Coordination structures of Ca2+ and Mg2+ in Akazara scallop troponin C in solution. FTIR spectroscopy of side-chain COO- groups.
FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO- antisymmetric stretch provides information about the coordination modes of COO- groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm-1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm-1 in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm-1 in the Mg2+-bound state was identical to that in the apo state. Therefore, the side-chain COO- group of Glu142 at the 12th position in the Ca2+-binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO- antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO- groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide-I' region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound TnC > Ca2+-bound TnC. These results suggest that the tertiary structures are different in the Ca2+- and Mg2+-bound states. The present study may provide direct evidence that the side-chain COO- groups in the Ca2+-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC.
Yumoto
F
F
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Nara
M
M
Kagi
H
H
Iwasaki
W
W
Ojima
T
T
Nishita
K
K
Nagata
K
K
Tanokura
M
M
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002942:Circular Dichroism; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008974:Mollusca / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D019209:Troponin C / Q000737:chemistry*
D012996:Solutions; D019209:Troponin C; D008274:Magnesium; D002118:Calcium