The Biochemical Kinetics Underlying Actin Movement Generated by One and Many Skeletal Muscle Myosin Molecules

Abstract

To better understand how skeletal muscle myosin molecules move actin filaments, we determine the motion-generating biochemistry of a single myosin molecule and study how it scales with the motion-generating biochemistry of an ensemble of myosin molecules. First, by measuring the effects of various ligands (ATP, ADP, and P(i)) on event lifetimes, tau(on), in a laser trap, we determine the biochemical kinetics underlying the stepwise movement of an actin filament generated by a single myosin molecule. Next, by measuring the effects of these same ligands on actin velocities, V, in an in vitro motility assay, we determine the biochemistry underlying the continuous movement of an actin filament generated by an ensemble of myosin molecules. The observed effects of P(i) on single molecule mechanochemistry indicate that motion generation by a single myosin molecule is closely associated with actin-induced P(i) dissociation. We obtain additional evidence for this relationship by measuring changes in single molecule mechanochemistry caused by a smooth muscle HMM mutation that results in a reduced P(i)-release rate. In contrast, we observe that motion generation by an ensemble of myosin molecules is limited by ATP-induced actin dissociation (i.e., V varies as 1/tau(on)) at low [ATP], but deviates from this relationship at high [ATP]. The single-molecule data uniquely provide a direct measure of the fundamental mechanochemistry of the actomyosin ATPase reaction under a minimal load and serve as a clear basis for a model of ensemble motility in which actin-attached myosin molecules impose a load.

Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

“…1). In the 1980s, several investigations established that elevated levels of organic phosphate (P i ) reduced muscular force, leading to the notion that P i release by myosin was closely associated with force generation (12) and the rotation of the lever arm (2). This hypothesis postulates that P i rebinds to myosin (M) in a state in which myosin is strongly bound to both actin (A) and ADP (AM.ADP) and, in one or more steps, reverses the forcegenerating step, leading to an increase in the population of the weakly bound myosin, in the M.ADP.P i state (32).…”

Hysteresis in cross-bridge models of muscle

“…24 Fitting low [ATP] velocity data to this equation (dashed lines, Fig. 4), we obtain values for k ÀD(+strain) of 55 s À1 for smooth and 174 s À1 for skeletal muscle myosin.…”

“…27 Strain-dependent kinetics are not unique to myosin V. It has long been argued that because there is no net force on an actin filament during unloaded sliding, the positive forces (and strain) generated by actin-myosin binding must be offset by negative forces (and strain) that resist actin movement. 23,24 According to most muscle models, this change in strain alters the rate of ADP release. However, whether the strain that affects the rate of ADP release is intrahead as described in Huxley-like models 23 or interhead like in Fig.…”

“…Over time (left to right), actin movement decreases this strain eventually pulling the myosin head so that it becomes negatively strained before detaching from actin (right). 24 This balance of forces is required in an unloaded motility assay at any ATP concentration. time, t on , is spent waiting for ADP to be released, or t on E t ÀD .…”

“…Unloaded actin filament sliding decreases the strain in all myosin heads bound to that filament, eventually pulling myosin heads into regions of negative force and strain, where they resist actin movement. 24 The effect of this variable strain on the kinetics of ADP release is historically described through arbitrarily defined strain-dependent kinetics, presumably as an estimate of the effects of strain on the active site of myosin. With these models an energetic link between myosin force-generating and force-sensing transitions is muddled.…”