Analysis of structural changes in the Kai complex during circadian oscillation

2021 Fiscal Year Final Research Report

• 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
• 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

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.

Free Research Field

生物物理

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。