表面带负电荷氨基酸残基的突变对细胞色素b5与细胞色素c结合和识别的影响

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Browniandynamicssimulations优化出的Glu48-Lys13,Glu56-Lys₈₇,Asp60-Lys86和heme6-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,E56/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,E56/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.

Effects of Surface Negatively Charged Amino Acid Mutation on Binding Between Cytochrome b5 and Cytochrome c

The binding between different Cytochrome b₅mutants and Cytochrome c has been studied by using of NMRmethod. 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 b₅surface 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 b₅complex, 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 b₅, do occur in the association between Cytochrome b₅and Cytochrome c. The competition between the ferricytochrome b₅mutant 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 b₅, still binds to the mutant with relatively weaker strength. Our results indicate that there are other docking domains between Cyt b₅and 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 b₅and Cyt c interface results in flexible association complexes.