Measurement of enzyme rotation in the ATP synthesis reaction using fluorescent molecule burning and fluorescence recovery

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K19210
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: HISABORI TORU 東京工業大学, 国際先駆研究機構, 特任教授 (40181094)
• Co-Investigator(Kenkyū-buntansha): 石内 俊一 東京工業大学, 理学院, 教授 (40338257)
• Project Period (FY): 2021-07-09 – 2024-03-31
• Keywords: ATP合成酵素 / 完全複合体 / 回転 / 蛍光標識 / 酸化還元制御 / ATP合成活性

Outline of Final Research Achievements

This study aimed to demonstrate the rotation of ATP synthase during ATP synthesis using fluorescent molecules that undergo photobleaching and fluorescence recovery. The following key findings were obtained. (1) ATP synthase from the green alga Chlamydomonas was isolated as a complete complex, and a method was established to embed the enzyme catalytic site outwardly oriented in artificial liposomes. (2) Cy3 molecules were introduced into the γ subunit as stable fluorescent labels. However, due to limitations in detection sensitivity, the current setup was unable to demonstrate enzyme rotation. (3) Molecular mechanism of the redox regulation of ATP synthesis reaction was revealed using ATP synthase incorporated into liposomes. Key amino acids on the α subunit, crucial for enzyme complex stability, were identified.

Free Research Field

植物生化学

Academic Significance and Societal Importance of the Research Achievements

生体内のATP合成反応は、生命の基幹エネルギー物質であるATPを生産する普遍的な反応である。生体膜に存在してこの反応を担うATP合成酵素の反応機構、および、特に光合成生物のATP合成酵素が特異的に持つ制御機構を解明することは、生体エネルギー分野長年の夢であり、極めて重要な研究と位置付けられる。ATPが生体内のあらゆる生命現象に対してエネルギーを供給しているその重要性を考えると、ATPの生産機構を総合的に解明することは、生物の生産性の向上などに直結する知見となるものであり、エネルギー獲得や食糧増産など人類社会に貢献する研究である。