Locking the Hydrophobic Loop 262−274 to G-Actin Surface by a Disulfide Bridge Prevents Filament Formation
| Autor: | Martin L. Phillips, Peter A. Rubenstein, Emil Reisler, Alexander Shvetsov, Runa Musib |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Phalloidine Polymers Phalloidin Mutant Magnesium Chloride macromolecular substances Protein Engineering Biochemistry Protein filament chemistry.chemical_compound Leucine Cysteine Disulfides Actin-binding protein Actin Alanine biology Chemistry Wild type Actins Peptide Fragments Protein Structure Tertiary Actin Cytoskeleton Dithiothreitol Crystallography Cross-Linking Reagents Polymerization Mutagenesis Site-Directed biology.protein Hydrophobic and Hydrophilic Interactions Oxidation-Reduction |
| Zdroj: | Biochemistry. 41:10787-10793 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi020205f |
| Popis: | 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) 2 CA (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 MgCl 2 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 MgCl 2 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. |
| Databáze: | OpenAIRE |
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