Center for Structure of Membrane Proteins

膜蛋白结构中心

• 批准号: 8529561
• 负责人: Robert M Stroud
• 金额: $ 155.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8529561
• 关键词: ATP-Binding Cassette Transporters; Accounting; Active Biological Transport; Affinity; Amino Acid Sequence; Antibody Affinity; Archives; Bacteriophages; Benchmarking; Benzodiazepine Receptor; Bioinformatics; Biological; Biological Assay; Biology; Carrier Proteins; Cells; Cloning; Collaborations; Communities; Complex; Computer software; Coupled; Cryoelectron Microscopy; Crystal Formation; Crystallization; Crystallography; Data; Data Collection; Detection; Detergents; Disease; Docking; Electron Diffraction Microscopy; Electron Microscopy; Electrophysiology (science); Elements; Environment; Equilibrium; Escherichia coli; Excision; Exclusion; Fab Immunoglobulins; Family; Family member; Fluorescence; GTP-Binding Proteins; Gene Library; Genes; Genome; Goals; Green Fluorescent Proteins; Heavy Metals; Human; Immune system; In Situ; In Vitro; In Vivo NMR Spectroscopy; Insecta; Institution; Integral Membrane Protein; Ion Channel; Ion Pumps; Ion-Exchange Chromatography Procedure; Kinetics; Label; Light; Lipid Bilayers; Lipids; Macromolecular Complexes; Mammalian Cell; Maps; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metabolic; Metals; Methodology; Methods; Minor; Modeling; Molecular; Molecular Chaperones; Molecular Sieve Chromatography; Monitor; Mus; Mutation; NMR Spectroscopy; Noise; Nuclear Pore Complex; Organism; Peptide Hydrolases; Peptide Sequence Determination; Peripheral; Pharmaceutical Preparations; Phase; Play; Precipitation; Principal Investigator; Process; Production; Proteins; Proteome; Refractive Indices; Research; Research Personnel; Resolution; Rhodobacter; Robotics; Roentgen Rays; Role; Saccharomyces cerevisiae; Sampling; Scheme; Sensory; Signal Transduction; Site; Solid; Solutions; Source; Spin Labels; Staging; Structure; Surface Plasmon Resonance; System; Technology; Temperature; Therapeutic; Thermus thermophilus; Training; Transmission Electron Microscopy; Triage; Vertebral column; Viola; Viscosity; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; Yeasts; analytical ultracentrifugation; ascorbate; base; beamline; cell transformation; combinatorial; comparative; density; design; detector; expression cloning; flexibility; high throughput screening; improved; in vivo; interest; light scattering; member; nucleobase; operation; permease; polypeptide; programs; protein complex; protein degradation; protein purification; protein structure; protein-histidine kinase; receptor; response; restraint; screening; solute; structural biology; success; therapeutic target; two-dimensional; vector

Integral membrane proteins account for ~30% of a proteome and play critical roles in metabolic, regulatory and intercellular processes. Human MPs are the targets for ~40% of all therapeutic drugs, but the number of MP structures is less than 0.5% of the number of soluble protein structures. The proposed Center brings together 11 Investigators at five US institutions to focus cooperatively on the overarching aim of determining integral MP structures of high biomedical impact. The Specific Aims balance multiple priorities. Aims 1-3 are extensive, seeking to obtain structures by providing many targets from (1) E. coli, (2) extremophiles, and (3) human. The broad target base is triaged by dynamic bioinformatics screening to direct focus on the most tractable set by the end of year 1. Aims 4 and 5 are intensive, targeting families of highest biomedical relevance and impact for which structures have generally not yet been obtained; Aim 4 concerns specific prokaryotic MPs; Aim 5 involves the most challenging eukaryotic MPs, including human therapeutic targets and components of the nuclear pore complex. Aim 6 leverages MP structures by comparative modeling developed specifically for MPs. Ten core capabilities implement the methods that support the aims and cover every aspect of structure determination, including target selection, cloning, expression, purification, crystallization, structure determination by X-ray crystallography, NMR spectroscopy or electron microscopy, and modeling. The cores provide multi-point entry to High-Throughput-Enabled Structural Biology Partnerships. Expression cores cover prokaryotic and eukaryotic (including HEKs) in vivo systems, one using green fluorescent protein detection of expression, and an E. coli based cell-free in vitro system optimized for MP expression. The protein purification core, aided by several characterization methods, provides pure homogeneous and stable proteins free of excess detergent. The electron microscopy core provides further characterization and 2D crystallization. Structure determination methods include X-ray diffraction and NMR spectroscopy, where cell-free expression has been harnessed to a combinatorial labeling strategy for rapid determination of backbone structures. The X-ray crystallography core provides robotic crystal trials and diffraction at the Advanced Light Source beam line 8.3.1, one of the world's most productive protein crystallography facilities. Overall, the combined expertise of principal investigators provides a unique environment to achieve the proposed aims.

整体膜蛋白占蛋白质组的约30%，在代谢、调节和细胞间过程中起关键作用。人类MPs是约40%的治疗药物的靶点，但MP结构的数量不到可溶性蛋白质结构数量的0.5%。拟议中的中心汇集了来自美国5个机构的11名研究人员，共同致力于确定具有高生物医学影响的整体MP结构的总体目标。具体目标平衡了多个优先事项。目标1-3是广泛的，寻求通过提供来自(1)大肠杆菌，(2)极端微生物和(3)人类的许多靶点来获得结构。广泛的目标基础是通过动态生物信息学筛选进行分类，以便在第1年年底直接关注最容易处理的一组。目标4和5是密集的，针对生物医学相关性和影响最高的家庭，其结构通常尚未获得；目标4涉及特定的原核MPs；Aim 5涉及最具挑战性的真核MPs，包括人类治疗靶点和核孔复合物的组分。目的6通过专门为MPs开发的比较模型来利用MP结构。十个核心功能实现了支持目标的方法，涵盖了结构确定的各个方面，包括目标选择、克隆、表达、纯化、结晶、通过x射线晶体学、核磁共振光谱或电子显微镜进行结构确定以及建模。这些核心为高通量结构生物学伙伴关系提供了多点入口。表达核心包括原核生物和真核生物（包括HEKs）体内系统，一个使用绿色荧光蛋白检测表达，一个基于大肠杆菌的体外无细胞系统优化MP表达。蛋白质纯化核心，借助于几种表征方法，提供纯均匀和稳定的蛋白质，无多余的洗涤剂。电子显微镜核心提供进一步的表征和二维结晶。结构测定方法包括x射线衍射和核磁共振波谱，其中无细胞表达已被利用到快速测定骨干结构的组合标记策略。x射线晶体学核心在先进光源光束线8.3.1上提供机器人晶体试验和衍射，这是世界上最多产的蛋白质晶体学设施之一。总的来说，主要研究人员的综合专业知识为实现所提出的目标提供了一个独特的环境。