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

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.

为了有效地提供结晶原料，发展了两种快速、高效的方法，以便于大规模纯化荚膜红杆菌细胞色素（cyt）bc_1复合物。该方法依赖于六组氨酸标签对Ni^2+ 2-次氮基三乙酸（NTA）琼脂糖的高亲和力。设计了两种类型的（His）_6-taged bc_1复合物的基因：一种融合在cyt c^1的C-末端（ct-taged），另一种融合在Rieske亚基的C-末端（Rieske-taged）。每个工程基因都被转移到bc^1复合物缺失突变体MT-RBCl中。对所得到的菌株及其产生的bc^1复合物进行了详细的分析，发现带有Rieske和c1^1标签的bc^1复合物都支持荚膜红球菌的光合生长。色素细胞膜中的酶的稳态活性分别约为野生型复合物的20%和35%，闪光诱导的单次转换动力学分别约为野生型复合物的70%和90%。标记的bcl ^1复合物可以通过Ni^2+-NTA-琼脂糖层析法容易地以高收率纯化。它们的结晶正在研究中。

项目成果

K.Fukuyama, K.Sato, H.Itakura, S.Takahashi & T.Hosoya: "Binding of Iodide to Atrhromyces ramosus Peroxidase Investigated with X-Ray Crystallo-graphic Analysis, ^1H and ^<127>I NMR Spectroscopy, and Steady-state Kinetics" J.Biol.Chem.272(9). 5752-5756 (199

K.福山、K.佐藤、H.板仓、S.高桥

A.Shibata, N.Nakagawa, M.Sugihara, R.Masui, R.Kato, S.Kuramitsu & K.Fukuyama: "Crystallization and Preliminary X-Ray Diffraction Studies of a DNA Excision Enzyme, UvrB,from Thermus thermophilus HB8" Acta Cryst.D55. 704-705 (1999)

A.柴田、N.中川、M.杉原、R.增井、R.加藤、S.Kuramitsu

H.Oh-oka, M.Iwaki & S.Itoh: "Viscosity Dependence of the Electron Transfer Rate from Bound Cytochrome c to P840 in the Photosynthetic Reaction Center of the Green Sulfur Bacterium Chlorobium tepidum" Biochemistry. 36(30). 9267-9272 (1997)

H.Oh-oka，M.Iwaki

Y.Oda, T.Matsunaga, K.Fukuyama, T.Miyazaki, T.Morimoto: "Tertiary and Quaternary Structure of 0.19 alpha-Amilase Inhibitor from Wheat Kernel Determined by X-Ray Analysis of the Complex at 2.06 * Resolution" Biochemistry. 36 (44). 13503-13511 (1997)

Y.Oda、T.Matsunaga、K.Fukuyama、T.Miyazaki、T.Morimoto：“通过以 2.06 * 分辨率对复合物进行 X 射线分析测定小麦籽粒中 0.19 α-淀粉酶抑制剂的三级和四级结构”生物化学。

K.Fukuyama: "Binding of Iodide to Atrhromyces ramosus Peroxidase Investigated with X-Ray Crystallo-graphic Analysis, ^1H and ^<127>INMR Spectroscopy, and Steady-state Kinetics" J.Biol.Chem.272(9). 5752-5756 (1997)

K.Fukuyama：“通过 X 射线晶体分析、^1H 和 ^<127>INMR 光谱以及稳态动力学研究碘化物与支枝节孢过氧化物酶的结合”J.Biol.Chem.272(9)。