Crystallographic and Genetic Study of Cytochrome bc1 Complex

为了有效地提供结晶材料，研究了两种快速高效的方法，以实现从荚膜红杆菌中大规模纯化细胞色素（cyt） bc_1配合物。该方法依赖于六组氨酸标签对Ni^<2+>2-硝基三乙酸（NTA）琼脂糖的高亲和力。设计了两种（His） _6标记bc_1复合体的基因：一种融合在cyt c^1的c端（ct标记），另一种融合在Rieske亚基的c端（Rieske标记）。每个工程基因被转移到一个没有bc^1复合物的突变体MT-RBCl中。详细分析了合成菌株及其产生的bc^1络合物。Rieske-和c1 ^1标记的bc^1复合物都支持荚膜霉的光合生长。染色质膜上酶的稳态活性分别为野生型的20%和35%，闪变诱导的单次周转动力学分别为野生型的70%和90%。用Ni^<2+>- nta -琼脂糖层析法纯化bcl^1配合物，收率高。他们的结晶正在研究中。

For efficient supply of crystallization materials, two fast and highly efficient methods were developed to facilitate large scale purification of the cytochrome (cyt) bc_1 complex from Rhodobacer capsulatus. The methods rely on the high affinity of hexa-histidine tag for Ni^<2+>2-nitrilotriacetic acid (NTA) agarose. Genes for two type of (His) _6-tagged bc_1 complex were engineered : one was fused at the C-terminus of cyt c^1 (ct-tagged), and another was at the C-terminus of Rieske subunit (Rieske-tagged). Each engineered gene was transferred into a bc^1complex-null mutant MT-RBCl. The resultant strains and the bc^1 complexes they produce were analyzed in detail.Both Rieske- and c1 ^1-tagged bc^1 complex supported photosynthetic growth of R.capsulatus. The enzymes in chromatophore membrane showed the steady-state activity of about 20% and 35%, and the flash-induced single turnover kinetics of about 70% and 90% of those of wild-type complex, respectively. The tagged bcl^1 complex can be readily purified with high yield by Ni^<2+>-NTA-agarose chromatography. Their crystallization are under study.