Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides - Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Effect of the N-terminal extension in myosin essential light chain A1 on the mechanism of actomyosin ATP hydrolysis.

Myosin essential light chains A1 and A2 are identical isoforms except for an extension of ∼40 amino acids at the N terminus of A1 that binds F-actin. The extension has no bearing on the burst hydrolysis rate (M-ATP → M-ADP-Pi) as determined by chemical quench flow (100 µM isoenzyme). Whereas actomyosin-S1A2 steady state MgATPase (low ionic strength, 20 °C) is hyperbolically dependent on concentration: Vmax 7.6 s-1, Kapp 6.4 µM (F-actin) and Vmax 10.1 s-1, Kapp 5.5 µM (native thin filaments, pCa 4), the relationship for myosin-S1A1 is bimodal; an initial rise at low concentration followed by a decline to one-third the Vmax of S1A2, indicative of more than one rate-limiting step and A1-enforced flux through the slower actomyosin-limited hydrolysis pathway. In double-mixing stopped-flow with an indicator, Ca(II)-mediated activation of Pi dissociation (regulatedAM-ADP-Pi → regulatedAM-ADP + Pi) is attenuated by A1 attachment to thin filaments (pCa 4). The maximum accelerated rates of Pi dissociation are: 81 s-1 (S1A1, Kapp 8.9 µM) versus 129 s-1 (S1A2, Kapp 58 µM). To investigate apomyosin-S1-mediated activation, thin filaments (EGTA) are premixed with a given isomyosin-S1 and double-mixing is repeated with myosin-S1A1 in the first mix. Similar maximum rates of Pi dissociation are observed, 44.5 s-1 (S1A1) and 47.1 s-1 (S1A2), which are lower than for Ca(II) activation. Overall, these results biochemically demonstrate how the longer light chain A1 can contribute to slower contraction and higher force and the shorter version A2 to faster contraction and lower force, consistent with their distribution in different types of striated muscle.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Investigation into the mechanism of thin filament regulation by transient kinetics and equilibrium binding: Is there a conflict?

Striated muscle contraction occurs when myosin undergoes a lever-type structural change. This process (the power stroke) requires ATP and is governed by the thin filament, a complex of actin, tropomyosin, and troponin. The authors have used a fast-mixing instrument to investigate the mechanism of regulation. Such (pre-steady-state kinetic) experiments allow biochemical intermediates in a working actomyosin cycle to be monitored. The regulatory focal point is demonstrated to be the step that involves the departure of inorganic phosphate (i.e., AM-ADP-Pi → AM-ADP). This part of the cycle, which lies on the main kinetic pathway and coincides with the drive stroke, is maximally accelerated ∼100-fold by the combined association of ligands (Ca[II] and rigor myosin heads) with the thin filament. However, the observed ligand dependencies of the rates of Pi dissociation that are reported herein are at variance with predictions of models derived from experiments where ATP hydrolysis is not taking place (and myosin exists in a nonphysiological form). It is concluded that the principal influence of the thin filament is in setting the rate of Pi dissociation and that physiological levels of regulation are dependent upon the liganded state of the thin filament as well as the conformation of myosin.

The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step.

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

Omecamtiv Mecarbil modulates the kinetic and motile properties of porcine ß-cardiac myosin.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C.

We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.

Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments.

Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30-36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.

Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

Mechanism of regulation of native cardiac muscle thin filaments by rigor cardiac myosin-S1 and calcium.

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.

The SAH domain extends the functional length of the myosin lever.

Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.

Stabilization of helical order in the thick filaments by blebbistatin: further evidence of coexisting multiple conformations of myosin.

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder<-->order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.

Switch 1 mutation S217A converts myosin V into a low duty ratio motor.

We have determined the kinetic mechanism and motile properties of the switch 1 mutant S217A of myosin Va. Phosphate dissociation from myosin V-ADP-Pi (inorganic phosphate) and actomyosin V-ADP-Pi and the rate of the hydrolysis step (myosin V-ATP-->myosin V-ADP-Pi) were all approximately 10-fold slower in the S217A mutant than in wild type (WT) myosin V, resulting in a slower steady-state rate of basal and filamentous actin (actin)-activated ATP hydrolysis. Substrate binding and ADP dissociation kinetics were all similar to or slightly faster in S217A than in WT myosin V and mechanochemical gating of the rates of dissociation of ADP between trail and lead heads is maintained. The reduction in the rate constants of the hydrolysis and phosphate dissociation steps reduces the duty ratio from approximately 0.85 in WT myosin V to approximately 0.25 in S217A and produces a motor in which the average run length on actin at physiological concentrations of ATP is reduced 10-fold. Thus we demonstrate that, by mutational perturbation of the switch 1 structure, myosin V can be converted into a low duty ratio motor that is processive only at low substrate concentrations.

Direct observation of the mechanochemical coupling in myosin Va during processive movement.

Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.

Smooth muscle myosin phosphorylated at single head shows sustained mechanical activity.

Smooth muscle contraction is regulated by the phosphorylation of myosin. It is well known that tonic smooth muscles can maintain force with low energy consumption (latch state); however, the molecular mechanism underlying this phenomenon is unresolved. Here we show that single-head phosphorylated smooth myosin (SHPMII) exhibits fast ( approximately 24 s(-1)) and slow prolonged ( approximately 1 s(-1)) actin interactions, whereas double-head phosphorylated myosin (DHPMII) predominantly exhibits the fast ( approximately 29 s(-1)) interaction, suggesting that the phosphorylated head of SHPMII is mechanically as active as that of DHPMII. Both the fast and the slow actin interactions of SHPMII support the positive net mechanical displacement of actin. The actin translocating velocity of SHPMII was much slower than that of DHPMII, which is consistent with the slow actin interaction of SHPMII. We propose that the "latch state" can be explained by the motor characteristics of SHPMII that is present during the sustained phase of contraction.

Kinetics of ADP dissociation from the trail and lead heads of actomyosin V following the power stroke.

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.

Real-time measurement of myosin-nucleotide noncovalent complexes by electrospray ionization mass spectrometry.

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 +/- 10 Da were obtained with the binding of ADP to the active site and by 530 +/- 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of approximately 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 muM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.