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J. Biol. Chem., Vol. 278, Issue 43, 41742-41748, October 24, 2003

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Cardiomyopathic Tropomyosin Mutations That Increase Thin Filament Ca²⁺ Sensitivity and Tropomyosin N-domain Flexibility^*

Mark J. Heller, Mahta Nili, Earl Homsher, and Larry S. Tobacman¶

From the Departments of Internal Medicine and Biochemistry, University of Iowa, Iowa City, Iowa 52242 and the Department of Physiology, UCLA, Los Angeles, California 90025

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of -striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 °C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 °C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0–1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca²⁺ binding sites of thin filament in two settings: Ca²⁺-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca²⁺ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca²⁺ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca²⁺ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca²⁺ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.

Received for publication, April 2, 2003 , and in revised form, July 29, 2003.

^* 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: University of Illinois at Chicago, Dept. of Medicine, MC 787, 840 W. Wood St., Rm. 317F CSN, Chicago, IL 60612.

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