Crystallographic and Genetic Study of Cytochrome bc1 Complex

细胞色素bc1复合物的晶体学和遗传学研究

• 批准号: 08044203
• 负责人: FUKUYAMA Keiichi
• 金额: $ 6.34万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044203/
• 关键词: Cytochrome; Histidine-tag; Rhodobacter capsulatus; Membrane Protein; 電子伝達系; 膜蛋白質複合体; Rhodobacter; 鉄硫黄センター; チトクロムbc1複合体; 迅速精製; 結晶化; 呼吸酵素; Cytochrome; Histidine-tag; Rhodobacter capsulatus; Membrane Protein; 電子伝達系; 膜蛋白質複合体; Rhodobacter; 鉄硫黄センター; チトクロムbc1複合体; 迅速精製; 結晶化; 呼吸酵素

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.

为了有效地供应结晶材料，开发了两种快速，高效的方法，以促进Rhodobacer Capsulatus的细胞色素（CYT）BC_1复合物的大规模纯化。这些方法依赖于Hexa-Histidine标签的高亲和力对Ni^<2+> 2-硝基三环酸（NTA）琼脂糖的高亲和力。设计了两种类型的（His）_6标记的BC_1复合物的基因：一个基因在Cyt C^1（CT标记）的C端融合，另一个是在Rieske Subunit（Rieske标签）的C端。每个工程基因被转移到BC^1 Complex-Null突变体MT-RBCL中。详细分析了它们产生的所得菌株和BC^1复合物。色谱膜中的酶的稳态活性约为20％和35％，闪存诱导的单个周转动力学分别为70％和90％的野生型复合物。标记的Bcl^1复合物可以通过Ni^<2+> - NTA-Agarose色谱很容易纯化。他们的结晶正在研究。