Abstract
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.
Highlights
Myosin is an ATP-driven linear molecular motor that produces force and unidirectional movement along actin filaments
The ‘‘swinging lever arm’’ hypothesis proposes that small nucleotidedependent movements at the catalytic ATPase active site are amplified by rotation of the myosin ‘‘lever arm,’’ or ‘‘neck,’’ light chain–binding domain that extends from the motor domain, or ‘‘head’’ [1,2]
We observed the ATP-dependent foot orientation and its stabilizing on individual myosin Va molecules in real time under an optical microscope; we show that the lifted foot of walking myosin Va is oriented in a ‘‘toe-down’’ conformation so that binding to a forward site on actin is preferred largely over backward or adjacent sites
Summary
Myosin is an ATP-driven linear molecular motor that produces force and unidirectional movement along actin filaments. The ‘‘recovery stroke’’ is the essential motion that primes, or ‘‘cocks,’’ the lever arm in the pre-power stroke position while myosin is detached from actin. These strokes are the basis for the physiological functions of all characterized myosin motors. Myosin Va moves processively along actin filament and takes unidirectional ‘‘steps’’ [5] in which it alternately places its two heads in forward positions ,72 nm away from a previous binding site [6], analogous to human bipedal walking. The fluctuations are random [7], the power stroke of the bound head [4,9] tilts the neck via ‘‘lever action’’ and moves the junction (i.e., the pivot point for the fluctuations) forward, thereby favoring binding of the detached head to a forward site
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