Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis.

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.     

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.     

Actomyosin interactions in the presence of ATP and the N-terminal segment of actin.

The binding of myosin subfragment 1 (S-1) to actin in the presence of ATP and the acto-S-1 ATPase activities of acto-S-1 complexes were determined at 5 degrees C under conditions of partial saturation of actin, up to 90%, by antibodies against the first seven N-terminal residues on actin. The antibodies [Fab(1-7)] inhibited strongly the acto-S-1 ATPase and the binding of S-1 to actin in the presence of ATP at low concentrations of S-1, up to 25 microM. Further increases in S-1 concentration resulted in a partial and cooperative recovery of both the binding of S-1 to actin and the acto-S-1 ATPase while causing only limited displacement of Fab(1-7) from actin. The extent to which the binding and the ATPase activity were recovered depended on the saturation of actin by Fab(1-7). The combined amounts of S-1 and Fab binding to actin suggested that the activation of the myosin ATPase activity was due to actin free of Fab. Examination of the acto-S-1 ATPase activities as a function of S-1 bound to actin at different levels of actin saturation by Fab(1-7) revealed that the antibodies inhibited the activation of the bound myosin. Thus, the binding of antibodies to the N-terminal segment of actin can act to inhibit both the binding of S-1 to actin in the presence of ATP and a catalytic step in ATP hydrolysis by actomyosin. The implications of these results to the regulation of actomyosin interaction are discussed.     

A unique ATP hydrolysis mechanism of single-headed processive myosin, myosin IX

Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.     

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.     

Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay

In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.     

Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism

Mutations of myosin VIIA cause deafness in various species from human and mice to Zebrafish and Drosophila. We analyzed the kinetic mechanism of the ATPase cycle of Drosophila myosin VIIA by using a single-headed construct with the entire neck domain. The steady-state ATPase activity (0.06 s(-1)) was markedly activated by actin to yield V(max) and K(ATPase) of 1.72 s(-1) and 3.2 microm, respectively. The most intriguing finding is that the ATP hydrolysis predominantly takes place in the actin-bound form (actin-attached hydrolysis) for the actomyosin VIIA ATPase reaction. The ATP hydrolysis rate was much faster for the actin-attached form than the dissociated form, in contrast to other myosins reported so far. Both the ATP hydrolysis step and the phosphate release step were significantly faster than the entire ATPase cycle rate, thus not rate-determining. The rate of ADP dissociation from actomyosin VIIA was 1.86 s(-1), which was comparable with the overall ATPase cycle rate, thus assigned to be a rate-determining step. The results suggest that Drosophila myosin VIIA spends the majority of the ATPase cycle in an actomyosin.ADP form, a strong actin binding state. The duty ratio calculated from our kinetic model was approximately 0.9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure.     

Molecular dynamics simulations of evolved collective motions of atoms in the myosin motor domain upon perturbation of the ATPase pocket

A crucial point for mechanical force generation in actomyosin systems is how the energy released by ATP hydrolysis in the myosin motor domain gives rise to the movement of the myosin head along the actin filament. We assumed the signal of the ATP hydrolysis to be transmitted as modulated atomic vibrations from the nucleotide-binding site throughout the myosin head, and carried out 1-ns all-atom molecular dynamics simulations for that signal transmission. We distributed the released energy to atoms located around the ATPase pocket as kinetic energies and examined how the effect of disturbance extended throughout the motor domain. The result showed that the disturbance signal extended over the motor domain in 150 ps and induced slowly varying collective motions of atoms at the actin-binding site and the junction with the neck, both of which are relevant to the movement of the myosin head along the actin filament. We also performed a principal component analysis of thermal atomic motions for the motor domain, and the first principal component was consistent with the response to the disturbance given to the ATPase pocket.     

Simulations of the myosin II motor reveal a nucleotide-state sensing element that controls the recovery stroke

During the recovery stroke, the myosin motor is primed for the next power stroke by a 60° rotation of its lever arm. This reversible motion is coupled to the activation of the ATPase function of myosin through conformational changes along the relay helix, which runs from the Switch-2 loop near the ATP to the converter domain carrying the lever arm. Via a hydrogen bond between the side-chain of Asn475 on the relay helix and the Gly457/Ser456 peptide group on the Switch-2, the rotation of the converter domain is coupled to the formation of a hydrogen bond between Gly457 and γ-phosphate that is essential for ATP hydrolysis. Here, molecular dynamics simulations of Dictyostelium discoideum myosin II in the two end conformations of the recovery stroke with different nucleotide states (ATP, ADP·Pi, ADP) reveal that the side-chain of Asn475 breaks away from Switch-2 upon ATP hydrolysis to make a hydrogen bond with Tyr573. This sensing of the nucleotide state is achieved by a small displacement of the cleaved γ-phosphate towards Gly457 which in turn pushes Asn475 away. The sensing plays a dual role by (i) preventing the wasteful reversal of the recovery stroke while the nucleotide is in the ADP·Pi state, and (ii) decoupling the relay helix from Switch-2, thus allowing the power stroke to start upon initial binding to actin while Gly457 of Switch-2 keeps interacting with the Pi (known to be released only later after tight actin binding). A catalytically important salt bridge between Arg238 (on Switch-1) and Glu459 (on Switch-2), which covers the hydrolysis site, is seen to form rapidly when ATP is added to the pre-recovery stroke conformer and remains stable after the recovery stroke, indicating that it has a role in shaping the ATP binding site by induced fit.     

Effect of trinitrophenylation on myosin ATPase

1. The enzymic and actin binding properties of myosins trinitrophenylated to different extents in the presence or absence of ATP have been studied.

2. The enzymic properties of myosin trinitrophenylated in the absence of ATP are different from those of myosin treated in the presence of ATP even on trinitrophenylating an equal number of lysyl residues. On trinitrophenylation in the absence of ATP the EDTA-(K⁺-)activated ATPase and Ca²⁺-activated ATPase decrease while the Mg²⁺-activated ATPase considerably increases. In the presence of ATP the enzymic properties of myosin are much less affected by trinitrophenylation.

3. The actin binding capacity of trinitrophenylated myosin does not change, although its enzymic properties may be greatly altered, and even if its property to be activated by actin is completely lost.     

Coupling of Lever Arm Swing and Biased Brownian Motion in Actomyosin

An important unresolved problem associated with actomyosin motors is the role of Brownian motion in the process of force generation. On the basis of structural observations of myosins and actins, the widely held lever-arm hypothesis has been proposed, in which proteins are assumed to show sequential structural changes among observed and hypothesized structures to exert mechanical force. An alternative hypothesis, the Brownian motion hypothesis, has been supported by single-molecule experiments and emphasizes more on the roles of fluctuating protein movement. In this study, we address the long-standing controversy between the lever-arm hypothesis and the Brownian motion hypothesis through in silico observations of an actomyosin system. We study a system composed of myosin II and actin filament by calculating free-energy landscapes of actin-myosin interactions using the molecular dynamics method and by simulating transitions among dynamically changing free-energy landscapes using the Monte Carlo method. The results obtained by this combined multi-scale calculation show that myosin with inorganic phosphate (P_i) and ADP weakly binds to actin and that after releasing P_i and ADP, myosin moves along the actin filament toward the strong-binding site by exhibiting the biased Brownian motion, a behavior consistent with the observed single-molecular behavior of myosin. Conformational flexibility of loops at the actin-interface of myosin and the N-terminus of actin subunit is necessary for the distinct bias in the Brownian motion. Both the 5.5–11 nm displacement due to the biased Brownian motion and the 3–5 nm displacement due to lever-arm swing contribute to the net displacement of myosin. The calculated results further suggest that the recovery stroke of the lever arm plays an important role in enhancing the displacement of myosin through multiple cycles of ATP hydrolysis, suggesting a unified movement mechanism for various members of the myosin family.     

Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins

The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.     

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.     

Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

Metal cation controls myosin and actomyosin kinetics

We have perturbed myosin nucleotide binding site with magnesium‐, manganese‐, or calcium‐nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time‐resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation‐specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.     

Mechanism of formation of actomyosin interface

Force generation in muscle results from binding of myosin to F-actin. ATP binding to myosin provides energy to dissociate actomyosin complex while the hydrolysis of ATP is needed for re-binding of myosin to F-actin. At the end of each cycle myosin and actin form a tight complex with a substantial interface area. We investigated the dynamics of formation of actomyosin interface in presence and absence of nucleotides by quenched flow cross-linking technique. We showed previously that myosin head (subfragment 1, S1) directly interacts with at least two monomers in the actin filament. The quenched flow cross-linking experiments revealed that the initial contact (in presence or absence of nucleotides) occurs between loop 635-647 of S1 and 1-12 N-terminal residues of one actin and, then, the second contact forms between loop 567-574 of S1 and the N terminus of the second actin. The distance between these two loops in S1 corresponds to the distance between N termini of two actins in the same strand (53 A) but is smaller than that between two actins from the different strands (102 A). The formation of the actomyosin complex proceeds in ordered sequence: S1 initially binds to one actin then binds with the second actin located in the same strand but probably closer to the barbed end of F-actin. The presence of nucleotides slows down the interaction of S1 with the second actin, which correlates with recently proposed cleft movement in a 50 kDa domain of S1. The sequential mechanism of formation of actomyosin interface starting from one end and developing towards the barbed end might be involved in force generation and directional movement in actin-myosin system.     

Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction

Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.     

Antibody against the amino terminus of alpha-actin inhibits actomyosin interactions in the presence of ATP

Actomyosin interactions in the presence of ATP were examined by using site-specific antibodies directed against the first seven N-terminal residues on skeletal alpha-actin. Fab fragments of these antibodies (S alpha N Fab) inhibited effectively the actin-activated ATPase of myosin subfragment 1 (S-1) at both 5 and 25 degrees C. Binding experiments carried out in the presence of ATP at 5 degrees C revealed that the catalytic inhibition was related to the inhibition of S-1 binding to actin by Fab. At equimolar ratios of Fab to actin, the binding of S-1 to actin and the activated ATPase were inhibited by 75 and 82%, respectively. These results, when contrasted with the small effect of Fab on rigor actomyosin binding, suggest ATP-induced changes at the interface of actin and myosin.     

Kinetic mechanism of human myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.