西坂 崇之

Mycoplasma mobile is one of the fastest gliding bacteria, gliding with a speed of 4.5 μm s-1. This gliding motility is driven by a concerted movement of 450 supramolecular motor units composed of three proteins, Gli123, Gli349, and Gli521, in the gliding motility machinery. With general experimental setups, it is difficult to obtain the information on how each motor unit works. This chapter describes strategies to decrease the number of active motor units to extract stepwise cell movements driven by a minimum number of motor units. We also describe an unforeseen motility mode in which the leg motions convert the gliding motion into rotary motion, which enables us to characterize the motor torque and energy-conversion efficiency by adding some more assumptions.

F1-ATPase is a world's smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F1-ATPase from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F1 molecule which consists of one mutant β and two wild type βs (hybrid F1). As a result, the new hybrid F1 showed two pausing angles which are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and give insights into the mechanism of driving unidirectional rotation.