Rotational dynamics of actin-bound intermediates in the myosin ATPase cycle.

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin subfragment one (MSL-S1) bound to actin in the presence of the ATP analogues AMPPNP (5'-adenylylimido diphosphate) and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)], which are believed to trap myosin in strongly and weakly bound intermediate states of the actomyosin ATPase cycle, respectively. Sedimentation binding measurements were used to determine the fraction of myosin heads bound to actin under ST-EPR conditions and the fraction of heads containing bound nucleotide. ST-EPR spectra were then corrected to obtain the spectrum corresponding to the ternary complex (actin.MSL-S1.nucleotide). The ST-EPR spectrum of MSL-S1.AMPPNP bound to actin is identical to that obtained in the absence of nucleotide (rigor complex), indicating no rotational motion of MSL-S1 relative to actin on the microsecond time scale. However, MSL-S1-ATP gamma S bound to actin is rotationally mobile, with an effective rotational correlation time (tau r) of 17 +/- 2 microseconds. This motion is similar to that observed previously for actin-bound MSL-S1 during the steady-state hydrolysis of ATP [Berger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8753-8757]. We conclude that, in solution, the weakly bound actin-attached states of the myosin ATPase cycle undergo microsecond rotational motions, while the strongly bound intermediates do not, and that these motions are likely to be involved in the molecular mechanism of muscle contraction.     

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP induces microsecond rotational motions of myosin heads crosslinked to actin.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to study the effect of ATP on the rotational dynamics of spin-labeled myosin heads crosslinked to actin (XLAS1). We have previously shown that ATP induces microsecond rotational motions in activated myofibrils or muscle fibers, but the possibility remained that the motion occurred only in the detached phase of the cross-bridge cycle. The addition of ATP to the crosslinked preparation has been shown to be a model system for active cross-bridges, presumably providing an opportunity to measure the motion in the attached state, without interference from unattached heads. In the absence of ATP, XLAS1 had very little microsecond rotational mobility, yielding a spectrum identical to that observed for uncrosslinked acto-S1. This suggests that all of the labeled S1 forms normal rigor complexes when crosslinked to actin. The addition of 5 mM ATP greatly increased the microsecond rotational mobility of XLAS1, and the effects were reversed upon depletion of ATP. The most plausible explanation for these results is that myosin heads undergo microsecond rotational motion while attached actively to actin during steady state ATPase activity. These results have important implications for the interpretation of spectroscopic data obtained during muscle contraction.     

Effects of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle cross-bridges.

We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to obtain high-resolution information about the orientational distribution, and saturation transfer EPR to measure microsecond rotational motion. At 4 degrees C and 100 mM ionic strength, we find that AMPPNP increases both the orientational disorder and the microsecond rotational motion of myosin heads. However, computer analysis of digitized spectra shows that no new population of probes is observed that does not match either rigor or relaxation in both orientation and motion. At 4 degrees C, under nearly saturating conditions of 16 mM AMPPNP (Kd = 3.0 mM, determined from competition between AMPPNP and an ADP spin label), 47.5 +/- 2.5% of myosin heads are dynamically disoriented (as in relaxation) without a significant decrease in rigor stiffness, whereas the remainder are rigidly oriented as in rigor. The oriented heads correspond to actin-attached heads in a ternary complex, and the disoriented heads correspond to detached heads, as indicated by EPR experiments with spin-labeled subfragment 1 (S1) that provide independent measurements of orientation and binding. We take these findings as evidence for a single-headed cross-bridge that is as stiff as the double-headed rigor cross-bridge. The data are consistent with a model in which, in the presence of saturating AMPPNP, one head of each cross-bridge binds actin about 10 times more weakly, whereas the remaining head binds at least 10 times more strongly, than extrinsic S1. Thus, although there is no evidence for heads being attached at nonrigor angles, the attached cross-bridge differs from that of rigor. The heterogeneous behavior of heads is probably due to steric effects of the filament lattice.     

Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction. Saturation transfer electron paramagnetic resonance.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin heads in bundles of skinned muscle fibers, under conditions of rigor, relaxation, and isometric contraction. Experiments were performed on fiber bundles perfused continuously with an ATP-regenerating system. Conditions were identical to those we have used in previous studies of myosin head orientation, except that the fibers were perpendicular to the magnetic field, making the spectra primarily sensitive to rotational motion rather than to the orientational distribution. In rigor, the high intensity of the ST-EPR signal indicates the absence of microsecond rotational motion, showing that heads are all rigidly bound to actin. However, in both relaxation and contraction, considerable microsecond rotational motion is observed, implying that the previously reported orientational disorder under these conditions is dynamic, not static, on the microsecond time scale. The behavior in relaxation is essentially the same as that observed when myosin heads are detached from actin in the absence of ATP (Barnett and Thomas, 1984), corresponding to an effective rotational correlation time of approximately 10 microseconds. Slightly less mobility is observed during contraction. One possible interpretation is that in contraction all heads have the same mobility, corresponding to a correlation time of approximately 25 microseconds. Alternatively, more than one motional population may be present. For example, assuming that the spectrum in contraction is a linear combination of those in relaxation (mobile) and rigor (immobile), we obtained a good fit with a mole fraction of 78-88% of the heads in the mobile state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.     

Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads

We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.     

Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation.

The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads.     

Reaction intermediates of myosin ATPase from scallop adductor muscles: nonidentical two-headed structure of striated adductor muscle myosin

To determine whether or not the two heads of myosin from striated adductor muscles of scallop are nonidentical and the main intermediate of the ATPase reaction, MADPP, is produced only on one of the two heads, the Pi-burst size, the amount of total bound nucleotides and the amount of bound ADP during the ATPase reaction were measured in this study. The Pi-burst size was 1 mol per mol in the presence of 0.1-5 mM Mg2+ ions. The amount of total nucleotides bound to myosin was 2 mol per mol. Both the amounts of bound ADP and ATP at sufficiently high ATP concentrations were 1 mol per mol of striated adductor myosin, and the affinity for ADP binding was higher than that for ATP binding. These findings indicate that MADPP or MATP is produced on each of the two heads of striated adductor myosin on its interaction with ATP. The fluorescence intensity at 340 nm of striated adductor myosin was enhanced by about 7% upon addition of ATP. The time for the half maximum fluorescence enhancement, tau 1/2, at 5 microM ATP was 0.25 s, which was almost equal to the tau 1/2 values for the Pi-burst and for the dissociation of actomyosin reconstituted from striated adductor myosin and skeletal muscle F-actin. The dependences on ATP concentration of the extent of the fluorescence enhancement and the dissociation of actomyosin could be explained by assuming that these changes are associated with the formation of MADPP on one of the two heads of myosin. The Pi-burst size and the amount of bound ADP of smooth adductor myosin were slightly but significantly larger than 1 mol per mol. Both ATPase reactions of striated and smooth adductor myofibrils showed the substrate inhibition. The extent of substrate inhibition of ATPase of smooth adductor myofibrils was less than that of striated adductor myofibrils. All the present findings support the view that the nonidentical two-headed structure is required for substrate inhibition of the actomyosin ATPase reaction.     

Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate

Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548].     

Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges

We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time range from 0.5 to 200 microsecond, indicating that they do not undergo rotational motion in this time range. In the presence of 5 mM MgATP, however, heads undergo complex rotational motion with correlation times of about 5 and 40 microsecond. The motion of heads in relaxed myofibrils is restricted out to 1 ms, as indicated by a nonzero value of the residual anisotropy. The anisotropy decay of eosin-labeled myosin, extracted from labeled myofibrils, also exhibits complex decay on the 200-microsecond time scale when assembled into synthetic thick filaments. The correlation times and amplitudes of heads in filaments (under the same ionic conditions as the myofibril experiments) are unaffected by MgATP and very similar to the values for heads in relaxed myofibrils. The larger residual anisotropy and longer correlation times seen in myofibrils are consistent with a restriction of rotational motion in the confines of the myofibril protein lattice. These are the first time-resolved measurements under physiological conditions of the rotational motions of cross-bridges in the microsecond time range.     

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Ca(2+)-activated myofibrillar ATPase: transient kinetics and the titration of its active sites.

The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase were studied in a buffer of near physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase and the cleavage and release of products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.     

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.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

Mechanism of action of myosin X, a membrane-associated molecular motor

We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.     

Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies.

The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.     

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.