Center for Structure of Membrane Proteins

膜蛋白结构中心

• 批准号: 8290668
• 负责人: Robert M Stroud
• 金额: $ 27.81万
• 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/8290668
• 关键词: ATP-Binding Cassette Transporters; Accounting; Active Biological Transport; Benchmarking; Benzodiazepine Receptor; Bioinformatics; Biological; Biology; Carrier Proteins; Cells; Complex; Coupled; Crystallization; Crystallography; Detection; Detergents; Electron Microscopy; Environment; Equilibrium; Escherichia coli; Family; Family member; Genome; Goals; Green Fluorescent Proteins; Heavy Metals; Human; Immune system; In Vitro; In Vivo NMR Spectroscopy; Institution; Instruction; Integral Membrane Protein; Ion Channel; Label; Light; Membrane Proteins; Metabolic; Methods; Minor; Modeling; Mus; NMR Spectroscopy; Nuclear Pore Complex; Organism; Peripheral; Pharmaceutical Preparations; Play; Principal Investigator; Process; Proteins; Proteome; Research Personnel; Resolution; Rhodobacter; Robotics; Roentgen Rays; Role; Saccharomyces cerevisiae; Sampling; Scheme; Screening procedure; Signal Transduction; Source; Spin Labels; Staging; Structure; System; Temperature; Therapeutic; Thermus thermophilus; Triage; Vertebral column; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; Yeasts; ascorbate; base; beamline; combinatorial; comparative; design; expression cloning; flexibility; improved; in vivo; interest; member; nucleobase; permease; protein purification; protein structure; protein-histidine kinase; receptor; response; restraint; solute; structural biology; success; therapeutic target

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% ofthe 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 bioinformatic 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 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 fijrther 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 beamline 8.3.1, one ofthe 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30％，在代谢中起关键作用 调节和细胞间过程。人MP是所有治疗药物中约40％的目标，但 MP结构的数量小于可溶性蛋白质结构数量的0.5％。提议的中心 汇集了美国五个机构的11位调查人员，以合作关注的总体目的 确定高生物医学影响的积分MP结构。 具体目标平衡了多个优先事项。目标1-3是广泛的，试图通过 提供了（1）大肠杆菌，（2）极端细胞和（3）人类的许多靶标。宽阔的目标基础是三角形的 通过动态生物信息学筛查，将重点关注到第1年底时最可牵引。目标4和 5是密集的，针对具有最高生物医学相关性和影响力的家庭 通常尚未获得； AIM 4关注特定的原核MP； AIM 5最涉及 具有挑战性的真核MP，包括人类的治疗靶标和核孔的组成部分 复杂的。 AIM 6通过专门为MPS开发的比较建模来利用MP结构。 十个核心能力实施了支持目标并涵盖结构各个方面的方法 确定，包括目标选择，克隆，表达，纯化，结晶，结构 通过X射线晶体学，NMR光谱或电子显微镜和建模测定。核心 提供高通量启用的结构生物学合作伙伴关系的多点条目。表达核 覆盖原核和真核生物（包括HEK）在体内系统中，一种使用绿色荧光 表达的蛋白质检测和用于MP表达的优化的基于大肠杆菌的体外系统。 蛋白质纯化核心在几种表征方法的帮助下提供了纯均匀和 稳定的蛋白质不含多余的洗涤剂。电子显微镜核心提供了fijrther的表征和 2D结晶。结构测定方法包括X射线衍射和NMR光谱，其中 无细胞表达已被利用为组合标记策略，以快速确定 骨干结构。 X射线晶体学核心提供机器人晶体试验和衍射 先进的轻源光束线8.3.1，世界上最有生产力的蛋白质晶体学设施之一。 总体而言，首席研究人员的综合专业知识提供了一个独特的环境，以实现 拟议的目标。