Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 7732998
• 负责人: DI S XIA
• 金额: $ 74.07万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732998
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Area; Bacteria; Binding; Biochemical; Biochemical Genetics; Biological; Biological Models; Biological Process; Cell Communication; Cell physiology; Cells; Classification; Complex; Condition; Copper; Crystallization; Cytochrome bc1 Complex; Databases; Deposition; Detergents; Development; Drug Delivery Systems; Drug Metabolic Detoxication; Electrons; Environment; Enzymes; Escherichia coli; Fostering; Genes; Genome; Goals; Homologous Gene; Housing; Human; Hydroquinones; Integral Membrane Protein; Intramural Research Program; Ion Transport; Laboratories; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Mitochondria; Modeling; Molecular; Numbers; Nutrient; Organism; P-Glycoprotein; P-Glycoproteins; Photosynthetic Complexes; Preparation; Proteins; Protons; Quinones; Range; Research; Resolution; Respiratory Chain; Risk; Role; Saccharomyces cerevisiae; Sampling; Scheme; Signal Transduction; Site; Solutions; Structure; Surface; System; Techniques; Testing; Transportation; Uncertainty; United States National Institutes of Health; Work; X-Ray Crystallography; Yeasts; benzoquinone; improved; inhibitor/antagonist; interest; mutant; oxidation; photosynthetic bacteria; protein expression; protein structure; receptor; research study; structural biology

Over the past few years, we have made significant progress in understanding the mechanism of function of the bc1 at atomic resolution by analyzing both native- and inhibitor-bound structures. We proposed a scheme for bc1 inhibitor classification and put forward mechanisms for quinone reduction at the QN site and quinol oxidation at the QP site. Most importantly, we have obtained experimental evidence to support our confomation switch model for the electron bifurcation at the quinol oxidation site, which is the key to the high proton translocation efficiency in the bc1 complex. Recently, we have successfully determined the crystal structures of the wild type and mutant bc1 complex from the photosynthetic bacterium R. sphaeroides (Rsbc1) in complex with various inhibitors, demonstrating our ability to reproducibly obtain atomic resolution structural information on the bacterial bc1 in various forms and our perseverance in pursuing difficult projects. This work accomplishes one of our goals in establishing a model system to systematically study the bc1 complex by combining structural, genetic, and biochemical techniques; it marks another milestone in the study of bc1 complex and in the field of membrane protein structural biology. The development of methodology for membrane protein expression, purification, and crystallization has been an integral part of our research on structure determinations of P-gp and its homologues. To this end, we have been exploring various expression systems to achieve consistent high-level protein expression for a few membrane proteins; those include yeast systems such as S. cerevisiae and P. pastoris expression systems, bacterial systems such as E. coli and L. lactis expression systems, and photosynthetic bacterum R. sphaeroides. We have extended the use of Blue-Native techniques to detecting monodispersity of membrane protein preparations. We have also developed and refined a multi-parameter kit to screen for conditions for stabilizing P-gp in solution. We have achieved high-level expressions for a number of integral membrane proteins. In addition to the bacterial bc1 complex, the human P-glycolprotein, bacterial ABC transporter LmrA, and bacterial CopB were purified in large quantities. To obtain monodispersed protein samples, we have been using the Blue-Native technique developed in house to screen for various detergents, which is very successful. For conformationally flexible membrane proteins such as ABC transporters, we tested mutants and Fab-complexed P-gp in crystallization experiments. Although we have yet to reach our goal of structure solutions of these membrane proteins, the methods developed here will be useful for other membrane proteins.

在过去的几年中，我们通过分析天然和抑制剂结合的结构来理解原子分辨率的BC1功能机理。我们提出了一种用于BC1抑制剂分类的方案，并提出了在QN位点减少奎因酮的机制，并在QP位点提出了奎诺醇氧化。 最重要的是，我们获得了实验证据，以支持奎诺醇氧化位点电子分叉的混合物开关模型，这是BC1复合物中高质子易位效率的关键。最近，我们成功地确定了来自光合细菌的野生型和突变体BC1复合物的晶体结构。这项工作实现了我们建立模型系统来通过结合结构，遗传和生化技术来系统研究BC1复合物的目标之一。它标志着BC1复合物研究和膜蛋白结构生物学领域的另一个里程碑。膜蛋白表达，纯化和结晶的方法的发展一直是我们在P-gp及其同源物结构测定方面的研究的一部分。为此，我们一直在探索各种表达系统，以实现一些膜蛋白的一致的高级蛋白表达。其中包括酵母系统，例如酿酒酵母和P. p. pastoris表达系统，细菌系统，例如大肠杆菌和乳酸乳乳杆菌表达系统，以及光合杆菌R. sphaeroides。我们已经扩展了蓝色技术来检测膜蛋白制剂的单分散性。我们还开发了并完善了一个多参数套件，以筛选稳定溶液中p-gp的条件。我们已经为许多积分膜蛋白实现了高级表达。除了细菌BC1复合物外，大量纯化了人类P-聚糖蛋白，细菌ABC转运蛋白LMRA和细菌COPB。为了获得单分散的蛋白质样品，我们一直在使用内部开发的蓝色技术来筛选各种洗涤剂，这非常成功。对于构象柔性的膜蛋白（例如ABC转运蛋白），我们在结晶实验中测试了突变体和Fab-oppox的P-gp。尽管我们尚未达到这些膜蛋白结构溶液的目标，但此处开发的方法将对其他膜蛋白有用。