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

作　　者：LIU Wen DUAN Lian ZHAO YaQin LIANG AiHua YANG BinSheng 

机构地区：[1]Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University , Taiyuan 030006, China [2]Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China

出　　处：《Chinese Science Bulletin》2010年第27期3118-3122,共5页

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)

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.

关 键 词：CENTRIN 金属离子 突变型 纤毛虫 结合特性 游仆虫 野生型 钙结合蛋白 

分 类 号：Q25[生物学—细胞生物学] O641.4[理学—物理化学]