The Effects of Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop on the Dynamics and Stability of Yeast G-actin*
- ‡ To whom correspondence should be addressed. Tel.: 319-335-7911; Fax: 319-335-9570.
Abstract
The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33°C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.
Footnotes
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↵* This work was supported by a grant from the National Institutes of Health GM33689 (to P. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- G- and F-actin
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globular and filamentous actin
- IAEDANS
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N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine
- WT
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wild-type
- ϵ-ATP
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1, N6-ethenoadenosine-5′-triphosphate
- Tm
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apparent melting temperature.
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- Received November 4, 1996.
- Revision received November 18, 1996.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
