Loop 2 of myosin is a force-dependent inhibitor of the rigor bond.

Myosin's actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state-the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.

Force spectroscopy reveals multiple "closed states" of the muscle thin filament.

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.