Microsecond resolved single molecule fluorescence measurements triggered by solution mixing

• Project/Area Number: 18H02382
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: Tohoku University
• Principal Investigator: Takahashi Satoshi 東北大学, 多元物質科学研究所, 教授 (30283641)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2020: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2018: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000)
• Keywords: 一分子蛍光分光法 / 溶液混合装置 / 溶液混合 / タンパク質フォールディング / ライン型共焦点顕微鏡 / ポリアラニン / ヘリックス・コイル構造転移 / 一分子蛍光観察 / タンパク質ダイナミクス / ライン共焦点顕微鏡 / 蛋白質ダイナミクス / 一分子蛍光測定 / 蛋白質フォールディング / タンパク質 / フォールディング

Outline of Final Research Achievements

We used single molecule fluorescence spectroscopy and revealed the structures and dynamics of biological macromolecules. We developed rapid solution mixing device that can mix two solutions quickly, and enabled the system to be used in combination with the ultrafast single molecule fluorescence detection techniques. We also developed the nano-second region fluorescence correlation spectroscopic method. Using the systems, we investigated the dynamics of various proteins including the wild type of F1-ATPase, the fluorescence intensity fluctuation in the poly-alanine peptide, and the structures of a droplet forming protein, the RGG domain of LAF1.

Academic Significance and Societal Importance of the Research Achievements

本研究において、揺らぎながら生体中で機能を果たすタンパク質の運動を一分子レベルで観察するための基礎技術を開発した。さらに、開発した技術を用いてさまざまなタンパク質の運動を解明した。このようなデータを積み重ねることで、生体分子の機能を正しく理解し、さらには予想することが可能になると思われる。さらには、薬剤と生体分子の相互作用などの解明に資すると考えられる。

Research Products

• [Journal Article] Unique Electronic Structures of the Highly Ruffled Hemes in Heme-Degrading Enzymes ofStaphylococcus aureus, IsdG and IsdI, by Resonance Raman and Electron Paramagnetic Resonance Spectroscopies (2020)
  • Author(s): Takahashi Satoshi、Nambu Shusuke、Matsui Toshitaka、Fujii Hiroshi、Ishikawa Haruto、Mizutani Yasuhisa、Tsumoto Kouhei、Ikeda-Saito Masao
  • Journal Title: Biochemistry Volume: 59 Issue: 40 Pages: 3918-3928
  • DOI: 10.1021/acs.biochem.0c00731
  • Peer Reviewed
• [Journal Article] Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains (2020)
  • Author(s): Kamagata Kiyoto、Kanbayashi Saori、Honda Masaya、Itoh Yuji、Takahashi Hiroto、Kameda Tomoshi、Nagatsugi Fumi、Takahashi Satoshi
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 580-580
  • DOI: 10.1038/s41598-020-57521-w
  • Peer Reviewed / Open Access
• [Journal Article] The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA (2020)
  • Author(s): Kamagata Kiyoto、Ouchi Kana、Tan Cheng、Mano Eriko、Mandali Sridhar、Wu Yining、Takada Shoji、Takahashi Satoshi、Johnson Reid C
  • Journal Title: Nucleic Acids Research Volume: 48 Issue: 19 Pages: 10820-10831
  • DOI: 10.1093/nar/gkaa799
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking (2020)
  • Author(s): Subekti D. R. G., Murata A., Itoh Y., Takahashi S., Kamagata K.
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 13697-13697
  • DOI: 10.1038/s41598-020-70763-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 一分子蛍光分光法を用いた天然変性タンパク質の構造計測 (2020)
  • Author(s): 髙橋 聡・小井川浩之
  • Journal Title: 現代化学増刊「相分離生物学の全貌」 Volume: 46 Pages: 373-377
• [Journal Article] Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution (2018)
  • Author(s): Takahashi Satoshi、Yoshida Aya、Oikawa Hiroyuki
  • Journal Title: Biophysical Reviews Volume: 印刷中 Issue: 2 Pages: 363-373
  • DOI: 10.1007/s12551-018-0405-8
  • Peer Reviewed / Open Access
• [Journal Article] Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors (2018)
  • Author(s): Hiroyuki Oikawa、Takumi Takahashi、Supawich Kamonprasertsukac、Satoshi Takahashi
  • Journal Title: Physical Chemistry Chemical Physics Volume: 20 Issue: 5 Pages: 3277-3285
  • DOI: 10.1039/c7cp06268k
  • Peer Reviewed
• [Journal Article] Intrinsically disordered domain of tumor suppressor p53 facilitates target search by ultrafast transfer between different DNA strands (2018)
  • Author(s): Itoh Yuji、Murata Agato、Takahashi Satoshi、Kamagata Kiyoto
  • Journal Title: Nucleic Acids Research Volume: 46 Issue: 14 Pages: 7261-7269
  • DOI: 10.1093/nar/gky586
  • Peer Reviewed / Open Access
• [Presentation] タンパク質のフォールディング研究：物性研究と生命研究の境界 (2020)
  • Author(s): 高橋 聡
  • Organizer: 東北大学生命科学研究科主催「勉強会：それ、生命に聴いてみよう！」
  • Invited
• [Presentation] Conformational properties of the intrinsically-disordered RGG domain of LAF-1 detected by single-molecule fluorescence spectroscopy (2020)
  • Author(s): Kimura, M.; Nakano, S.; Takahashi, Y.; Oikawa, H.; Takahashi, S.
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] 天然変性タンパク質 LAF-1 RGGドメインの一分子蛍光分光測定による構造特性評価 (2020)
  • Author(s): 木村美智子, 中野沙耶, 高橋泰人, 小井川浩之, 高橋 聡
  • Organizer: 分子科学会オンライン討論会
• [Presentation] Single Molecule Fluorescence Tracking at 10-μs Resolution: Application to Protein Folding and Functional Dynamics (2019)
  • Author(s): Satoshi Takahashi, Hiroyuki Oikawa
  • Organizer: Tohoku University's Chemistry Summer School
  • Invited
• [Presentation] Dynamics of the Helix-Coil Transition of Alanine-based Polypeptides Detected by Nanosecond Region Fluorescence Correlation Spectroscopy (2019)
  • Author(s): Supawich Kamonprasertsuk, Hiroyuki Oikawa and Satoshi Takahashi
  • Organizer: Indo-Japan mini-workshop, Kobe Japan
  • Int'l Joint Research / Invited
• [Presentation] 一分子測定によって明らかにする液液相分離のメカニズム (2019)
  • Author(s): 木村美智子，小井川浩之, 高橋聡
  • Organizer: 日本生物物理学会 東北支部会2019
• [Presentation] Observation of Tumor Suppressor p53 Search Dynamics using Sub-millisecond ResolvedSingle-molecule Fluorescence Microscopy (2019)
  • Author(s): Dwiky Rendra Graha Subekti, Agato Murata, Yuji Itoh, Satoshi Takahashi, Kiyoto Kamagata
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Dynamics of the helix-coil transition of alanine-based polypeptides detected by nanosecond region fluorescence correlation spectroscopy (2019)
  • Author(s): Supawich Kamonprasertsuk, Hiroyuki Oikawa, Satoshi Takahashi
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Engineering of genome editing protein Cas9 that slides along DNA faster and might enable efficient target search (2019)
  • Author(s): Trishit Banerjee, Dwiky Rendra Graha Subekti, Hiroto Takahashi, Satoshi Takahashi, Kiyoto Kamagata
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Microsecond-resolved observation of F1-ATPase conformational changes by single molecular fluorescence spectroscopy (2019)
  • Author(s): Hiroki Senmaru, Hiroyuki Oikawa, Mitsuhiro Sugawa, Satoshi Takahashi
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Dynamics of the helix-coil transition of alanine-based polypeptides detected by nanosecond region fluorescence correlation spectroscopy (2019)
  • Author(s): Supawich Kamonprasertsuk, 小井川浩之, 高橋聡
  • Organizer: 第13回分子科学討論会
• [Presentation] Single Molecule Fluorescence Tracking at 10-μs Resolution: Application to Protein Folding and Functional Dynamics (2018)
  • Author(s): Satoshi Takahashi, Hiroyuki Oikawa
  • Organizer: Indo-Japan mini-workshop
  • Int'l Joint Research / Invited
• [Presentation] Development of single molecule fluorescence detection system having the 10-μs time resolution: Application to protein folding and functional dynamics (2018)
  • Author(s): Satoshi Takahashi, Hiroyuki Oikawa
  • Organizer: IMRAM Taipei Tech Symposium
  • Int'l Joint Research / Invited
• [Presentation] Development of single molecule fluorescence detection system having 10-μs time resolution: Application to protein folding and functional dynamics (2018)
  • Author(s): Satoshi Takahashi and Hiroyuki Oikawa
  • Organizer: 第91回生化学会年会
  • Invited
• [Presentation] Single molecule fluorescence tracking at 10-μs resolution: Application to protein folding and functional dynamics (2018)
  • Author(s): Satoshi Takahashi and Hiroyuki Oikawa
  • Organizer: 日本生物物理学会第56回年
  • Invited