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 contain s only a single Ca2+ binding site. The region of the COO- antisymmetric str etch provides information about the coordination modes of COO- groups to th e metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were obs erved at 1584 and 1567 cm(-1) in the apo state, whereas additional bands we re 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 sidechain 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 s light upshift of COO- antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO- groups o f Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordina tion structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remai ned unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallo p 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 la rger than that upon Mg2+ binding. Moreover, gel-filtration experiments indi cated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound Tn C > 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 a re directly involved in the functional on/off mechanism of the activation o f Akazara scallop TnC.