Characterization of the Ca2+-coordination structures of L- and T-plastins in combination with their synthetic peptide analogs by FTIR spectroscopy

Abstract

FTIR spectroscopy was employed to characterize the coordination structures of divalent cations (M²⁺ = Ca²⁺ or Mg²⁺) bound by L- and T-plastins, which contain two EF-hand motifs. We focused on the N-terminal headpieces in the L- and T-plastins to analyze the regions of COO^- stretching and amide-I in solution. The spectral profiles indicated that these headpieces have EF-hand calcium-binding sites because bands at 1551 cm^-1 and 1555 cm^-1 were observed for the bidentate coordination mode of Glu at the 12th position of the Ca²⁺-binding site of Ca²⁺-loaded L-plastin and T-plastin, respectively. The amide-I profile of the Mg²⁺-loaded L-plastin headpiece was identical with that of the apo L-plastin headpiece, meaning that L-plastin has a lower affinity for Mg²⁺. The amide-I profiles for apo, Mg²⁺-loaded and Ca²⁺-loaded T-plastin suggested that aggregation was generated in protein solution at a concentration of 1 mM. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of data obtained for synthetic peptide analogs corresponding to the Ca²⁺-binding site.