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J. Biol. Chem., Vol. 277, Issue 51, 49716-49726, December 20, 2002

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Engineering Competitive Magnesium Binding into the First EF-hand of Skeletal Troponin C^*

Jonathan P. Davis, Jack A. Rall, Peter J. Reiser^¶, Lawrence B. Smillie, and Svetlana B. Tikunova^**

From the Departments of  Physiology and Cell Biology, ^** Molecular and Cellular Biochemistry, and ^¶ Oral Biology, The Ohio State University, Columbus, Ohio 43210 and the  Department of Biochemistry, the University of Alberta, Edmonton, Alberta T6G 2H7, Canada

The goal of this study was to examine the mechanism of magnesium binding to the regulatory domain of skeletal troponin C (TnC). The fluorescence of Trp²⁹, immediately preceding the first calcium-binding loop in TnC^F29W, was unchanged by addition of magnesium, but increased upon calcium binding with an affinity of 3.3 µM. However, the calcium-dependent increase in TnC^F29W fluorescence could be reversed by addition of magnesium, with a calculated competitive magnesium affinity of 2.2 mM. When a Z acid pair was introduced into the first EF-hand of TnC^F29W, the fluorescence of G34DTnC^F29W increased upon addition of magnesium or calcium with affinities of 295 and 1.9 µM, respectively. Addition of 3 mM magnesium decreased the calcium sensitivity of TnC^F29W and G34DTnC^F29W ~2- and 6-fold, respectively. Exchange of G34DTnC^F29W into skinned psoas muscle fibers decreased fiber calcium sensitivity ~1.7-fold compared with TnC^F29W at 1 mM [magnesium]_free and ~3.2-fold at 3 mM [magnesium]_free. Thus, incorporation of a Z acid pair into the first EF-hand allows it to bind magnesium with high affinity. Furthermore, the data suggests that the second EF-hand, but not the first, of TnC is responsible for the competitive magnesium binding to the regulatory domain.

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