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Direct observation of the myosin-Va power stroke and its reversal.

ABSTRACT: Complex forms of cellular motility, including cell division, organelle trafficking or signal amplification in the auditory system, require strong coordination of the myosin motors involved. The most basic mechanism of coordination is via direct mechanical interactions of individual motor heads leading to modification of their mechanochemical cycles. Here we used an optical trap-based assay to investigate the reversibility of the force-generating conformational change (power stroke) of single myosin-Va motor heads. By applying load to the head shortly after binding to actin, we found that, at a certain load, the power stroke could be reversed, and the head fluctuated between an actin-bound pre- and a post-power stroke conformation. This load-dependent mechanical instability might be critical to coordinate the heads of processive, dimeric myosin-Va. Nonlinear response to load leading to coordination or oscillations amongst motors might be relevant for many cellular functions.

SUBMITTER: Sellers JR

PROVIDER: S-EPMC3487478 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Direct observation of the myosin-Va power stroke and its reversal.

Sellers James R JR Veigel Claudia C

Nature structural & molecular biology 20100425 5

Complex forms of cellular motility, including cell division, organelle trafficking or signal amplification in the auditory system, require strong coordination of the myosin motors involved. The most basic mechanism of coordination is via direct mechanical interactions of individual motor heads leading to modification of their mechanochemical cycles. Here we used an optical trap-based assay to investigate the reversibility of the force-generating conformational change (power stroke) of single myo ...[more]

PMID: 20418880

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Project description: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.

Dataset Information

Direct observation of the myosin-Va power stroke and its reversal.

Publications

Direct observation of the myosin-Va power stroke and its reversal.

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