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Biophys. J. BioFAST: First Published December 2, 2005. doi:10.1529/biophysj.105.064634
© 2005 by the Biophysical Society.

A more recent version of this article appeared on February 15, 2006.
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MUSCLE AND CONTRACTILITY

Differential Interaction of Cardiac, Skeletal Muscle and Yeast Tropomyosins with Fluorescent (Pyrene235) Yeast Actin

Weizu Chen 1, Kuo-Kuang Wen 1, Ashley E. Sens 1 and Peter A. Rubenstein 1*

1 University of Iowa

* To whom correspondence should be addressed. E-mail: peter-rubenstein{at}uiowa.edu.

Submitted on April 14, 2005
Revised on May 18, 2005
Accepted on 7 November 2005


  Abstract
To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, while that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca+2-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin ± troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.

Key Words: fluorescence, mutation, pyrene, tropomyosin, troponin, yeast actin




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Copyright © 2005 by the Biophysical Society.