A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR) 2 FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.FRET | myosin | power stroke | phosphate release | structural kinetics M yosin family proteins use ATP hydrolysis to generate force and movement required for normal physiology. They drive muscle contraction, help control cell division and cellular motility, move organelles through the cytoplasm, and are important elements of the cellular mechanical-sensing machinery (1, 2). The key to understanding how myosin and related enzymes function in cells, and how to modulate their activity to treat disease, is to determine how the protein's structural dynamics and biochemical kinetics are coupled. Although high-resolution crystal structures provide best-guess snapshots of protein structure over a range of biochemical states, determining the physiological relevance of these snapshots remains one of the central challenges of structural biophysics.How myosin generates force remains debated despite more than 50 y of intense research (1-3). The most popular current model (2, 4) proposes that after ATP hydrolysis, myosin interacts weakly with actin and this interaction initiates an ordered series of structural and biochemical transitions that culminate in the dissociation of hydrolyzed phosphate, followed by the isomerization of the actin-binding interface to a state that binds actin with nanomolar affinity and then the rotation of the myosin lightchain domain (LCD) toward the plus end of the actin filament. This rotation converts the thermodynamic energy of phosphate release and actin binding into mechanical energy that performs work. A number of results question this model, however, including spectroscopic data showing that a structural transition in the myosin relay helix, hypothesized to be coupled to LCD rotation, precedes P i release (5) and force development precedes P i release in muscle fibers (6).Determining how these events take place in solution and in cells is an important question, because (i) differences in the mechanics of different myosins likely reflect differences in how the biochemical and structural transitions described above are coordinated (4); (ii) disea...

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.

Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.heart failure | omecamtiv mecarbil | FRET | myosin | phosphate release H eart failure is the leading cause of mortality in the United States (1). A primary defect in heart failure is a loss in cardiac contractility (2) resulting from a range of molecular factors: the sarcoplasmic reticulum's inability to sequester Ca 2+ , dysfunction of excitation-contraction coupling, altered metabolism, changes in gene expression levels, and mutations in sarcomeric proteins (3). Treatments for heart failure include lifestyle changes, surgeries, medical devices, heart transplant, renin-angiotensin and β-adrenergic modulators, and inotropes that increase contractility. Despite these interventions, life expectancy remains low, and half of the patients diagnosed with heart failure die within 5 y (1).Omecamtiv mecarbil (OM) is a small-molecule β-cardiac myosin effector in clinical trials for the treatment of systolic heart failure. OM was developed from lead compounds identified in a highthroughput calcium-regulated and thin-filament-activated ventricular cardiac myosin ATPase activity screen (4). A high-resolution X-ray crystal structure (5) and a photoreactive cross-linking study (4) both suggest that OM binds near the interface of several of myosin's key conserved structural elements: the seven-stranded β-sheet, the C terminus of the relay helix, the SH1 helix, and the interface between the N-terminal and converter domains. Movements in these elements are coupled to the weak-to-strong actin-binding transition, rotation of the myosin light chain domain, actin-induced phosphate and ADP release, and subsequently to force generation (6). Despite a number of recent studies, however (4, 5, 7-11), the structural basis for how OM alters force generation in the heart remains enigmatic.Mechanically active myosins all use changes in the Gibbs free energy associated with myosin binding to actin, ATP, ADP, and inorganic phosphate (P i ) to drive force-generating structural transitions, most notably a lever a...

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force generating lever arm in myosin V. We directly compared the FRET results with single molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), while our simulations suggest the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.