Perturbations of functional interactions with myosin induce long-range allosteric and cooperative structural changes...

The role of the rotational dynamics of actin filaments in their interaction with myosin was studied by comparing the effect of myosin subfragment 1 (S1) with two other structural perturbations, which have substantial inhibitory effects on activation of myosin ATPase and in vitro motility of F-actin: (1) binding of the antibody fragment Fab(1-7) against the first seven N-terminal residues and (2) copolymerization with monomers treated with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), referred to as EDC-actin. The rotational motion of actin was measured by time-resolved phosphorescence anisotropy (TPA) of erythrosin iodoacetamide (ErIA) attached to Cys 374 on actin. The binding of S1 in a rigor complex (no nucleotide) induced intramonomer (allosteric) and intermonomer (cooperative) structural changes that increased the residual anisotropy of labeled F-actin, indicating a conformational change in the region of the C terminus. Similar allosteric and cooperative changes were induced by binding of Fab(1-7) and by copolymerization of the ErIA-labeled actin monomers with EDC-actin. This suggests that the functional perturbations transform actin to a form resembling the rigor actomyosin complex. The correlation of the perturbation-induced changes in TPA of actin with the functional effects suggests that the actomyosin interaction can be inhibited by stabilization of actin in one of its structural intermediates.