Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation

Alexander Shvetsov; Runa Musib; Martin Phillips; Peter A Rubenstein; Emil Reisler

doi:10.1021/bi020205f

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Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation

Journal article

Peer reviewed

Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation

Alexander Shvetsov, Runa Musib, Martin Phillips, Peter A Rubenstein and Emil Reisler

Biochemistry (Easton), Vol.41(35), pp.10787-10793

09/03/2002

DOI: 10.1021/bi020205f

PMID: 12196017

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Abstract

Models of F-actin structure predict the importance of hydrophobic loop 262-274 at the interface of subdomains 3 and 4 to interstrand interactions in filaments. If this premise is correct, prevention of the loop conformational change--its swinging motion--should abort filament formation. To test this hypothesis, we used site-directed mutagenesis to create yeast actin triple mutant (LC)2CA (L180C/L269C/C374A). This mutation places two cysteine residues in positions potentially enabling the locking of loop 262-274 to the monomer surface via disulfide formation. Exposure of the purified mutant to oxidation catalysts resulted in an increased electrophoretic mobility of actin on SDS PAGE and a loss of two cysteines by DTNB titrations, consistent with disulfide formation. The polymerization of un-cross-linked mutant actin by MgCl2 was inhibited strongly but could be restored to wild type actin levels by phalloidin and improved greatly through copolymerization with the wild-type actin. Light scattering measurements revealed nonspecific aggregation of the cross-linked actin under the same conditions. Electron microscopy confirmed the absence of filaments and the presence of amorphous aggregates in the cross-linked actin samples. Reduction of the disulfide bond by DTT restored normal actin polymerization in the presence of MgCl2 and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop 262-274 in the polymerization of actin into filaments.

Magnesium Chloride - chemistry

Protein Engineering - methods

Actin Cytoskeleton - chemistry

Actins - ultrastructure

Actins - genetics

Cysteine - genetics

Alanine - genetics

Disulfides - chemistry

Actins - chemistry

Dithiothreitol - chemistry

Leucine - genetics

Peptide Fragments - genetics

Actin Cytoskeleton - genetics

Phalloidine - chemistry

Peptide Fragments - ultrastructure

Saccharomyces cerevisiae Proteins - ultrastructure

Cross-Linking Reagents - chemistry

Mutagenesis, Site-Directed

Oxidation-Reduction

Saccharomyces cerevisiae Proteins - antagonists & inhibitors

Protein Structure, Tertiary - genetics

Saccharomyces cerevisiae Proteins - genetics

Peptide Fragments - chemistry

Actins - antagonists & inhibitors

Peptide Fragments - antagonists & inhibitors

Hydrophobic and Hydrophilic Interactions

Polymers - chemistry

Actin Cytoskeleton - ultrastructure

Saccharomyces cerevisiae Proteins - chemistry

Details

Title: Subtitle: Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation
Creators: Alexander Shvetsov - Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA
Runa Musib
Martin Phillips
Peter A Rubenstein
Emil Reisler
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.41(35), pp.10787-10793
Publisher: United States
DOI: 10.1021/bi020205f
PMID: 12196017
ISSN: 0006-2960
eISSN: 1520-4995
Grant note: AR 22031 / NIAMS NIH HHS R01 AR022031 / NIAMS NIH HHS GM 33689 / NIGMS NIH HHS
Language: English
Date published: 09/03/2002
Academic Unit: Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984025267302771

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