Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 8175305
• 负责人: di s xia
• 金额: $ 88.21万
• 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/8175305
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Active Biological Transport; Affinity; Area; Bacteria; Belief; Binding; Biochemical; Biological; Biological Models; Biological Process; Cattle; Cell Communication; Cell Line; Cell membrane; Cell physiology; Cells; Cisplatin; Classification; Cloning; Complex; Coupled; Coupling; Crystallization; Cytochrome bc1 Complex; Databases; Deposition; Detergents; Development; Drug Delivery Systems; Drug Metabolic Detoxication; Electron Transport; Electrons; Elements; Energy Metabolism; Environment; Enzymes; Escherichia coli; Fostering; Genes; Genetic; Genome; Goals; Homologous Gene; Housing; Human; Hydroquinones; Integral Membrane Protein; Intramural Research Program; Iron-Sulfur Proteins; Knowledge; Laboratories; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Mitochondria; Modeling; Molecular; Molecular Conformation; Monoclonal Antibodies; Motion; Movement; Nutrient; One-Step dentin bonding system; Organism; P-Glycoprotein; P-Glycoproteins; Pharmaceutical Preparations; Physiological; Preparation; Proteins; Protons; Quinones; Recombinants; Research; Resistance; Resolution; Respiratory Chain; Rhodobacter sphaeroides; Roentgen Rays; Role; Saccharomyces cerevisiae; Sampling; Scheme; Signal Transduction; Site; Solutions; Staging; Structure; Structure-Activity Relationship; Surface; System; Techniques; Testing; Transfection; Transportation; Uncertainty; United States National Institutes of Health; Work; X-Ray Crystallography; Yeasts; base; cDNA Library; cancer cell; flexibility; high risk; inhibitor/antagonist; interest; mutant; oxidation; photosynthetic bacteria; polyclonal antibody; protein complex; protein expression; protein structure; receptor; research study; structural biology; therapeutic target; ubiquinol

My group has been working on the mechanisms of function of three integral membrane proteins: proton transporting cyt bc1 complexes, multidrug ABC transporters and putative cisplatin resistance (CP-r) associated protein TMEM205. These projects are currently at different stages with respect to our understanding of their mechanisms of function. For the bc1 complex, we have a very good understanding of its mechanism of coupling. We have determined the crystal structures of bc1 complexes from the bovine mitochondria (Mtbc1) and from the photosynthetic bacterium Rhodobacter sphaeroides (Rsbc1) in both apo and inhibitor-bound forms. We have identified critical structural elements that are essential for the coupling of electron transfer and proton translocation. We have proposed a surface-affinity modulated iron-sulfur protein (ISP) motion control hypothesis to explain the bifurcated electron transfer in bc1. For the multidrug ABC transporters, in particular human P-glycoprotein (hP-gp), advances are being made towards its structure solution with respect to its over-expression in a number of eukaryotic systems, its purification and complex formation with monoclonal antibodies (mAb). We also made significant progress in obtain diffraction quality crystals of TMEM205 and its structure solution is on the way. 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 surface modulated conformation 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. We have also 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. Recently, we have achieved a long-sought after structure of ubiquinol, the substrate, bound at the quinol oxidation site of bc1, which is a one step forward toward experimental verification of our hypothesis on bifurcated electron transfer under physiological conditions. Development of CP-r in cancer cells appears to be a consequence of multifaceted alterations involved in various cellular processes. Recently, it was found that expression of the hypothetical transmembrane protein TMEM205 is associated with cisplatin resistance, first detected by functional cloning from a retroviral cDNA library made from human CP-r cells. Using a polyclonal antibody, it was found that TMEM205 expression is increased in our CP-r cell lines. Stable transfection of the TMEM205 gene confers resistance to cisplatin by approximately 2.5-fold. Recombinant over-expression of TMEM205 in yeast directs the protein to cytoplasmic membrane, which was purified to homogeneity and in large quantity suitable for crystallographic studies. Crystals of TMEM205 were grown and diffracted X-rays to 3.4 resolution. Structural solution is in progress and it is expected that it will help to unveil the structural basis of TMEM205 involvement in cisplatin resistant. 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, its homologues and TMEM205. 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 membrane proteins 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, human TMEM205, 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.

我的小组一直在研究三种整合膜蛋白功能的机理：质子转运Cyt BC1配合物，多药ABC转运蛋白和假定的顺铂耐药性（CP-R）相关蛋白TMEM205。这些项目目前处于我们对其功能机制的理解方面的不同阶段。对于BC1复合体，我们对其耦合机制有很好的了解。我们已经确定了来自牛线粒体（MTBC1）的BC1复合物的晶体结构，以及来自Apo和抑制剂结合形式的光合细菌Sphaeroides（RSBC1）的光合细菌。我们已经确定了关键的结构元素，这些元素对于电子转移和质子易位耦合至关重要。我们提出了表面亲和力调制的铁硫蛋白（ISP）运动控制假设，以解释BC1中分叉的电子转移。对于多药ABC转运蛋白，尤其是人P-糖蛋白（HP-GP），在许多真核系统中的过表达方面正在对其结构解决方案取得进展，其纯化和与单克隆抗体（MAB）的纯化和复杂形成。我们还在获得TMEM205的衍射质量晶体方面取得了重大进展，其结构解决方案正在途中。 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，以支持奎诺氧化位点电子分叉的表面调制构象开关模型，这是BC1复合物中高质子易位效率的关键。我们还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。最近，我们已经实现了在BC1的奎诺醇氧化位点结合的泛素醇的长期追求的结构，这是在生理条件下对分叉电子转移的假设迈出的一步。 癌细胞中CP-R的发展似乎是各种细胞过程中涉及的多方面变化的结果。最近，发现假设的跨膜蛋白TMEM205的表达与顺铂耐药有关，首先是通过由人CP-R细胞制成的逆转录病毒cDNA库来检测的。使用多克隆抗体，发现在我们的CP-R细胞系中TMEM205表达增加。 TMEM205基因的稳定转染使对顺铂的抗性约为2.5倍。 TMEM205在酵母中的重组过表达将蛋白质引导到细胞质膜，该蛋白被纯化为同质性，并且大量适合晶体学研究。生长TMEM205的晶体，并衍射为X射线至3.4分辨率。结构解决方案正在进行中，预计它将有助于揭示TMEM205参与顺铂耐药性的结构基础。 膜蛋白表达，纯化和结晶的方法的发展一直是我们在P-gp，其同源物和TMEM205结构测定研究的研究的一部分。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。我们还开发了一个多参数套件，以筛选溶液中稳定膜蛋白的条件。 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，还大量纯化了人类P-聚糖蛋白，人TMEM205，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。