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

基本信息

  • 批准号:
    08044203
  • 负责人:
  • 金额:
    $ 6.34万
  • 依托单位:
  • 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

项目摘要

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

期刊论文数量(16)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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。
  • DOI:
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    0
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  • 通讯作者:
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)。
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    0
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FUKUYAMA Keiichi其他文献

FUKUYAMA Keiichi的其他文献

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{{ truncateString('FUKUYAMA Keiichi', 18)}}的其他基金

Elucidation of mechanisms of bilin synthesizing enzyme and photo-adaptation regulating protein
胆素合成酶和光适应调节蛋白机制的阐明
  • 批准号:
    23370052
  • 财政年份:
    2011
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Elucidation of molecular mechanism of bilin synthesis by ferredoxin-dependent bilin reductases
阐明铁氧还蛋白依赖性胆素还原酶合成胆素的分子机制
  • 批准号:
    20370037
  • 财政年份:
    2008
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Elucidation of reaction mechanism ofthe enzymes involved in the syntbssis of photosynthetic pigments
光合色素合成酶反应机制的阐明
  • 批准号:
    18570105
  • 财政年份:
    2006
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Functional analysis of proteins based on the tertiary structures focussing on hydrogen atom positions
基于以氢原子位置为重点的三级结构的蛋白质功能分析
  • 批准号:
    14580674
  • 财政年份:
    2002
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Mechanism of DNA excision repair based on the three-dimensional structure of UvrABC endonuclease
基于UvrABC核酸内切酶三维结构的DNA切除修复机制
  • 批准号:
    12680659
  • 财政年份:
    2000
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Analysis of assembly mechanism of spherical virus based on its high resolution three-dimensional structure
基于高分辨率三维结构的球形病毒组装机制分析
  • 批准号:
    10680634
  • 财政年份:
    1998
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Study on the reaction mechanism of peroxidase based on its tertiary structure
基于过氧化物酶三级结构的反应机理研究
  • 批准号:
    06680654
  • 财政年份:
    1994
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Studies on the reaction mechanism of peroxidase based on its tertiary structure
基于过氧化物酶三级结构的反应机理研究
  • 批准号:
    04680055
  • 财政年份:
    1992
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
X-Ray Crystallographic Analysis of Spherical Plant Viruses
球形植物病毒的 X 射线晶体学分析
  • 批准号:
    61580049
  • 财政年份:
    1986
  • 资助金额:
    $ 6.34万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
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