STRUCTURAL CONNECTIVITY IN ACTIN - EFFECT OF C-TERMINAL MODIFICATIONSON THE PROPERTIES OF ACTIN

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 c oupling between the C-terminus and the DNaseI loop in subdomain II is observed in proteolytic digestion experiments in which a new C-termina l cleavage site is exposed upon DNaseI binding. The functional consequ ences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene act in, labeled at Cys-374, activates S-1 ATPase activity only half as wel l as control actin. This reduction is attributed to a lower V-max valu e because the affinity of pyrene actin to S-1 is not significantly alt ered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the V-m ax of acto-S-1 ATPase activity and the in vitro sliding velocity by ap proximately 25%. These results are discussed in terms of conformationa l coupling between distant regions in actin and the functional implica tions of the interactions of actin-binding proteins with the C-terminu s of actin.