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J. Biol. Chem., Vol. 279, Issue 34, 35341-35352, August 20, 2004

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Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C^*

Svetlana B. Tikunova and Jonathan P. Davis

From the Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210

Cardiac troponin C belongs to the EF-hand superfamily of calcium-binding proteins and plays an essential role in the regulation of muscle contraction and relaxation. To follow calcium binding and exchange with the regulatory N-terminal domain (N-domain) of human cardiac troponin C, we substituted Phe at position 27 with Trp, making a fluorescent cardiac troponin C^F27W. Trp²⁷ accurately reported the kinetics of calcium association and dissociation of the N-domain of cardiac troponin C^F27W. To sensitize the N-domain of cardiac troponin C^F27W to calcium, we individually substituted the hydrophobic residues Phe²⁰, Val⁴⁴, Met⁴⁵, Leu⁴⁸, and Met⁸¹ with polar Gln. These mutations were designed to increase the calcium affinity of the N-domain of cardiac troponin C by facilitating the movement of helices B and C (BC unit) away from helices N, A, and D (NAD unit). As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C^F27W 2.1–15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6–8.7-fold faster) rather than to slower calcium dissociation rates (1.2–2.9-fold slower). The regulatory N-domains of cardiac troponin C^F27W and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2–2.7 mM). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.

Received for publication, May 14, 2004 , and in revised form, June 11, 2004.

^* This work was supported in part by National Institutes of Health Grants AR20792 (to Jack A. Rall) and HL073600 (to S. B. T.) and by an award from the American Heart Association, Ohio Valley Affiliate (to J. P. D.). 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 Physiology and Cell Biology, The Ohio State University, 209 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-4467; Fax: 614-292-4888; E-mail: tikunova1{at}hotmail.com.

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