Structural effects of cofilin on longitudinal contacts in F-actin.
Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)<or=0.05 microM for both CaATP-G-actin and F-actin. Cofilin binding enhanced the fluorescence of dansyl ethylenediamine (DED) attached to Gln41 on the DNase I binding loop of skeletal muscle F-actin and decreased the fluorescence of AEDANS at Cys41 on yeast Q41C/C374S mutant F-actin. However, cofilin had no effect on the spectral properties of DED or AEDANS on CaATP-G-actin. Fluorescence energy transfer (FRET) from tryptophan residues to DED at Gln41 on skeletal muscle actin and to AEDANS at Cys41 on yeast Q41C/C374S actin was decreased by cofilin binding to F- but not to G-actin. Cofilin inhibited strongly the rate of interprotomer disulfide cross-linking of Cys41 to Cys374 on yeast Q41C mutant F-actin. Binding of cofilin enhanced excimer formation between pyrene probes attached to Cys41 and Cys374 on Q41C F-actin. These results indicate that cofilin alters the interface between subdomains 1 and 2 and shifts the DNase I binding loop away from subdomain 1 of an adjacent actin protomer. Cofilin reduced FRET from tryptophan residues to 4-azido-2-nitrophenyl-putrescine (ANP) at Gln41 in skeletal muscle F-but not in G-actin. However, following the interprotomer cross-linking of Gln41 to Cys374 in F-actin by ANP, cofilin binding did not change FRET from the tryptophan residues to ANP. This suggests that cofilin binding and the conformational effect on F-actin are not coupled tightly. Overall, this study provides solution evidence for the weakening of longitudinal, subdomain 2/1 contacts in F-actin by cofilin.
Bobkov
Andrey A
AA
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA. abobkov@burnham.org
Muhlrad
Andras
A
Kokabi
Kaveh
K
Vorobiev
Sergey
S
Almo
Steven C
SC
Reisler
Emil
E
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D003619:Dansyl Compounds; D003850:Deoxyribonuclease I / Q000378:metabolism; D051345:Destrin; D004220:Disulfides; D005456:Fluorescent Dyes; D005973:Glutamine; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D015003:Yeasts / Q000737:chemistry
D051339:Actin Depolymerizing Factors; D000199:Actins; D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D051345:Destrin; D004220:Disulfides; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D005973:Glutamine; D003850:Deoxyribonuclease I; D003545:Cysteine