FLUORESCENCE PROBING OF YEAST ACTIN SUBDOMAIN-3 4 HYDROPHOBIC LOOP-262-274 - ACTIN-ACTIN AND ACTIN-MYOSIN INTERACTIONS IN ACTIN-FILAMENTS/

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 stabi lization, To assess directly the behavior of this loop, we mutated Ser (265) Of yeast actin to cysteine (S265C) and created another mutant (S 265C/C374A) by changing Cys(374) Of S265C actin to alanine, These chan ges allowed us to attach a pyrene maleimide stoichiometrically to eith er Cys(374) Or Cys(265), These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal st ability of labeled or unlabeled G-actin. The presence of the loop cyst eine, 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 cons iderably faster than wild-type (WT) actin, although the critical conce ntration was not affected, Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C3 74A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was similar to 50% less than the sum of the fluorescence of t he individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins, An excimer peak was present in t he emission spectrum of labeled S265C F-actin and in the labeled S265C /C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly i nteracts with the loop pyrene of neighboring monomers, bringing the tw o cysteine sulfurs to within 18 Angstrom of one another, Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe, Both proteins h ad no effect on excimer fluorescence, These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.