Myosin-V stepping kinetics: A molecular modelfor processivityMatthias Rief*, Ronald S. Rock*, Amit D. Mehta†, Mark S. Mooseker‡, Richard E. Cheney§, and James A. Spudich*¶*Department of Biochemistry, Stanford University Medical Center, Stanford, CA 94305;†Howard Hughes Medical Institute, The Rockefeller University, 1230York Avenue, New York, NY 10021-6399;‡Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; and§Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599Contributed by James A. Spudich, June 7, 2000Myosin-V is a molecular motor that moves processively along itsactin track. We have used a feedback-enhanced optical trap toexamine the stepping kinetics of this movement. By analyzing thedistribution of time periods separating discrete ⬇36-nm mechan-ical steps, we characterize the number and duration of rate-limitingbiochemical transitions preceding each such step. These data showthat myosin-V is a tightly coupled motor whose cycle time is limitedby ADP release. On the basis of these results, we propose a modelfor myosin-V processivity.Class-V myosins, two-headed actin-based motors (1), havebeen implicated in several forms of organelle transport (2).The various roles of molecular motors require special kineticadaptations (3). Unlike muscle myosin-II, which assembles inlarge arrays, myosin-V is a processive motor (4), meaning thatone molecule can undergo multiple productive catalytic cyclesand associated mechanical steps before it detaches from its track.To understand the mechanism for chemomechanical transduc-tion, one must decipher the kinetic scheme underlying ATPturnover and movement. Presteady-state kinetic studies havehelped clarify such mechanisms in many motor proteins (5). Inthe case of myosin-V, kinetic characterization of truncatedsingle-headed constructs in bulk studies has contributed impor-tant insights into the myosin-V ATPase cycle (6, 7). However, tounderstand the mechanism for myosin-V processivity, it isessential to study the full-length double-headed dimer throughthe course of its movement. In the present study, we used a forcefeedback-enhanced laser trap to measure the stepping rate ofmyosin-V molecules purified from brain. This allowed us tocharacterize the rate-limiting transition in the turnover cycle.Materials and MethodsBead Preparation. One-microliter Polystyrene beads (Ø 356 nm,Polysciences, 2.5% solid) were incubated for 15 min in 99lofbuffer (25 mM imidazole HCl, pH 7.4兾25 mM KCl兾1mMEGTA兾10 mM DTT兾4 mM MgCl2) containing 10g兾ml BSA(to preblock the surface), 1g兾ml tetramethyl rhodamine-labeled BSA, and 30 pM tissue-purified chick-brain myosin-V[purification as described in (8)]. Buffer conditions during theexperiment were as in ref. 4.Optical Trap. Beads were optically trapped and positioned near afluorescently labeled biotinylated actin filament immobilizedonto an avidin-coated coverslip. Imaging and trap steering wereas described (9–11). A feedback loop (M44 DSP-board, Inno-vative Integration, West Lake Village, CA) maintains a constantseparation between the bead and trap centers. This distancescales with the load experienced by the molecule as it steps alongthe actin filament. The trap stiffness was calibrated for eachtrapped bead from the amplitude of the thermal diffusion. Forsome beads, it was also calibrated by measurement of the beadrise time in response to sudden trap displacement and by the3-dB corner frequency in the diffusion power spectrum. Thethree methods gave consistent results.Results and DiscussionPolystyrene beads, sparsely coated with myosin-V molecules,were optically trapped in a focused laser beam and positionednear a surface-immobilized actin filament (Fig. 1A). To confirmthat a single molecule is sufficient to generate the movementobserved, we examined the fraction of beads that bind and moveprocessively on an actin filament as a function of myosin-V兾beadSee commentary on page 9357.¶To whom reprint requests should be addressed. E-mail: [email protected] publication costs of this article were defrayed in part by page charge payment. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.§1734 solely to indicate this fact.Fig. 1. (A) Experimental scheme for single-molecule myosin-V experiments.(B) The fraction of beads moving continuously (⬎four steps) along actin isrecorded as a function of the molar ratio of incubated beads to myosin-Vmolecules. Values are displayed as mean ⫾公[f䡠(1-f)兾N]. The probability that abead carries one or more motors is 1-exp(⫺c), where c is the molar ratio ofmyosin-V molecules to beads during the incubation andis a fit parameter(12) accounting for the fact that not all myosin-V molecules incubated to-gether with the beads will find a bead or adsorb in a functional conformation.The data can be well fit by this functional form (solid line,⫽ 0.2, reduced2⫽0.04), showing that a single molecule is sufficient to move a bead. The datacannot be fit assuming that two or more molecules per bead are required formovement (dashed line, reduced2⫽ 0.98). When the trap was turned offduring processive stepping, the bead continued to advance for ⬎1m beforedissociating. (C) Experimental scheme of the force feedback enhanced lasertrap. A feedback loop keeps the distance between the bead center (gray curve)and the trap center (lower black curve) constant as the myosin-V moleculesteps along the actin filament. Thus the myosin-V molecule is always keptunder constant load. The thin black line within the gray curve is a filtered beadposition signal (box filter, 15 ms).9482–9486兩PNAS兩August 15, 2000兩vol. 97兩no. 17stoichiometry. Observed Poisson statistics (Fig. 1B) confirm thatsingle myosin-V molecules are sufficient to move beads (12). Theexperiments described here used protein-to-bead stoichiome-tries low enough that less than 33% of the beads were moving.Assuming a myosin-V molecule has 100 nm of reach, ⬎95% ofmoving beads should be driven by only one molecule (13).If the motor moves the bead against a stationary optical trap,two problems complicate the experiment: compliant linkagescan absorb some of the protein displacement, making theobserved bead advance less than the motor (4, 14–18), and themotor can take relatively few steps before facing prohibitiveresistance (4). To circumvent these
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