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J. Biol. Chem., Vol. 276, Issue 21, 18442-18449, May 25, 2001

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Tropomyosin-Troponin Regulation of Actin Does Not Involve Subdomain 2 Motions^*

Jack H. Gerson, Eldar Kim, Andras Muhlrad^§, and Emil Reisler^¶

From the  Department of Chemistry and Biochemistry and the ^¶ Molecular Biology Institute, University of California, Los Angeles, California 90095 and the ^§ Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem 91120, Israel

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln⁴¹-labeled -actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C_AEDANS/C374S and D51C_AEDANS/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp³⁴⁰ and Trp³⁵⁶ to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys⁴¹ or Cys⁵¹ of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (Ca²⁺) and closed (+Ca²⁺) states. Ca²⁺ also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys⁴¹ and Cys³⁷⁴. ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca²⁺ nor S1 binding to regulated -actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked -actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.

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