Characterization of single actomyosin rigor bonds: Load dependence of lifetime and mechanical properties

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling th e barbed end of the actin filament with optical tweezers. For S1, the relat ionship between the lifetime (tau) and the externally imposed load (F) at a bsolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to sing le-headed binding and the slow component to double-headed binding. These la rge interaction distances, which may be a common characteristic of motor pr oteins, are attributed to the geometry for applying an external load. The p ulling experiment has also allowed direct estimation of the number of myosi n molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indi cating a low torsional stiffness for HMM. From these results, we discuss th e characteristics of interaction between actin and myosin, with the focus o n the manner of binding of myosin.