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Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262–274: ACTIN-ACTIN AND ACTIN-MYOSIN INTERACTIONS IN ACTIN FILAMENTS
Journal article   Open access   Peer reviewed

Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262–274: ACTIN-ACTIN AND ACTIN-MYOSIN INTERACTIONS IN ACTIN FILAMENTS

Li Feng, Eldar Kim, Wei-Lih Lee, Carl J Miller, Bing Kuang, Emil Reisler and Peter A Rubenstein
The Journal of biological chemistry, Vol.272(27), pp.16829-16837
07/04/1997
DOI: 10.1074/jbc.272.27.16829
PMID: 9201989
url
https://doi.org/10.1074/jbc.272.27.16829View
Published (Version of record) Open Access

Abstract

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 (S265C) and created another mutant (S265C/C374A) by changing Cys374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys374 or Cys265. These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. 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 wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was approximately 50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the 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 interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 A 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 had 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.

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