Structure-Function of Hetero-oligomeric Integral Membrane Proteins

异源寡聚整合膜蛋白的结构-功能

• 批准号: 7319520
• 负责人: William A. Cramer
• 金额: $ 33.59万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7319520
• 关键词: 1,2-diacylglycerol; 4-hydroxyquinoline; Abbreviations; Amino Acids; Bacillus cereus; Bacillus subtilis; Benzoquinones; Beta Carotene; Binding; Binding Sites; Bioenergetics; Cations; Choline; Complex; Coupled; Coupling; Crystallization; Cyanobacterium; Cytochrome c Reductase; Cytochromes; Cytochromes b; Data; Databases; Diffusion; Diglycerides; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Electrophoresis; Ethanolamines; Family; Ferredoxin; Ferredoxin-NADP Reductase; Future; Generations; Glycerol; Goals; Gram-Positive Bacteria; Grant; Heme; Hydroquinones; Immunoglobulin Fragments; Integral Membrane Protein; Iron; Iron-Sulfur Proteins; Lecithin; Ligands; Link; Lipid Bilayers; Lipids; Mass Spectrum Analysis; Membrane; Membrane Proteins; Mitochondria; Molecular; Molecular Conformation; Molecular Weight; NADH dehydrogenase (ubiquinone); Numbers; Oxidation-Reduction; Oxides; Oxygen; Pathway interactions; Pharmaceutical Preparations; Phase; Photosynthesis; Photosynthetic Reaction Centers; Plastocyanin; Plastoquinone; Polymerase Chain Reaction; Prosthesis; Proteins; Purpose; Quinones; Rate; Reactive Oxygen Species; Reducing Agents; Relative (related person); Research Personnel; Resolution; Role; Route; Ruthenium; Side; Simulate; Site; Site-Directed Mutagenesis; Sodium; Source; Structure; Structure-Activity Relationship; Sulfur; Superoxides; Techniques; Testing; Ubiquinone; Vitamin K 2; analog; benzoquinone; cadmium ion; chlorophyll a; chlorophyll a' ; cytochrome b6f; digalactosyldiacylglycerol; dimer; electron donor; ethanolamine; improved; inhibitor/antagonist; membranous labyrinth; molecular dynamics; monomer; nonyl-4-hydroxyquinoline-N-oxide; novel; oxidation; phenyltin; polyacrylamide; progesterone 11-hemisuccinate-(2-iodohistamine); programs; research study; respiratory; stigmatellin; triplet state; tyramine-deoxysorbitol; ubiquinol; uptake

DESCRIPTION (provided by applicant): The cytochrome b6f complex is one of a small number (presently, 17) of hetero-oligomeric integral membrane proteins whose crystal structure has been solved to a resolution better than 3.0 A. The structure is a dimer that encloses a large (10,000 A3) lipophilic inter-monomer 'quinone exchange cavity that exchanges quinone/quinol with the lipid bilayer, and connects the quinone reduction site on the electrochemically negative n-side of one monomer with the oxidation site at the [2Fe-2S] cluster on the positive p-side. The quinone pathway between n- and p-sides involves passage through anllAx!2A portal. We have solved four b6f structures from the thermophilic cyanobacterium, M. laminosus, (i) a native structure with resolution recently improved to 2.95 A in the presence of Cd2+ cations, and three structures of complexes with quinone- analogue inhibitors, p-side TDS and DBMIB, and n-side NQNO. These structures have provided markers for the route of quinone passage across the complex and diffusion within it. Further understanding of the structure and dynamics of quinone transfer is relevant to understand transfer of hydrophobic drugs and metabolites across membrane proteins, and will be pursued through higher resolution structures, site-directed mutagenesis and molecular/steered dynamics. The complex contains 8 subunits: 4 of these form a core of 'large' subunits that bind 8 prosthetic groups. Each b6f monomer contains 4 hemes, one [2Fe-2S] cluster, one plastoquinone, and 3 unique prosthetic groups: (i) a novel heme en with one covalent linkage to a Cys residue of cytochrome b, and no amino acid side chains as axial ligands; (ii) one chlorophyll a, for which two H2O have been resolved as its 5th ligand; (iii) one beta-carotene to which the Chi, can transfer excited triplet state energy in spite of their 14A separation. These prosthetic groups unique to b6f raise new questions about redox function in be complexes. We will focus on the unique heme en, whose functions are linked to the ability of the b6f complex to carry out ferredoxin-dependent cyclic electron transport and of the photosynthetic membrane to evolve O2. A unique coupling between heme en and the nearby (4 A) heme bn was established, which implies an ability of hemes bn - en to serve as a 2 electron donor, thus providing a new mechanism to protect against formation of superoxide and reactive oxygen species (ROS). Studies on a second hetero- oligomeric integral membrane protein, Ndh-1 from the cyanobacterial membrane that also reduces quinone and generates ROS, have masses of 12 of the 14-15 subunits, and in which the goal is to obtain a crystal structure.

描述（由申请人提供）：细胞色素b6 f复合物是少数（目前为17种）异源寡聚整合膜蛋白之一，其晶体结构已解析至优于3.0 A的分辨率。该结构是一种二聚体，其封闭了一个大的（10，000 A3）亲脂性单体间醌交换空腔，该空腔与脂质双层交换醌/醌醇，并将一个单体的电化学负n侧上的醌还原位点与正p侧上的[2Fe-2S]簇的氧化位点连接起来。n-和p-侧之间的醌途径涉及通过anllAx！2A传送门。我们已经解决了四个b6 f结构的嗜热蓝藻，M。laminosus，（i）最近在Cd 2+阳离子存在下分辨率提高到2.95 A的天然结构，以及与醌类似物抑制剂、p侧TDS和DBMIB以及n侧NQNO的络合物的三种结构。这些结构提供了标记的途径醌通过复杂的和扩散内it. Further了解醌转移的结构和动力学是相关的了解疏水药物和代谢物跨膜蛋白的转移，并将通过更高的分辨率结构，定点诱变和分子/转向动力学追求。该复合物包含8个亚基：其中4个形成一个“大”亚基的核心，结合8个辅基。每个b6 f单体含有4个血红素、一个[2 Fe-2 S]簇、一个质体醌和3个独特的辅基：（i）一个新的血红素烯，它与细胞色素B的一个Cys残基共价连接，没有氨基酸侧链作为轴向配体;（ii）一个叶绿素a，它的两个H 2 O已被解析为它的第5个配体;（iii）一种β-胡萝卜素，尽管它们的14 A分离，但Chi可以向其转移激发三重态能量。这些b6 f特有的辅基提出了关于be复合物中氧化还原功能的新问题。我们将专注于独特的血红素en，其功能与B6 F复合物进行铁氧还蛋白依赖的循环电子传递和光合膜进化O2的能力有关。血红素en与邻近的（4A）血红素bn之间建立了独特的偶联，这意味着血红素bn-en能够充当2电子供体，从而提供了防止超氧化物和活性氧物种（ROS）形成的新机制。对第二种异源寡聚体整合膜蛋白的研究，来自蓝藻膜的Ndh-1，其也还原醌并产生ROS，具有14-15个亚基中的12个的质量，并且其中目标是获得晶体结构。