Center for Structure of Membrane Proteins

膜蛋白结构中心

基本信息

批准号：
8308507
负责人：
Robert M Stroud
金额：
$ 160.85万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
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/8308507
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 Screening procedure 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 solute structural biology success therapeutic target two-dimensional vector

项目摘要

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30％，在代谢，调节和细胞间过程中起关键作用。人MP是所有治疗药物中约40％的靶标，但MP结构的数量小于可溶性蛋白质结构数量的0.5％。拟议的中心汇集了美国五个机构的11位调查人员，以合作地关注确定高生物医学影响的整体MP结构的总体目的。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（1）大肠杆菌，（2）极端粒子和（3）人类的许多目标来获得结构。动态生物信息学筛查可以将广泛的目标基础分为三角形，以直接关注到第1年底的最可诉讼。目标4和5是密集的，针对具有生物医学相关性最高的家族和尚未获得结构的最高生物医学相关性和影响。 AIM 4关注特定的原核MP； AIM 5涉及最具挑战性的真核MP，包括人类的治疗靶标和核孔复合体的组成部分。 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确定的各个方面的方法，包括目标选择，克隆，表达，纯化，结晶，结构测定，通过X射线晶体学，NMR光谱或电子显微镜以及建模。核心为高通量启用的结构生物学合作伙伴关系提供了多点条目。表达核心覆盖原始系统中的原核和真核系统（包括HEK），一种使用绿色荧光蛋白表达检测，而基于大肠杆菌的基于大肠杆菌的细胞在体外系统中优化了用于MP表达的体外系统。蛋白质纯化核心在几种表征方法的帮助下提供了纯均匀且稳定的蛋白质，没有过多的洗涤剂。电子显微镜核心提供了进一步的表征和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了机器人晶体试验和高级光源光束线8.3.1的衍射，这是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为实现拟议目标提供了独特的环境。