Structural Implications of the Chemical Modification of Cys10 on Actin

Cys10 is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys10 was modified with fluorescence probes N-(iodoacetyl)N′-(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of Drewes and Faulstich (1991, J. Biol. Chem. 266:5508–5513). The specificity of Cys10 modification was verified by showing that the 33-kDa subtilisin fragment of actin (residues 48–375), which contains all of the actin thiols but Cys10, is not fluorescent. Cys10 modification exposed a new site on actin to subtilisin cleavage. Edman degradation revealed this site to be between Ala19 and Gly20. The modification slightly increased the rate of ϵATP-ATP exchange and decreased the rates of G-actin ATPase and polymerization. The activation of S1 ATPase by Cys10-modified F-actin showed small probe-dependent changes in the values of Vmax and KM. The sliding speed of actin filaments in the in vitro motility assay remained unchanged upon modification of Cys10. These results indicate that although the labeling of Cys10 perturbs the structure of subdomain 1, the modified actin remains fully functional. The binding of S1 to actin filaments decreases the accessibility of Cys10 probes to acrylamide and nitromethane quenchers. Because Cys10 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.