| 八肋游仆虫中心蛋白N端半分子Loop区首个氨基酸的作用(英文) |
| Roles of Asp37 and Asp73 in the Loop of N-Terminal Domain of Ciliate Euplotes Octocarinatus Centrin |
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| 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loop Ⅰ区37位的天冬氨酸突变为赖氨酸后，loop Ⅰ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loop Ⅱ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loop Ⅰ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loop Ⅱ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。 |
| 关键词: 中心蛋白 N端半分子 Tb3+ Ca2+ TNS |
| 基金项目: |
| Abstract: 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)×104 L·mol-1 and KⅡ=(0.94±0.12)×102 L·mol-1 with Tb3+ and Ca2+, respectively, and the order for the metal-binding affinity of the two sites in N-EoCen is Ⅰ>Ⅱ. |
| Keywords: centrin N-terminal Tb3+ Ca2+ TNS |
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| 摘要点击次数: 1389 |
| 全文下载次数: 1999 |
| 赵 冰,段 炼,刘 文,赵亚琴,杨斌盛.八肋游仆虫中心蛋白N端半分子Loop区首个氨基酸的作用(英文)[J].无机化学学报,2011,27(2):245-250. |
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