【摘要】 利用核磁共振方法研究表面带不同负电荷氨基酸残基突变后的细胞色素 b5与细胞色素 c的结合与识别 .结果表明 ,静电作用在细胞色素 b5与细胞色素 c的结合过程中有着重要的贡献 ,而且这些静电贡献在一定程度上具有累加性 ,E48的贡献略大于 E44.同时还证明 Brownian dynamics simulations优化出的Glu48— Lys1 3 ,Glu5 6— Lys87,Asp60— Lys86和 heme 6-propionate-Tml72 (细胞色素 b5的残基排在前面 )的结合方式在溶液中的确存在 .细胞色素 b5突变体 (E48,E5 6/ A,D60 / A)及 [Cr(oxalate) 3]3-对细胞色素 c的表面结合竞争实验表明 ,细胞色素 c表面结合区 Site 仍然同细胞色素 b5突变体 (E48,E5 6/ A,D60 / A)有结合作用 ,只是结合强度上相对于野生细胞色素 b5同细胞色素 c的结合有所降低 .这表明除上述的Brownian dynamics simulations模型外 ,尚有其它如 Salemme模型等的结合方式 ,这也揭示出细胞色素 b5和细胞色素 c之间的结合是比较动态的更多还原

【Abstract】 The binding between different Cytochrome b 5 mutants and Cytochrome c has been studied by using of NMR method. The result shows that electrostatic effect plays an important role in maintaining the stability and specificity of the complex formed. The differences in association constants demonstrate the electrostatic contributions of Cytochrome b 5 surface negatively charged residues, which are suggested to be involved in the complex formation in the Brownian dynamics simulations and Salemme models, and to have a significant degree of additivity to the stability of Cyt c Cyt b 5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry obtained from Brownian dynamics simulations by Northrup et al , which was involved in the participation of Glu48, Glu56, Asp60 and heme propionate of Cytochrome b 5, do occur in the association between Cytochrome b 5 and Cytochrome c. The competition between the ferricytochrome b 5 mutant Ⅰ(E48, E56/A, D60/A) and [Cr(oxalate) 3] 3- for ferricytochrome c shows that site Ⅲ of Cytochrome c, which is a strong binding site to wild type Cytochrome b 5, still binds to the mutant with relatively weaker strength. Our results indicate that there are other docking domains between Cyt b 5 and Cyt c different from the above mentioned one from Brownian dynamics simulations, at the same time, it also implies that electrostatic interaction on the wild type Cyt b 5 and Cyt c interface results in flexible association complexes.更多还原