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J. Biol. Chem., Vol. 277, Issue 51, 49716-49726, December 20, 2002
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From the Departments of 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
TnCF29W, was unchanged by addition of magnesium, but
increased upon calcium binding with an affinity of 3.3 µM. However, the calcium-dependent increase
in TnCF29W 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 TnCF29W, the fluorescence of
G34DTnCF29W 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
TnCF29W and G34DTnCF29W ~2- and 6-fold,
respectively. Exchange of G34DTnCF29W into skinned
psoas muscle fibers decreased fiber calcium
sensitivity ~1.7-fold compared with TnCF29W 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.
Engineering Competitive Magnesium Binding into the First
EF-hand of Skeletal Troponin C*
,,
, and**
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
*
This work was supported in part by National Institutes of
Health Grants AR20792 (to J. A. R.) and DK33727 (to R. A. A.), by a grant from the Canadian Institutes for Health
Research (to L. B. S.), and 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. The 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, 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-292-6137; Fax: 614-292-4888;
E-mail: davis.812@osu.edu.
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