Travis J. Stewart

The myosin duty ratio tunes the calcium sensitivity and cooperative activation of the thin filament.

In striated muscle, calcium binding to the thin filament (TF) regulatory complex activates actin-myosin ATPase activity, and actin-myosin kinetics in turn regulates TF activation. However, a quantitative description of the effects of actin-myosin kinetics on the calcium sensitivity (pCa50) and cooperativity (nH) of TF activation is lacking. With the assumption that TF structural transitions and TF-myosin binding transitions are inextricably coupled, we advanced the principles established by Kad et al. [Kad, N., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16990-16995] and Sich et al. [Sich, N. M., et al. (2011) J. Biol. Chem. 285, 39150-39159] to develop a simple model of TF regulation, which predicts that pCa50 varies linearly with duty ratio and that nH is maximal near physiological duty ratios. Using in vitro motility to determine the calcium sensitivity of TF sliding velocities, we measured pCa50 and nH at different myosin densities and in the presence of ATPase inhibitors. The observed effects of myosin density and actin-myosin duty ratio on pCa50 and nH are consistent with our model predictions. In striated muscle, pCa50 must match cytosolic calcium concentrations and a maximal nH optimizes calcium responsiveness. Our results indicate that pCa50 and nH can be predictably tuned through TF-myosin ATPase kinetics and that drugs and disease states that alter ATPase kinetics can, through their effects on calcium sensitivity, alter the efficiency of muscle contraction.

Sucrose increases the activation energy barrier for actin-myosin strong binding.

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin-myosin (A-M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A-M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A-M strong binding by 37±3%, indicating that sucrose inhibits the rate of A-M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A-M rigor binding (71%) are comparable in magnitude to sucroses effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A-M strong bond formation significantly influences both kcat and V.

The Relative Influence of Actin-Myosin Attachment and Detachment Kinetics on Actin Sliding Velocities is Modulated by Myosin Density

In an in vitro motility assay, actin sliding velocities, V, exhibit a multi-phasic response to increasing myosin densities, N. At low N, V increases with increasing N reaching a peak value before decreasing. The mechanism underlying this biphasic response remains unclear. Here we propose a simple model in which at low myosin numbers, the working step of a myosin head, which occurs upon actin-myosin attachment, is free to displace an actin filament, resulting in a V that is attachment limited. At high myosin numbers, actin-bound myosin heads impose a load against which the working step of a myosin head cannot move, resulting in a detachment limited V. We test this hypothesis by measuring the effects of perturbations to attachment- and detachment-kinetics on the N-dependence of V. The resulting data are accurately fit to our simple model. These results provide insights into the basic mechanism of muscle contraction with implications for how in general multiple motors work together to move cargo. Specifically, these results suggest that V measured in conventional in vitro motility assays is influenced by both attachment and detachment kinetics.

The Effects of Ions and Water on Actin-Myosin Binding Energetics and Mechanics

Myosins discrete lever arm rotation upon strong actin binding is capable of generating large forces, but in ensemble myosin systems the average force generated by this transition is limited by the free energy for actin-myosin binding. Yet when looking for insights into mechanisms of muscle force generation, myosin structures are studied while factors that affect actin-myosin binding energetics are seldom considered. Here we use in vitro motility assays, AM binding assays, and stopped flow kinetics to study the effects of water and KCl on actin-myosin binding. First, we show that sucrose dramatically inhibits actin-myosin binding kinetics through a mechanism other than crowding or viscous damping, implying that sucrose has a desolvation effect. Second, we show that the effects of sucrose are ionic strength dependent, having little effect at low ionic strength. Our data support a simple model in which the strength of weak actin-myosin bonds is reduced by the shielding effect of ions, and sucrose enhances this effect possibly by displacing ordered waters around actin and myosin. Our data suggest that water solvation and ionic shielding may have a significant influence on muscle mechanics.

Actin Sliding Velocities Are Influenced By The Chemical Driving Force Of Actin-myosin Binding

Actin sliding velocities are influenced by the chemical driving force of actin-myosin bindingRyan Smith, Steve Shannon, Travis Stewart, Olivia John, Josh BakerUniversity of Nevada Reno School of Medicine, Department of Biochemistry and Molecular Biology, Reno, NV 89557It is widely assumed that muscle shortening velocity, V, is solely limited by actin-myosin detachment kinetics; however, it was recently shown (Hooft et al., 2007) that V is influenced by a driving force modulated by actin-myosin attachment kinetics and energetics. To further test this hypothesis, we have developed a novel in vitro motility and force assay that allows us to correlate changes in internal driving force with changes in V. We alter the driving force by varying inorganic phosphate, Pi, and blebbistatin concentrations using an in vitro motility assay to measure corresponding changes in V. We estimate changes in driving force by calculating the rate, kbreak, at which actin filaments break during this assay. We observe that at 30 μM ATP, both V and kbreak decrease by approximately 50% (V from 1.5 to 0.88 μm·sec−1 and kbreak from 0.04 to 0.02 sec−1), demonstrating that actin-myosin driving forces in an in vitro motility assay decrease with V upon addition of Pi. Similarly upon addition of 50 μM blebbistatin, a small molecule known to decrease actin-myosin mechanics by inhibiting actin-myosin binding kinetics, both V and kbreak decrease by approximately 50% (V from 1.5 to 0.62 and kbreak from 0.04 to 0.02 sec−1). These results support the hypothesis that in an in vitro motility assay internal forces modulated by actin-myosin binding energetics influence actin sliding velocities, supporting a new paradigm for the mechanism of muscle shortening.