Fourier transform infrared spectroscopic study on the binding of Mg^2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca^2+-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca^2+, while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca^2+ at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca^2+. Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg^2+ bound in a mutant Akazara scallop TnC (E142Q) in D2O solution. The result showed that the side-chain COO- groups of Asp 131 and Asp 133 in the Ca^2+-binding site of E142Q bind to Mg^2+ in the pseudo-bridging mode. Mg^2+ titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm^-1, which is due to the pseudo-bridging Asp COO- groups. As a result, the binding constants of them for Mg^2+ were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO- group of Glu 142 of the wild type has no relation to the Mg^2+ ligation. The effect of Mg^2+ binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg^2+ as well as Ca^2+.