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Single Molecule Energetics of F₁-ATPase Motor

Eiro Muneyuki ^*, Takahiro Watanabe-Nakayama , Tetsuya Suzuki , Masasuke Yoshida , Takayuki Nishizaka   and Hiroyuki Noji  ^¶

^* Department of Physics, Faculty of Science and Technology, Chuo University, Tokyo, Japan; Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan; Department of Physics, Gakushuin University, Tokyo, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan; and ^¶ The Institute of Scientific and Industrial Research (ISIR) Osaka University, Osaka, Japan

Correspondence: Address reprint requests to Eiro Muneyuki, Dept. of Physics, Faculty of Science and Technology, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan. Tel.: 81-3-3817-1769; Fax: 81-3-3817-1792; E-mail: emuneyuk{at}phys.chuo-u.ac.jp.

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P_i in its individual actions at a single molecule level. The F₁ portion of ATP synthase, also called F₁-ATPase, is a rotary molecular motor in which the central -subunit rotates against the ₃ß₃ cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of G_ATP. The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.