944
  • DOI: 10.1021/acsomega.7b00424
  • Peer Reviewed / Open Access
• [Journal Article] Conformational states of HAMP domains interacting with sensory 1 rhodopsin membrane 2 systems: An integrated all-atom and coarse-grained molecular dynamics simulation approach (2017)
  • Author(s): Sahoo B.R., and Fujiwara T.
  • Journal Title: Molecular BioSystems Volume: 13 Issue: 1 Pages: 193-207
  • DOI: 10.1039/c6mb00730a
  • Peer Reviewed
• [Journal Article] Structure determination of uniformly 13C, 15N labeled protein using qualitative distance restraints from MAS solid-state 13C-NMR observed paramagnetic relaxation enhancement (2016)
  • Author(s): H. Tamaki, A. Egawa, K. Kido, T. Kameda, M. Kamiya, T. Kikukawa, T. Aizawa, T. Fujiwara, M. Demura
  • Journal Title: Journal of Biomolecular NMR Volume: 64 Issue: 1 Pages: 87-101
  • DOI: 10.1007/s10858-015-0010-0
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Advanced instrumentation for DNP-enhanced MAS NMR for higher magnetic fields and lower temperatures (2016)
  • Author(s): Matsuki Y., Idehara T, Fukazawa J., and Fujiwara T.
  • Journal Title: Journal of Magnetic Resonance Volume: 264 Pages: 107-115
  • DOI: 10.1016/j.jmr.2016.01.022
  • Peer Reviewed
• [Journal Article] Membrane Mediated Antimicrobial and Antitumor Activity of Cathelicidin 6: Structural Insights from Molecular Dynamics Simulation on Multi-microsecond Scale (2016)
  • Author(s): Sahoo B.R., and Fujiwara T.
  • Journal Title: POLOS ONE Volume: 11(7) Issue: 7 Pages: e0158702-e0158702
  • DOI: 10.1371/journal.pone.0158702
  • Peer Reviewed
• [Journal Article] Closed-cycle cold helium magic-angle spinning for sensitivity-enhanced multi-dimensional solid-state NMR (2015)
  • Author(s): Yoh Matsuki; Shinji Nakamura; Shigeo Fukui; Hiroto Suematsu and Toshimichi Fujiwara
  • Journal Title: J. Magn. Reson. Volume: 259 Pages: 76-81
  • DOI: 10.1016/j.jmr.2015.08.003
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] High-Speed FrequencyModulation of a 460-GHz Gyrotron for Enhancement of 700-MHz DNP-NMR Spectroscopy (2015)
  • Author(s): T. Idehara, E. M. Khutoryan, Y. Tatematsu, Y. Yamaguchi, A. N. Kuleshov, O. Dumbrajs, Y. Matsuki, T. Fujiwara
  • Journal Title: Journal of Infrared, Millimeter, and Terahertz Waves Volume: 36 Issue: 9 Pages: 819-829
  • DOI: 10.1007/s10762-015-0176-2
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The crystal structure of the plant small GTPase OsRacl reveals its mode of binding to NADPH oxidase. (2015)
  • Author(s): Kosami KI, Ohki I, Nagano M, Furuita F, Sugiki T, Kawano Y, Kawasaki T, Fujiwara T, Nakagawa A, Shimamoto K, Kojima C
  • Journal Title: The Journal of Biological Chemistry Volume: 289 Issue: 41 Pages: 28569-78
  • DOI: 10.1074/jbc.m114.603282
  • Description: (in press)(印刷中)
  • Peer Reviewed
• [Journal Article] Active-Site Structure of Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR (2014)
  • Author(s): Kang S, Todokoro Y, Yumen I, Shen B, Iwasaki I, Suzuki T, Miyagi A, Morikawa K, Yoshida M, Fujiwara T, Akutsu H
  • Journal Title: Biophys J Volume: 106 Issue: 2 Pages: 390-398
  • DOI: 10.1016/j.bpj.2013.12.005
  • Peer Reviewed / Open Access
• [Journal Article] Broadband Continuously Frequency Tunable Gyrotron for 600 MHz DNP-NMR Spectroscopy (2014)
  • Author(s): Ryosuke Ikeda, Yuusuke Yamaguchi, Yoshinori Tatematsu, Toshitaka Idehara, Isamu Ogawa, Teruo Saito, Yoh Matsuki and Toshimichi Fujiwara
  • Journal Title: Plasma and Fusion Research: Rapid Commun. Volume: 9 Pages: 1206058-1206058
  • NAID: 130005089499
  • Peer Reviewed / Open Access
• [Journal Article] ^<1>H, ^<15>N and ^<13>C resonance assignments of the conserved region in the middle domain of S. pombe Sin1 protein. (2014)
  • Author(s): Kataoka S, Furuita K Hattori Y, Kobayashi N, Ikegami T, Shiozaki K, Fujiwara T, Kojima C
  • Journal Title: Biomolecular NMR Assignments Volume: (印刷中) Issue: 1 Pages: 89-92
  • DOI: 10.1007/s12104-014-9550-6
  • Peer Reviewed
• [Presentation] rization with double 460 GHz gyrotrons for the sensitivity enhancement of magic-angle-spinning NMR at 30 K (2017)
  • Author(s): 藤原敏道
  • Organizer: Asia Pacific NMR Symposium
  • Place of Presentation: バンガロール(インド）
  • Year and Date: 2017-02-16
  • Int'l Joint Research / Invited
• [Presentation] High-Field DNP and Cellular Solid-State NMR Using Paramagnetism under Low Temperatures (2017)
  • Author(s): Toshimichi Fujiwara
  • Organizer: Taiwan-Japan Biomedical Symposium on Magnetic Resonance
  • Int'l Joint Research / Invited
• [Presentation] High-Field DNP and Cellular Solid-State NMR Using Paramagnetism under Low Temperatures (2017)
  • Author(s): Toshimichi Fujiwara
  • Organizer: Telluride workshop on DNP and Paramagnetic NMR
  • Int'l Joint Research / Invited
• [Presentation] High-Field DNP and Quantitative Cellular Solid-State NMR under Low Temperatures (2017)
  • Author(s): Toshimichi Fujiwara
  • Organizer: The 20th International Society of Magnetic Resonance
  • Int'l Joint Research
• [Presentation] 定量固体NMRを用いた単一大腸菌細胞内で合成される分子種の同定および分子数の計測 (2016)
  • Author(s): 山田和哉
  • Organizer: NMR討論会（55）
  • Place of Presentation: 広島国際会議場（広島県，広島市中区）
  • Year and Date: 2016-11-15
• [Presentation] In-cell solid-state NMR spectroscopy: New technology for probing atomic structure and location of macromolecule in living cells (2016)
  • Author(s): Khampa Chalermpon
  • Organizer: National Tsing Hua University - Osaka University Joint Symposium
  • Place of Presentation: 新竹市（台湾）
  • Year and Date: 2016-09-22
  • Int'l Joint Research
• [Presentation] Solid State NMR Spectroscopy for Exploring the Location of Macromolecules in Living Cells (2016)
  • Author(s): Khampa Chalermpon
  • Organizer: International Conference on Magnetic Resonance in Biological Systems (ICMRBS2016)
  • Place of Presentation: 国立京都国際会館（京都府，京都市左京区）
  • Year and Date: 2016-08-21
  • Int'l Joint Research
• [Presentation] Quantitative in-cell solid-state NMR for counting the number of molecules synthesized in a ubiquitin-overexpressed intact Escherichia coli cell”, (2016)
  • Author(s): 山田和哉
  • Organizer: Experimental Nuclear Magnetic Resonance Conference
  • Place of Presentation: アメリカ（ピッツバーグ）
  • Year and Date: 2016-04-10
  • Int'l Joint Research
• [Presentation] High-Field DNP and Cellular Solid-State NMR Using Paramagnetism under Low Temperatures (2016)
  • Author(s): 藤原敏道
  • Organizer: lnternational Conference on Magnetic Resonance in Biological Systems 2016
  • Place of Presentation: 国立京都国際会館（京都府，京都市左京区）
  • Int'l Joint Research / Invited
• [Presentation] Development of high-field DNP instrumentation for magic-angle spinning 13C-NMR at 16.4 T and 30 K (2016)
  • Author(s): 藤原敏道
  • Organizer: The 4th Awaji lnternational Workshop on Electron Spin Science & Technology
  • Place of Presentation: 夢舞台（兵庫県，淡路市）
  • Int'l Joint Research / Invited
• [Presentation] Dynamic nuclear polarization with double 460 GHz gyrotrons for the sensitivity enhancement of magic-angle-spinning NMR at 30 K (2016)
  • Author(s): 藤原敏道
  • Organizer: Symposium on Novel Magnetic Resonance Techniques in Millimeter and Terahertz Waves and their Applications to Bioscience
  • Place of Presentation: 神戸大学（兵庫県，神戸市灘区）
  • Int'l Joint Research / Invited
• [Presentation] 高磁場 DNP 法開発と生体系マジック角回転クライオ NMR への応用,ミニシンポ 1 生命をささえるタンパク質分子の電子スピン科学 (2016)
  • Author(s): 藤原敏道
  • Organizer: 電子スピンサイエンス学会年会（55）
  • Place of Presentation: 大阪市立大学（大阪府，大阪市住吉区）
  • Invited
• [Presentation] Development of the Quantitative Solid-state NMR and its Application to the Counting of the Number of Molecules in an Intact Escherichia Coli Cell”, The 27th International Conference on Magnetic Resonance in Biological Systems (2016)
  • Author(s): 山田和哉
  • Organizer: International Conference on Magnetic Resonance in Biological Systems (ICMRBS2016)
  • Place of Presentation: 国立京都国際会館（京都府，京都市左京区）
  • Int'l Joint Research
• [Presentation] Solid-state NMR analysis of proteins by spectral simulation (2015)
  • Author(s): Toshimichi Fujiwara
  • Organizer: Pacifichem 2015, Advances in Biological SS NMR
  • Place of Presentation: Royal Hawaiian, Honolulu
  • Year and Date: 2015-12-15
  • Int'l Joint Research / Invited
• [Presentation] Development of high-field DNP instrumentation for magic-angle spinning 13C-NMR at 16.4 T and 30 K andsolid-state NMR analysis of proteins (2015)
  • Author(s): Toshimichi Fujiwara
  • Organizer: International Society of Magnetic Resonance
  • Place of Presentation: Shanghai, China
  • Year and Date: 2015-08-16
  • Int'l Joint Research / Invited
• [Presentation] Development of High-Field DNP Instrumentation for Magic-Angle Spinning 13C-NMR at 16.4 T and 30 K (2015)
  • Author(s): Toshimichi Fujiwara
  • Organizer: 57th Rocky Mountain Conference on Magnetic Resonance
  • Place of Presentation: Snowbird, Utah, USA
  • Year and Date: 2015-07-26
  • Int'l Joint Research / Invited
• [Presentation] Structural analysis of proteins by solid-state NMR and sensitivity enhancement by DNP (2015)
  • Author(s): Toshimichi Fujiwara
  • Organizer: The 4th International Symposium on Drug Discovery and Design by NMR
  • Place of Presentation: RIKEN Yokohama Institute Main Lecture Hall
  • Year and Date: 2015-02-04 – 2015-02-05
  • Invited
• [Presentation] 単一細胞あたりの特定タンパク質の分子数計測のための定量固体NMR法の開発 (2014)
  • Author(s): 山田 和哉, 江川 文子, 藤原 敏道
  • Organizer: 第53回NMR討論会
  • Place of Presentation: 大阪大学コンベンションセンター
  • Year and Date: 2014-11-04 – 2014-11-06
• [Presentation] 固体NMRを用いた単一細胞あたりの特定タンパク質の分子数計測 (2014)
  • Author(s): 山田 和哉, 江川 文子, 藤原 敏道
  • Organizer: 第52回生物物理学会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Conformational states of HAMP domains interacting with SRII-membrane systems: A molecular dynamics approach (2014)
  • Author(s): Bikash Sahoo, Toshimichi Fujiwara
  • Organizer: 第52回生物物理学会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2014-09-25 – 2014-09-27
• [Book] Experimental Approaches of NMR Spectroscopy, Methodology and Application to Life Science and Materials Science (2017)
  • Author(s): Yoh Matsuki and Toshimichi Fujiwara
  • Total Pages: 636
  • Publisher: Springer
  • ISBN: 9789811059667
• [Remarks] 大阪大学蛋白質研究所機能構造計測学研究室
  • URL: http://www.protein.osaka-u.ac.jp/biophys/bussei.html
• [Remarks] 大阪大学 蛋白質研究所機能計測学研究室
  • URL: http://www.protein.osaka-u.ac.jp/biophys/
• [Patent(Industrial Property Rights)] NMR試料管 (2018)
  • Inventor(s): 松木陽,藤原敏道,遠藤由宇生,中村新治,根本貴宏
  • Industrial Property Rights Holder: 日本電子株式会社
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2018-081242
  • Filing Date: 2018
• [Patent(Industrial Property Rights)] NMR プローブ (2016)
  • Inventor(s): 中村新冶，松木陽，藤原敏道
  • Industrial Property Rights Holder: 中村新冶，松木陽，藤原敏道
  • Industrial Property Rights Type: 特許
  • Filing Date: 2016-01-28
  • Overseas