Structural implications of the chemical modification of Cys(10) on actin

Cys(10) is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys(10) w as modified with fluorescence probes N-(iodoacetyl)N'-(5-sulfo-1-naphthyl)e thylene diamine (IAEDANS), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylc oumarin (CPM), or monobromo bimane (MBB) by the method of Drewes and Faulst ich (1991, J. Biol. Chem. 266:5508-5513). The specificity of Cys(10) modifi cation was verified by showing that the 33-kDa subtilisin fragment of actin (residues 48-375), which contains all of the actin thiols but Cys(10), is not fluorescent. Cys(10) modification exposed a new site on actin to subtil isin cleavage. Edman degradation revealed this site to be between Ala(19) a nd Gly(20). The modification slightly increased the rate of epsilon ATP-ATP exchange and decreased the rates of G-actin ATPase and polymerization. The activation of S1 ATPase by Cys(10)-modified F-actin showed small probe-dep endent changes in the values of V-max and K-M. The sliding speed of actin f ilaments in the in vitro motility assay remained unchanged upon modificatio n of Cys(10). These results indicate that although the labeling of Cys(10) perturbs the structure of subdomain 1, the modified actin remains fully fun ctional. The binding of S1 to actin filaments decreases the accessibility o f Cys(10) probes to acrylamide and nitromethane quenchers. Because Cys(10) does not participate directly in either actin polymerization or S1 binding, our results indicate that actin-actin and actin-myosin interactions induce dynamic, allosteric changes in actin structure.