Conformational dynamics of loop 262-274 in G- and F-actin

Alexander Shvetsov, John D. Stamm, Martin Phillips, Dora Warshaviak, Christian Altenbach, Peter A. Rubenstein, K. Hideg, Wayne L. Hubbell, Emil Reisler

Research output: Contribution to journalArticle

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Abstract

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)2CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)2CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 Å) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 Å) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance (∼10 Å) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.

Original languageEnglish
Pages (from-to)6541-6549
Number of pages9
JournalBiochemistry
Volume45
Issue number20
DOIs
Publication statusPublished - May 23 2006

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Actins
Disulfides
Actin Cytoskeleton
Myosin Subfragments
Phalloidine
Tropomyosin
Cross Reactions
Electron Spin Resonance Spectroscopy
Protein Subunits
Polymerization
Copper
Electron Microscopy
Yeasts
Air
Yeast
Electron microscopy
Paramagnetic resonance
Conformations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Shvetsov, A., Stamm, J. D., Phillips, M., Warshaviak, D., Altenbach, C., Rubenstein, P. A., ... Reisler, E. (2006). Conformational dynamics of loop 262-274 in G- and F-actin. Biochemistry, 45(20), 6541-6549. https://doi.org/10.1021/bi052558v

Conformational dynamics of loop 262-274 in G- and F-actin. / Shvetsov, Alexander; Stamm, John D.; Phillips, Martin; Warshaviak, Dora; Altenbach, Christian; Rubenstein, Peter A.; Hideg, K.; Hubbell, Wayne L.; Reisler, Emil.

In: Biochemistry, Vol. 45, No. 20, 23.05.2006, p. 6541-6549.

Research output: Contribution to journalArticle

Shvetsov, A, Stamm, JD, Phillips, M, Warshaviak, D, Altenbach, C, Rubenstein, PA, Hideg, K, Hubbell, WL & Reisler, E 2006, 'Conformational dynamics of loop 262-274 in G- and F-actin', Biochemistry, vol. 45, no. 20, pp. 6541-6549. https://doi.org/10.1021/bi052558v
Shvetsov A, Stamm JD, Phillips M, Warshaviak D, Altenbach C, Rubenstein PA et al. Conformational dynamics of loop 262-274 in G- and F-actin. Biochemistry. 2006 May 23;45(20):6541-6549. https://doi.org/10.1021/bi052558v
Shvetsov, Alexander ; Stamm, John D. ; Phillips, Martin ; Warshaviak, Dora ; Altenbach, Christian ; Rubenstein, Peter A. ; Hideg, K. ; Hubbell, Wayne L. ; Reisler, Emil. / Conformational dynamics of loop 262-274 in G- and F-actin. In: Biochemistry. 2006 ; Vol. 45, No. 20. pp. 6541-6549.
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