Cooperative Conformational Dynamics of Ring-Shaped ATPase studied by High-Speed AFM/Fluorescence Microscopy

2014 Fiscal Year Final Research Report

• Project/Area Number: 24241048
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Nanomaterials/Nanobioscience
• Research Institution: Kanazawa University
• Principal Investigator: UCHIHASHI TAKAYUKI 金沢大学, 数物科学系, 教授 (30326300)
• Co-Investigator(Kenkyū-buntansha): IINO Ryota 大学共同利用機関法人自然科学研究機構, 岡崎統合バイオサイエンスセンター, 教授 (70403003) ; WATANABE Yo-hei 甲南大学, 理工学部, 准教授 (40411839)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: 一分子イメージング / 走査型プローブ顕微鏡 / 高速原子間力顕微鏡 / タンパク質 / 分子シャペロン / 構造変化

Outline of Final Research Achievements

We observed conformational dynamics of a molecular chaperone ClpB which disaggregates denatured proteins. We found that the hexameric ring of the TClpB dynamically changes between symmetric, asymmetric and broken ring. Also it was indicated that the dynamic fluctuation of the ring structure is essential for the chaperon activity of ClpB.
We also improved combined system high-speed AFM and fluorescent microscopy. The combined system enabled simultaneous single-molecule imaging with high-speed AFM and fluorescence microscopy. We succeeded in observing processive movement of enzymatic chitinase with fluorophores along a chitin fibril and further conformational change of F1-ATPase and binding/dissociation of fluorescent-ATP analogue. We have applied high-speed AFM to investigate cooperative conformational change of V1-ATPase without the shaft (DF subunit). We found that conformational change of the A subunit propagates unidirectionally in the hexameric ring even without the shaft.

Free Research Field

生物物理