| Acidic residues on actin's subdomain 1 are postulated to mediate the binding and docking of the myosin head (subfragment-1, S1) on the actin filament in the presence of ATP, the weakly bound cross-bridge. We studied the effects of charge density and the location of acidic residues in actin subdomain 1 using yeast actin mutants, and linked groups of actin residues to specific functions in the cross-bridge cycle. The deletion of charged residues at actin's NH2-terminus resulted in lowered Vmax values, as well as a three-fold decrease in weak S1 binding. The loss of acidic residues elsewhere in actin subdomain 1 (20--29 and 93--100 loops) resulted in similar decreases in the activation of S1 ATPase, weak binding of S1, as well as force development. The relocation of acidic residues within these three sites on actin subdomain 1 (NH2-terminus residues 1--4, and 24/25 and 99/100 asp-asp or glu-glu pairs) showed that the weak state actomyosin functions were non-specific to the exact location of acidic residues in actin subdomain 1. There is, instead, a dependence of the weak S1 binding, its activation by actin, and the force development on the charge density in actin subdomain 1. Increasing the number of acidic residues within this region of actin at the NH2-terminus and in loop 93--100 revealed a specific contribution of the NH2-terminal site to the activation of S1 ATPase activity.;NH2-terminal actin residues believed to interact with troponin I (Tn-I) were also investigated by monitoring the Ca 2+-dependent regulation of actomyosin in the in vitro motility assays using thin filaments reconstituted with yeast actin mutants. We found that the inhibition of the actomyosin interaction and the Ca2+-sensitivity of the system were unaffected by the loss or addition of putatitve Tn-I binding sites on actin, suggesting limited, if any, role of these sites in the regulation process.;Recent genetic mutations discovered in actin and linked to the development of human cardiomyopathy disease were created in yeast actin. One such actin mutant, E361G, in actin subdomain 3, exhibited decreased binding to alpha-actinin, pointing to a mechanism for impaired transduction of force in sarcomeres, a phenotype of dilated cardiomyopathy. |
