The F 1 motor is a rotating molecular motor that ensures a tight chemomechanical coupling between ATP hydrolysis/synthesis reactions and rotation steps. However, the mechanism underlying this tight coupling remains to be elucidated. In this study, we used electrorotation in single-molecule experiments using an F 1 bE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F 1 , indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type. Experiments on the ATPase activity after heat treatment and gel filtration of the a 3 b 3 -subcomplex revealed the unstable structure of the bE190D mutant. Our results suggest that the tight chemomechanical coupling of the F 1 motor relies on the structural stability of F 1 . We also discuss the difference between the stepping torque and the stall torque.

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F1-ATPase is a motor protein driven by ATP hydrolysis. It converts chemical free energy change into the kinetic energy of the rotation. Toyabe examined the external torque dependency of the rotation rate of wild type F1-ATPase (PANS 2011). He found the maximum work estimated from stall torque is nearly equal to the free energy change of ATP hydrolysis, reached conclusion of high energy efficiency. Thus, interest issue is to find out the mechanism of this high efficiency. In the previous annual meeting, we reported that βE190D mutant exhibited response to external torque different from WT. Furthermore, we found that the stall torque was smaller than that of WT. Here, we discuss origin of the characteristic response of βE190D mutant to the external torque. The MS-ring is a part of the bacterial flagellar basal structure embedded in the inner membrane and composed of membrane protein FliF. It has two transmembrane segments and a large periplasmic region. When Vibrio alginolyticus fliF is overexpressed in Escherichia coli, FliF is equally detected from soluble and membrane fractions. Size exclusion chromatography showed that soluble FliF behaves as an oligomer. 1P164 軸糸外腕ダイニンによるトルク発生Here we examined several conditions for Vibrio FliF purification and found that it was eluted at smaller molecular size from a gel filtration chromatography, when solubilized by detergent at an alkaline condition (pH 11). We will optimize the purification procedures to purify FliF as a monomer and crystallize. In the last decade, the in vivo biophysical research techniques have been developed. However most methods for structural biology have been limited to the in vitro level. Hence, we revealed the structure of proteins in cells with cryo-ET. We focus on the intracellular structure of filopodia, and we successfully obtained the 3D volumes of filopodia with cryo-ET. In filopodia, actin filaments were bundled tightly and parallelly. They were bundled by actin bundling protein, presumably assigned as fascia. It bound to actin filaments in 36 nm period, which is equal to the half pitch of actin filaments. 1P168 クライオ電子線トモグラフィー法を用いたフィロポディア内 構造解析In the next step, we are introducing 3D subtomogram averaging techniques to improve the resolution of intracellular protein structures.