摘要
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.
基金
supported by the National Natural Science Foundation of China(20771068,20901048)
the Ph.D Programs Foundation of Ministry of Education of the People’s Republic of China(20091401110007)
the Natural Science Foundation of Shanxi Province(2007011024)
