八肋游仆虫中心蛋白N端半分子Loop区首个氨基酸的作用  

Roles of Asp37 and Asp73 in the Loop of N-Terminal Domain of Ciliate Euplotes Octocarinatus Centrin

作　　者：赵冰[1] 段炼[1] 刘文[1] 赵亚琴[1] 杨斌盛[1] 

机构地区：[1]山西大学分子科学研究所,太原030006

出　　处：《无机化学学报》2011年第2期245-250,共6页Chinese Journal of Inorganic Chemistry

基　　金：国家自然科学基金(No.2077106820901048);山西省自然科学基金(No.20901048)资助项目

摘　　要：本文通过分子生物学方法将八肋游仆虫中心蛋白N端半分子loop区的首个氨基酸,天冬氨酸Asp37和Asp73,分别突变为带相反电荷的赖氨酸。使用铽敏化荧光、TNS疏水探针研究了八肋游仆虫中心蛋白N端半分子loop区的首个氨基酸的作用。结果表明:当中心蛋白loopⅠ区37位的天冬氨酸突变为赖氨酸后,loopⅠ丧失了金属离子结合能力,进而影响了中心蛋白依赖于金属离子的构象变化;而loopⅡ区73位的天冬氨酸突变为赖氨酸后仍保持金属离子结合能力,依赖于金属离子的构象变化减小。中心蛋白发挥大部分生物功能都依赖于金属离子,这就表明loopⅠ区37位的天冬氨酸在中心蛋白发挥生物功能时起着重要作用,是不可缺少的。在10 mmol·L-1 Hepes、pH 7.4、20 mmol·L-1 KCl条件下,八肋游仆虫中心蛋白N端半分子loopⅡ与金属离子Tb3+和Ca2+的结合常数分别为:KⅡ(Tb3+)=(8.31±0.18)×104 L·mol-1和KⅡ(Ca2+)=(0.94±0.12)×102 L·mol-1,中心蛋白N端半分子的两个金属结合部位结合能力顺序为:Ⅰ>Ⅱ。Ciliate Euplotes octocarinatus centrin（EoCen） is a member of the EF-hand superfamily of calcium-binding proteins,which often associated with the centrosomes and basal bodies.To explore the importance of Asp37 and Asp73 in the loop of N-terminal of EoCen,the mutants of N-D37K and N-D73K（D is aspartic acid,and K is lysine） were obtained by molecular biological methods.The experiments of aromatic residue-sensitized Tb3＋ energy transfer demonstrate that：N-D73K can bind two equivalents Tb3＋,which is similar to N-EoCen（WT-EoCen binds four equiv.Tb3＋）,and N-D37K can only bind one equivalent Tb3＋.Using 2-p-toluidinylnaphthalene-6-sulfonate（TNS） as a hydrophobic probe,exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated N-D37K mutant.The results indicate that Asp37 plays an important role in the biological functions of N-terminal domain of EoCen.Meanwhile the conditional binding constants of the N-EoCen loop Ⅱ were quantitatively found to be KⅡ=（8.31±0.18）×10^4 L·mol^-1 and KⅡ=（0.94±0.12）×10^2 L·mol^-1 with Tb^3＋ and Ca^2＋,respectively,and the order for the metal-binding affinity of the two sites in N-EoCen is Ⅰ〉Ⅱ.

关 键 词：中心蛋白 N端半分子 TB3+ CA2+ TNS 

分 类 号：O641.1[理学—物理化学] O614.341[理学—化学]