Myosin V stepping mechanism

Abstract

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.

Fig. 1.

Schematic view of a single-molecule bead assay in TWT. The bead trajectory is recorded along the two directions x and z, respectively parallel and perpendicular to the actin filament.

Fig. 2.

Myosin V stepping. (A) A single myosin V moving against the optical tweezers. In this run the measured stall force is 1.6 pN. (B–D) Examples of single 36-nm steps, extracted from the curve in A. (E and F) Bead motion during a single step. The trajectory is projected along the x axis (parallel to the actin filament) and the z axis (perpendicular to the actin filament).

Fig. 3.

Average step. (A) Parallel motion (blue curve). The step requires a few milliseconds to be completed and presents a small kink (0 → 5 nm) before the steeper slope. We note the transitory increase of the mean deviation (red curve) in the last part of the step. This indicates that the steps differ in this part. This effect seems to be due to the diffusive search for the next binding site. (B) The mean distance between the bead and the surface increases when the trailing head unbinds.

Fig. 4.

Average step of myosin V for two ATP concentrations. The last part of the steps behaves similarly at different concentration of ATP. In contrast, the prestep lasts several milliseconds with 10 μM ATP (red curve) and only a few hundred microseconds at saturating ATP (blue curve).

Fig. 5.

Myosin V stepping model. (Upper) Example of a step, in which only the first transition (A ⇄ B) is reversible, whereas B → C and C → A′ are irreversible. (Lower) This schematic view of the myosin dimer stages during the step was suggested by electron micrographs (32) and is consistent with our experimental data.