Sun Y L, Xie Y, Wang Y H, Xiao G T, Huang Z X
Department of Chemistry, Fudan University, Shanghai, PRC.
Protein Eng. 1996 Jul;9(7):555-8. doi: 10.1093/protein/9.7.555.
The gene encoding trypsin-solubilized bovine liver microsomal cytochrome b5 (82 residues in length) has been mutated, in which the codons of Glu44 and Glu56 were changed to those of Ala. The mutated genes were expressed in Escherichia coli successfully and three mutant proteins (E44A, E56A and E44/56A) were obtained. The UV-visible, CD and 1H NMR spectra of proteins have been studied. The results show that the mutagenesis at surface residues does not alter the secondary and tertiary structures of cytochrome b5 significantly. The interactions between recombinant cytochrome b5 and its mutants with cytochrome c were studied by using optical difference spectra. The results demonstrated that both Glu44 and Glu56 of cytochrome b5 participate in the formation of a complex between cytochrome b5 and cytochrome c.
编码胰蛋白酶可溶解的牛肝微粒体细胞色素b5（长度为82个氨基酸残基）的基因已发生突变，其中第44位和第56位谷氨酸的密码子被替换为丙氨酸的密码子。突变基因在大肠杆菌中成功表达，获得了三种突变蛋白（E44A、E56A和E44/56A）。对这些蛋白的紫外可见光谱、圆二色光谱和一维核磁共振光谱进行了研究。结果表明，表面残基的诱变对细胞色素b5的二级和三级结构没有显著影响。利用光差光谱研究了重组细胞色素b5及其突变体与细胞色素c之间的相互作用。结果表明，细胞色素b5的第44位和第56位谷氨酸均参与细胞色素b5与细胞色素c之间复合物的形成。
