The characterization for the binding of calcium and terbium to Euplotes octocarinatus centrin

Centrin is a member of the EF-hand superfamily that plays critical role in the centrosome duplication and separation. In the present paper, we characterized properties of metal ions binding to Euplotes octocarinatus centrin (EoCen) by fluorescence spectra and circular dichroism (CD) spectra. Changes of fluorescence spectra and α-helix contents of EoCen proved that Tb3+ and Ca2+ induced great conformational changes of EoCen resulting in exposing hydrophobic surfaces. At pH 7.4, Ca2+ (and Tb3+) bond with EoCen at the ratio of 4:1. Equilibrium experiment indicated that Ca2+ and Tb3+ exhibited different binding capabilities for C- and N-terminal domains of protein. C-terminal domain bond with Ca2+ or Tb3+ ∼ 100-fold more strongly than N-terminal. Aromatic residue-sensitized Tb3+ energy transfer suggested that site IV bond to Tb3+ or Ca2+ more strongly than site III. Based on fluorescence titration curves, we reckoned the conditional binding constants of EoCen site IV quantitatively to be KIV = (1.23 ± 0.51) × 108 M−1 and KIV = (6.82 ± 0.33) × 105 M−1 with Tb3+ and Ca2+, respectively. Metal ions bond to EoCen in the order of IV > III > II, I.