Molecular motors such as kinesin, myosin, and F(1)-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems. Here, we describe a simple dark-field imaging system that employs objective-type evanescent illumination to selectively illuminate a thin layer on the coverslip and thus yield images with high signal/noise ratios. Only by substituting the dichroic mirror in conventional objective-type total internal reflection fluorescence microscope with a perforated mirror, were nanometer spatial precision and microsecond temporal resolution simultaneously achieved. This system was applied to the study of the rotary mechanism of F(1)-ATPase. The fluctuation of a gold nanoparticle attached to the gamma-subunit during catalytic dwell and the stepping motion during torque generation were successfully visualized with 9.1-mus temporal resolution. Because of the simple optics, this system will be applicable to various biophysical studies requiring high spatial and temporal resolution in vitro and also in vivo.

In recent years, theories of nonequilibrium statistical mechanics such as the fluctuation theorem (FT) and the Jarzynski equality have been experimentally applied to micro and nanosized systems. However, so far, these theories are seldom applied to autonomous systems such as motor proteins. In particular, representing the property of entropy production in a small system driven out of equilibrium, FT seems suitable to be applied to them. Hence, for the first time, we employed FT in the single molecule experiments of the motor protein F1-adenosine triphosphatase (F1), in which the rotor γ subunit rotates in the stator α3β3 ring upon adenosine triphosphate hydrolysis. We found that FT provided the better estimation of the rotary torque of F1 than the conventional method.

Enzymatic hydrolysis of nucleotide triphosphate (NTP) plays a pivotal role in protein functions. In spite of its biological significance, however, the chemistry of the hydrolysis catalysis remains obscure because of the complex nature of the reaction. Here we report a study of the molecular mechanism of hydrolysis of adenosine triphosphate (ATP) in F(1)-ATPase, an ATP-driven rotary motor protein. Molecular simulations predicted and single-molecule observation experiments verified that the rate-determining step (RDS) is proton transfer (PT) from the lytic water molecule, which is strongly activated by a metaphosphate generated by a preceding P(γ)-O(β) bond dissociation (POD). Catalysis of the POD that triggers the chain activation of the PT is fulfilled by hydrogen bonds between Walker motif A and an arginine finger, which commonly exist in many NTPases. The reaction mechanism unveiled here indicates that the protein can regulate the enzymatic activity for the function in both the POD and PT steps despite the fact that the RDS is the PT step.

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis͞hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80°substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of Ϸ30 M and a Vmax of Ϸ350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 ؋ 10 7 M ؊1 ⅐s ؊1 . At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5-[␥-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of Ϸ80°. The rotational behavior of FoF1 resembles that of F1. This finding indicates that ''friction'' in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.ATP hydrolysis ͉ binding change mechanism ͉ membrane protein ͉ single-molecule imaging