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J. Biol. Chem., Vol. 280, Issue 2, 1696-1703, January 14, 2005

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An Intermediate Form of ADP-F-actin^*

Keith E. Bryan and Peter A. Rubenstein

From the Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

With yeast actin, contrary to other actins, filament formation, ATP hydrolysis, and P_i release are concurrent at low actin concentrations, the condition usually employed to assess actin polymerization. This observation leads to a question concerning the conformation of the filament barbed end that might be recognized by specific actin-binding proteins. To try to detect possible new actin polymer conformations that might be intermediate in the pathway leading to mature F-actin, we monitored the change in intrinsic tryptophan fluorescence of yeast and muscle actins polymerized at pH 6 to accelerate the rate of filament formation. This allowed temporal resolution of the P_i release process from the slower process of polymerization. With both actins, we detected a biphasic instead of the usual monophasic fluorescence change, a rapid decrease that tracks with filament formation followed by a slower rebound (the second phase). This second phase postpolymerization conformational change requires P_i release and occurs nearly coincident with its release. The addition of P_i causes this second phase response to disappear, and the inclusion of P_i during polymerization prevents its appearance. At pH 7.5, with higher yeast actin concentrations to accelerate polymerization, a two-phase fluorescence change is also observed. In this case, the second phase change lags substantially behind P_i release. P_i release could also be resolved from polymer formation. V159N yeast actin, hypothesized previously as remaining in a postpolymerization ATP-like state, exhibits the same two-phase intrinsic tryptophan fluorescence behavior as wild-type yeast actin. Together, these observations demonstrate the presence of an intermediate filament state between ADP-P_i and mature ADP-F-actin.

Received for publication, September 3, 2004 , and in revised form, November 5, 2004.

^* This work was supported by a National Institutes of Health Grant GM33689 (to P. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Iowa College of Medicine, Bowen Science Bldg., 51 Newton Rd., Iowa City, IA 52242. Tel.: 319-335-7911; Fax: 319-335-9570; E-mail: peter-rubenstein{at}uiowa.edu.

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