Structural effects of cofilin on longitudinal contacts in F-actin

Andrey A. Bobkov, Andras Muhlrad, Kaveh Kokabi, Sergey Vorobiev, Steven C. Almo, Emil Reisler

Research output: Contribution to journalArticle

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Abstract

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 Kd ≤ 0:05 μM 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.

Original languageEnglish (US)
Pages (from-to)739-750
Number of pages12
JournalJournal of Molecular Biology
Volume323
Issue number4
DOIs
StatePublished - 2002

Fingerprint

Actin Depolymerizing Factors
Actins
ethylenediamine
Yeasts
Fluorescence
Putrescine
Energy Transfer
Skeletal Muscle
Tryptophan
Deoxyribonuclease I
Adenosine Triphosphate

Keywords

  • Actin
  • ADF
  • Cofilin
  • DNase I binding loop
  • Longitudinal contacts

ASJC Scopus subject areas

  • Virology

Cite this

Structural effects of cofilin on longitudinal contacts in F-actin. / Bobkov, Andrey A.; Muhlrad, Andras; Kokabi, Kaveh; Vorobiev, Sergey; Almo, Steven C.; Reisler, Emil.

In: Journal of Molecular Biology, Vol. 323, No. 4, 2002, p. 739-750.

Research output: Contribution to journalArticle

Bobkov, AA, Muhlrad, A, Kokabi, K, Vorobiev, S, Almo, SC & Reisler, E 2002, 'Structural effects of cofilin on longitudinal contacts in F-actin', Journal of Molecular Biology, vol. 323, no. 4, pp. 739-750. https://doi.org/10.1016/S0022-2836(02)01008-2
Bobkov, Andrey A. ; Muhlrad, Andras ; Kokabi, Kaveh ; Vorobiev, Sergey ; Almo, Steven C. ; Reisler, Emil. / Structural effects of cofilin on longitudinal contacts in F-actin. In: Journal of Molecular Biology. 2002 ; Vol. 323, No. 4. pp. 739-750.
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AU - Bobkov, Andrey A.

AU - Muhlrad, Andras

AU - Kokabi, Kaveh

AU - Vorobiev, Sergey

AU - Almo, Steven C.

AU - Reisler, Emil

PY - 2002

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N2 - 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 Kd ≤ 0:05 μM 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.

AB - 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 Kd ≤ 0:05 μM 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.

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KW - Longitudinal contacts

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