The Two-Dimensional Kinetics of Binding and Unbinding are Both Regulated by Myosin's Actin-Binding Loop

Abstract

Binding of myosin to actin is a multi-step, ionic strength-sensitive process for which the kinetic properties of bond formation and rupture have been studied in solution. However, the solution (three dimensional) environment is a poor model of the pseudo-two dimensional geometry in which myofilament proteins interact in vivo. We therefore investigated the rate of actomyosin bond formation and rupture in a two-dimensional setting using dynamic force spectroscopy at physiological and reduced ionic strength. We previously showed that catch bond rupture is slowed at physiologic ionic strength (145 mM KCl) when compared to low ionic strength (25 mM), and that bond lifetime is maximal at the isometric force generated by a single myosin molecule. In the current study we confirmed this result and sought to determine whether this difference is mirrored in the rate of actomyosin bond formation. A laser trap was used to measure the time to bond formation (tb) between actin filaments and nucleotide-free heavy meromyosin (HMM) over a range of compressive loads. Two dimensional on-rates were determined from tb and were increasingly force-dependent with decreasing ionic strength. We examined the effect of ionic strength on the initial long-range ionic interaction constituting the first step of actomyosin binding by targeted tryptic digestion of HMM’s actin-binding loop. Our data at physiologic ionic strength suggest that the actin-binding loop contributes to the decreased 2D rate and load-dependence of bond formation. This is matched by reduced rates of actomyosin unbinding. These data provide useful parameters for modeling studies and suggest that the actin-binding loop of myosin contributes to both the binding and unbinding of myosin from actin.