Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments.
The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin.
Department of Chemistry and Biochemistry, University of California, Los Angeles 90095, USA.
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Kim
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Reisler
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D003300:Copper; D003545:Cysteine; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005033:Ethylmaleimide; D005456:Fluorescent Dyes; D007700:Kinetics; D018482:Muscle, Skeletal; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C032377:dansylethylenediamine; D003300:Copper; D017301:Ca(2+) Mg(2+)-ATPase; D003545:Cysteine; D005033:Ethylmaleimide