*Li Feng, Eldar Kim, Wei-Lih Lee, Carl Miller, Emil Reisler, and *Peter A. Rubenstein
*Department of Biochemistry , University of Iowa College of Medicine, Iowa City, Iowa 52242, and Department of Chemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095
Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser265 of yeast actin to cysteine (SC) and created another mutant (SCCA) by changing Cys374 of SC actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either C374 or C265. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than WT actin although the critical concentration was not affected. While the fluorescence of the C-terminal probe increased during polymerization, that of the loop (SCCA) probe decreased, and the fluorescence of the doubly labeled actin (SC) was less than the sum of the fluorescence of the individual fluorophores. This quenching was also observed in copolymers of labeled WT and SCCA actins. An excimer peak was present in the emission spectrum of labeled F-SC actin and in the labeled SCCA/ WT copolymer. These results show that in the filament, the C-terminal pyrene of one monomer directly interacts with the loop pyrene of a neighboring monomer and that in this interaction, the two cysteine sulfurs are within 18 Å of one another. Cu on the C-terminal cysteine of a WT monomer decreased the fluorescence of the loop pyrene of a neighboring labeled SCCA monomer in F-actin. Finally, when bound to labeled SCCA F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. These results help establish the orientation of monomers in F-actin and show that binding of S1 on actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.