Infrared spectroscopic study on Ca2+ binding to Akazara scallop troponin C in comparison with peptide analogues of its Ca2+-binding Site IV

Fourier-transform infrared spectroscopy (FT-IR) was applied to study the coordination structure of Ca2+ bound in Akazara scallop troponin C (TnC) and its site-directed mutant possessing inactivated Site IV (E142D mutant) in D2O solution. The COO− antisymmetric stretching region provides information about the coordination modes of a COO− group to a metal ion. The wild type exhibits a band at 1543 cm−1 in the Ca2+-bound state, indicating that the side-chain COO− group of Glu142 (the position 12 of Site IV) serves as the ligand for Ca2+ in the bidentate coordination mode [F. Yumoto, M. Nara, H. Kagi, W. Iwasaki, T. Ojima, K. Nishita, K. Nagata, M. Tanokura, Eur. J. Biochem. 268 (2001) 6284-6290]. However, the E142D mutant showed no band around 1543 cm−1 in the Ca2+-loaded state, indicating that the side-chain COO− group of Asp142 does not bind to Ca2+ in the bidentate coordination mode. This result suggests that the absence of a methylene group is critical for the Ca2+ coordination structure of Akazara scallop TnC. The Ca2+-ligand interaction at Site IV is discussed in comparison with the results of synthetic peptide analogues of Site IV of Akazara scallop TnC.