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; Kálmán Hideg; Wayne L Hubbell; Emil Reisler

doi:10.1021/bi052558v

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

Journal article

Peer reviewed

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, Kálmán Hideg, Wayne L Hubbell and Emil Reisler

Biochemistry (Easton), Vol.45(20), pp.6541-6549

05/23/2006

DOI: 10.1021/bi052558v

PMID: 16700564

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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)(2)CA], 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)(2)CA 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 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) 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 ( approximately 10 A) 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.

Disulfides - metabolism

Saccharomyces cerevisiae - genetics

Actins - metabolism

Mesylates - metabolism

Tropomyosin - pharmacology

Cysteine - genetics

Myosin Subfragments - pharmacology

Phalloidine - metabolism

Actin Cytoskeleton - drug effects

Disulfides - chemistry

Actins - chemistry

Cysteine - metabolism

Tropomyosin - metabolism

Cross-Linking Reagents

Bridged Bicyclo Compounds - pharmacology

Electron Spin Resonance Spectroscopy

Myosin Subfragments - metabolism

Cysteine - chemistry

Saccharomyces cerevisiae - chemistry

Saccharomyces cerevisiae - cytology

Actins - antagonists & inhibitors

Mesylates - pharmacology

Bridged Bicyclo Compounds - metabolism

Hydrophobic and Hydrophilic Interactions

Protein Conformation

Actin Cytoskeleton - ultrastructure

Details

Title: Subtitle: Conformational dynamics of loop 262-274 in G- and F-actin
Creators: Alexander Shvetsov - Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA
John D Stamm
Martin Phillips
Dora Warshaviak
Christian Altenbach
Peter A Rubenstein
Kálmán Hideg
Wayne L Hubbell
Emil Reisler
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.45(20), pp.6541-6549
Publisher: United States
DOI: 10.1021/bi052558v
PMID: 16700564
ISSN: 0006-2960
eISSN: 1520-4995
Grant note: AR 22031 / NIAMS NIH HHS R01 AR022031 / NIAMS NIH HHS T32 EY07026 / NEI NIH HHS EY 05216 / NEI NIH HHS GM 33689 / NIGMS NIH HHS
Language: English
Date published: 05/23/2006
Academic Unit: Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984024558702771

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