Analysis of structural changes in the Kai complex during circadian oscillation

• Project/Area Number: 18K14667
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: Fukuoka University
• Principal Investigator: Mutoh Risa 福岡大学, 理学部, 助教 (10622417)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: 時計タンパク質 / 藍色細菌 / 核磁気共鳴法 / 電子スピン共鳴法 / 生物時計 / NMR / ESR / スピンラベル

Outline of Final Research Achievements

In this study, we used a site-specific spin-labeled electron spin resonance (SDSL-ESR) method. An arbitrary position on KaiB was substituted by Cys residue and a spin label (MTSSL) was introduced into Cys residue. The KaiA interaction site on KaiB was determined by detecting released MTSSL from KaiB when the labeled KaiB and KaiA C-terminal domain proteins were mixed and reacted at physiological temperature. Next, ESR measurements were performed in the presence of the KaiABC mixture to verify when the spin label was released. As a result, the spin label was released 12 hours after the start of the reaction. This result indicates that the KaiA-KaiB interaction starts around 12 hours.

Academic Significance and Societal Importance of the Research Achievements

これまでにも、KaiABC三者共存下で、複合体が経時的に変化していることは報告されていた。また、電子顕微鏡解析によりKaiABC複合体構造も明らかにされた。本研究では、具体的にいつ、どこでKaiA-KaiBが相互作用するのかを明らかにし、またKaiAのN末端ドメインの構造変化を示唆するデータを得た。SDSL-ESR法を用いた本解析では、全長タンパク質を用いており、より生理的条件に近い状態を反映している。

Research Products

• [Journal Article] Energy transfer fluctuation observed by single-molecule spectroscopy of red-shifted bacteriochlorophyll in the homodimeric photosynthetic reaction center (2022)
  • Author(s): Kondo Toru、Mutoh Risa、Arai Shun、Kurisu Genji、Oh-oka Hirozo、Fujiyoshi Satoru、Matsushita Michio
  • Journal Title: The Journal of Chemical Physics Volume: 156 Issue: 10 Pages: 105102-105102
  • DOI: 10.1063/5.0077290
  • Peer Reviewed
• [Journal Article] Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy (2021)
  • Author(s): Suetake Isao、Nakazawa Shigeaki、Sato Kazunobu、Mutoh Risa、Mishima Yuichi、Kawakami Toru、Takei Toshiki、Watanabe Mikio、Sakai Norio、Fujiwara Toshimichi、Takui Takeji、Miyata Makoto、Shinohara Akira、Hojo Hironobu、Arata Toshiaki
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 567 Pages: 42-48
  • DOI: 10.1016/j.bbrc.2021.06.010
  • Peer Reviewed / Open Access
• [Journal Article] Rhythmic adenosine triphosphate release from the cyanobacterial circadian clock protein KaiC revealed by real‐time monitoring of bioluminescence using firefly luciferase (2021)
  • Author(s): Mutoh Risa、Iwata Keita、Iida Takahiro、Ishiura Masahiro、Onai Kiyoshi
  • Journal Title: Genes to Cells Volume: 26 Issue: 2 Pages: 83-93
  • DOI: 10.1111/gtc.12825
  • Peer Reviewed
• [Journal Article] Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin (2021)
  • Author(s): Tohda Rei、Tanaka Hideaki、Mutoh Risa、Zhang Xuhong、Lee Young-Ho、Konuma Tsuyoshi、Ikegami Takahisa、Migita Catharina T.、Kurisu Genji
  • Journal Title: Journal of Biological Chemistry Volume: 296 Pages: 100217-100217
  • DOI: 10.1074/jbc.ra120.016271
  • Peer Reviewed / Open Access
• [Journal Article] Cryogenic Single-Molecule Spectroscopy of the Primary Electron Acceptor in Photosynthetic Reaction Center (2020)
  • Author(s): T. Kondo, R. Mutoh, H. Tabe, G. Kurisu, H. Oh-oka, S. Fujiyoshi, and M. Matsushita
  • Journal Title: J. Phys. Chem. Lett. Volume: － Issue: 10 Pages: 3980-3986
  • DOI: 10.1021/acs.jpclett.0c00891
  • Peer Reviewed / Open Access
• [Journal Article] The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii (2020)
  • Author(s): Matsuo Takuya, Iida Takahiro, Ohmura Ayumi, Gururaj Malavika, Kato Daisaku, Mutoh Risa, Ihara Kunio, Ishiura Masahiro
  • Journal Title: PLOS GENETICS Volume: 16 Issue: 6 Pages: e1008814-e1008814
  • DOI: 10.1371/journal.pgen.1008814
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L. (2019)
  • Author(s): Grabsztunowicz Magda、Mulo Paula、Baymann Frauke、Mutoh Risa、Kurisu Genji、Setif Pierre、Beyer Peter、Krieger‐Liszkay Anja
  • Journal Title: The Plant Journal Volume: - Issue: 2 Pages: 245-256
  • DOI: 10.1111/tpj.14319
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 光化学系I-アナログフェレドキシン複合体の構造解析 (2019)
  • Author(s): 武藤梨沙、河合（久保田）寿子、池上貴久
  • Journal Title: 生物物理 Volume: 59 Pages: 32-33
  • NAID: 130007557845
  • Peer Reviewed
• [Journal Article] X-ray structure of an asymmetrical trimeric ferredoxin-photosystem I complex (2018)
  • Author(s): Kubota-Kawai Hisako、Mutoh Risa、Shinmura Kanako、Setif Pierre、Nowaczyk Marc M.、Rogner Matthias、Ikegami Takahisa、Tanaka Hideaki、Kurisu Genji
  • Journal Title: Nature Plants Volume: 4 Issue: 4 Pages: 218-224
  • DOI: 10.1038/s41477-018-0130-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Rhythmic ATP release from the cyanobacterial circadian clock protein KaiC revealed by real-time monitoring of bioluminescence (2021)
  • Author(s): Mutoh R, Iida T., Onai K.
  • Organizer: 第59回日本生物物理学会年会
  • Invited
• [Presentation] Structural study of heterochromatin protein 1 toward chemical synthesis (2021)
  • Author(s): Mutoh R., So M., Kawakami T., Takei T., Hojo H., Fujiwara T., Arata T., Suetake I.
  • Organizer: ISMAR-APNMR2021
  • Int'l Joint Research
• [Presentation] The determination of the interacting time between KaiA and KaiB during circadian oscillation. (2020)
  • Author(s): Mutoh R, Iida T., Mino H.
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] 概日リズム発振中のKaiA-KaiB相互作用時刻の決定 (2020)
  • Author(s): 武藤梨沙、飯田高広、三野広幸
  • Organizer: 電子スピンサイエンス学会2020
• [Presentation] Structural analyses of photosystem I-ferredoxin complex (2019)
  • Author(s): Mutoh, R., Kubota-Kawai, H., Muraki, N., Tanaka, H., Ikegami, T., Kurisu, G.
  • Organizer: The 6th Awaji International Workshop on “Electron Spin Science & Technology: Biological and Materials Science Oriented Applications”
  • Int'l Joint Research / Invited
• [Presentation] Protein interactions in the in vitro cyanobacterial circadian clock system revealed by SDSL-ESR (2019)
  • Author(s): Mutoh, R., Iida, T., Mino, H., Ishiura, M.
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Isolation of the Rieske/cytchrome b complex from green sulfur bacteria and interaction of the Rieske protein with cytochrome c-556 (2019)
  • Author(s): Kishimoto, H., Nagaoka, T., Azai, C., Mutoh, R., Tanaka, H., Miyanoiri, Y., Kurisu, G., Oh-oka, H.
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] 強光下における光合成膜内タンパク質の動的挙動解析 (2019)
  • Author(s): 太田明香音、武藤梨沙、得津隆太郎、皆川純、山本大輔
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] 時計タンパク質Kai複合体の機能と構造解析 (2019)
  • Author(s): 岩戸将貴、飯田高広、三島正規、池上貴久、武藤梨沙
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] ESR法を用いた藍色細菌時計タンパク質間相互作用部位の同定と経時的構造変化の解析 (2019)
  • Author(s): 武藤梨沙
  • Organizer: サントリー生有研シンポジウム2019
  • Invited
• [Presentation] 光化学系I-フェレドキシン複合体の構造解析 (2018)
  • Author(s): 武藤梨沙、河合（久保田）寿子、村木則史、池上貴久、栗栖源嗣
  • Organizer: NMR若手研究会
  • Invited
• [Presentation] X-ray structure of the type-I reaction center from Heliobacterium modesticaldum at 3.2 Å resolution (2018)
  • Author(s): T. Nakaniwa, R.Mutoh, K. Fushimi, A. Yasuda, T. Mizoguchi, H. Tamiaki, C. Azai, H. Tanaka, S. Itoh, H. Oh-oka, G. Kurisu
  • Organizer: 第56回日本生物物理学会年会
  • Invited