Single-molecule pull-out manipulation of the shaft of the rotary motor F1-ATPase.

F1-ATPase is a rotary motor protein in which the central γ-subunit rotates inside the cylinder made of α3ß3 subunits. To investigate interactions between the γ shaft and the cylinder at the molecular scale, load was imposed on γ through a polystyrene bead by three-dimensional optical trapping in the direction along which the shaft penetrates the cylinder. Pull-out event was observed under high-load, and thus load-dependency of lifetime of the interaction was estimated. Notably, accumulated counts of lifetime were comprised of fast and slow components. Both components exponentially dropped with imposed loads, suggesting that the binding energy is compensated by the work done by optical trapping. Because the mutant, in which the half of the shaft was deleted, showed only one fast component in the bond lifetime, the slow component is likely due to the native interaction mode held by multiple interfaces.

A change in the radius of rotation of F1-ATPase indicates a tilting motion of the central shaft.

F(1)-ATPase is a water-soluble portion of F(o)F(1)-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit γ has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit γ between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a ~4° outward tilting of the γ-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the γ-subunit and provides a new constraint condition of the ATP-waiting conformation of F(1)-ATPase, which has not been determined as an atomic structure from x-ray crystallography.