Abstract
In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.