Engineering competitive magnesium binding into the first EF-hand of skeletal troponin C.
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 Trp(29), 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 microm. 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 microm, respectively. Addition of 3 mm magnesium decreased the calcium sensitivity of TnC(F29W) and G34DTnC(F29W) approximately 2- and 6-fold, respectively. Exchange of G34DTnC(F29W) into skinned psoas muscle fibers decreased fiber calcium sensitivity approximately 1.7-fold compared with TnC(F29W) at 1 mm [magnesium](free) and approximately 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.
Davis
Jonathan P
JP
Departments of Physiology and Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA. davis.812@osu.edu
Rall
Jack A
JA
Reiser
Peter J
PJ
Smillie
Lawrence B
LB
Tikunova
Svetlana B
SB
D000818:Animals; D001667:Binding, Competitive; D001842:Bone and Bones / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D020832:EF Hand Motifs; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism
D019209:Troponin C; D004533:Egtazic Acid; D008274:Magnesium; D002118:Calcium