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J. Biol. Chem., Vol. 279, Issue 18, 18203-18209, April 30, 2004

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Tropomyosin Exon 6b Is Troponin-specific and Required for Correct Acto-myosin Regulation^*

Robin Maytum, Friederike Bathe¶, Manfred Konrad||, and Michael A. Geeves

From the University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, United Kingdom, ^¶Adolf-Butenandt-Institut, 80336 Munich, Germany, and ^||Max-Planck-Institut fur Biophysikalische Chemie, D-37070 Göttingen, Germany

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT₁-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1–0.5 µM) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin·Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin·Tm·Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin·Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.

Received for publication, October 23, 2003 , and in revised form, January 23, 2004.

^* 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.

Supported by Wellcome Trust Grant No. 055881. To whom correspondence should be addressed. Tel.: 44-1227-823950; Fax: 44-1227-763912; E-mail: rmm{at}ukc.ac.uk.

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