Metal ion-binding properties of wild-type and mutant D37K of ciliate Euplotes octocarinatus centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin.The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue.The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen.Aromatic-sensitized Tb3+fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation.Based on fluorescence titration curves of Lu2-D37K,the conditional binding constants of the EoCen loop II were quantitatively found to be KII=(1.61±0.04)×105 L mol-1 and KII=(3.52±0.08)×102 L mol-1 with Tb3+ and Ca2+,respectively.Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe,exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.

Ciliate Euplotes octoearinatus centrin （EoCen） is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. The first amino acid of the Ca2＋-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue. The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen. Aromatic-sensitized Tb3＋ fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation. Based on fluorescence titration curves of Lu2-D37K, the conditional binding constants of the EoCen loop II were quantitatively found to be KII = （1.61± 0.04） ×10 5 L mol-1 and KII = （3.52 ± 0.08） × 10 2L mol-l with Tb3＋ and Ca2＋, respectively. Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe, exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.