Myosin V stepping mechanism.

We observe the myosin V stepping mechanism by traveling wave tracking. This technique, associated with optical tweezers, allows one to follow a scattering particle in a two-dimensional plane, with nanometer accuracy and a temporal resolution in the microsecond range. We have observed that, at the millisecond time scale, the myosin V combines longitudinal and vertical motions during the step. Because at this time scale the steps appear heterogeneous, we deduce their general features by aligning and averaging a large number of them. Our data show that the 36-nm step occurs in three main stages. First, the myosin center of mass moves forward 5 nm; the duration of this short prestep depends on the ATP concentration. Second, the motor performs a fast motion over 23 nm; this motion is associated to a vertical movement of the myosin center of mass, whose distance from the actin filament increases by 6 nm. Third, the myosin head freely diffuses toward the next binding site and the vertical position is recovered. We propose a simple model to describe the step mechanism of the dimeric myosin V.