Interaction of myosin with F-actin: Time-dependent changes at the interface are not slow

The kinetics of formation of the actin-myosin complex have been reinvestiga ted on the minute and second time scales in sedimentation and chemical cros s-linking experiments. With the sedimentation method, we found that the bin ding of the skeletal muscle myosin motor domain (S1) to actin filament alwa ys saturates at one S1 bound to one actin monomer (or two S1 per actin dime r), whether S1 was added slowly (17 min between additions) or rapidly (10 s between additions) to an excess of F-actin. The carbodiimide (1-ethyl-3-(3 -dimethylaminopropyl) carbodiimide, EDC)-induced cross-linking of the actin -S1 complex was performed on the subsecond time scale by a new approach tha t combines a two-step cross-linking protocol with the rapid flow-quench tec hnique. The results showed that the time courses of S1 cross-linking to eit her of the two actin monomers are identical: they are not dependent on the actin/S1 ratio in the 0.3-20-s time range. The overall data rule out a mech anism by which myosin rolls from one to the other actin monomer on the seco nd or minute time scales. Rather, they suggest that more subtle changes occ ur at the actomyosin interface during the ATP cycle.