Myosins are
ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale
nucleotide-dependent conformational changes, termed "
strokes"; the "power
stroke" is the force-generating swinging of the
myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery
stroke" is the necessary initial motion that primes, or "cocks,"
myosin while detached from actin.
Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power
stroke of the attached head that moves the joint forward. However, the power
stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery
stroke, dictate actin binding site accessibility. Here, we directly observe the recovery
stroke dynamics and fluctuations of
myosin Va using a novel, transient
caged ATP-controlling system that maintains constant
ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric
myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head.
ATP induces a transient swing of the neck to the post-recovery
stroke conformation, where it remains for ∼40 s, until
ATP hydrolysis products are released. Angle distributions indicate that the post-recovery
stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery
stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery
stroke contributes to unidirectional stepping of
myosin Va.