Cross-linking constraints on F-actin structure

Eldar Kim; Willy Wriggers; Martin Phillips; Kevin Kokabi; P A Rubenstein; Emil Reisler

doi:10.1006/jmbi.2000.3727

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Cross-linking constraints on F-actin structure

Journal article

Peer reviewed

Cross-linking constraints on F-actin structure

Eldar Kim, Willy Wriggers, Martin Phillips, Kevin Kokabi, P A Rubenstein and Emil Reisler

Journal of molecular biology, Vol.299(2), pp.421-429

06/02/2000

DOI: 10.1006/jmbi.2000.3727

PMID: 10860749

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Abstract

The DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274), and the C terminus region are among the structural elements of monomeric (G-) actin proposed to form the intermonomer interface in F-actin. To test the proximity and interactions of these elements and to provide constraints on models of F-actin structure, cysteine residues were introduced into yeast actin either at residue 41 or 265. These mutations allowed for specific cross-linking of F-actin between C41 and C265, C265 and C374, and C41 and C265 using dibromobimane and disulfide bond formation. The cross-linked products were visualized on SDS-PAGE and by electron microscopy. Model calculations carried out for the cross-linked F-actins revealed that considerable flexibility or displacement of actin residues is required in the disulfide cross-linked segments to fit these filaments into model F-actin structures. The calculated, cross-linked structures showed a better fit to the Holmes rather than the refined Lorenz model of F-actin. It is predicted on the basis of such calculations that image reconstruction of electron micrographs of disulfide cross-linked C41-C374 F-actin should provide a conclusive test of these two similar models of F-actin structure.

Disulfides - metabolism

Biopolymers - chemistry

Pliability

Electrophoresis, Polyacrylamide Gel

Actins - metabolism

Actins - ultrastructure

Models, Molecular

Yeasts - chemistry

Copper Sulfate - metabolism

Microscopy, Electron

Biopolymers - metabolism

Actins - genetics

Cysteine - genetics

Cross-Linking Reagents - metabolism

Biopolymers - genetics

Mutagenesis, Site-Directed - genetics

Bridged Bicyclo Compounds - metabolism

Computer Simulation

Protein Structure, Quaternary

Actins - chemistry

Protein Binding

Amino Acid Substitution - genetics

Cysteine - metabolism

Binding Sites

Details

Title: Subtitle: Cross-linking constraints on F-actin structure
Creators: Eldar Kim - Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, CA, 90095, USA
Willy Wriggers
Martin Phillips
Kevin Kokabi
P A Rubenstein
Emil Reisler
Resource Type: Journal article
Publication Details: Journal of molecular biology, Vol.299(2), pp.421-429
Publisher: England
DOI: 10.1006/jmbi.2000.3727
PMID: 10860749
ISSN: 0022-2836
eISSN: 1089-8638
Grant note: R01 AR022031 / NIAMS NIH HHS
Language: English
Date published: 06/02/2000
Academic Unit: Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984025398702771

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