Center for Structure of Membrane Proteins

膜蛋白结构中心

基本信息

批准号：
8246543
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
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/8246543
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

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% 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.

整合膜蛋白约占蛋白质组的 30%，在代谢、调节和细胞间过程。人类 MP 是约 40% 的治疗药物的靶标，但 MP结构的数量小于可溶性蛋白质结构数量的0.5%。拟议的中心汇集了来自 5 个美国机构的 11 名研究人员，共同致力于实现以下目标：确定具有高生物医学影响的完整 MP 结构。具体目标平衡了多个优先事项。目标 1-3 范围广泛，寻求通过以下方式获得结构：提供来自 (1) 大肠杆菌、(2) 极端微生物和 (3) 人类的许多目标。对广泛的目标基础进行分类通过动态生物信息筛选，在年底前直接关注最容易处理的一组。目标 4 和 5 个是密集型的，针对具有最高生物医学相关性和影响力的家庭，这些家庭的结构一般尚未获得；目标 4 涉及特定的原核 MP；目标 5 涉及最多具有挑战性的真核 MP，包括人类治疗靶点和核孔成分复杂的。目标 6 通过专门为 MP 开发的比较模型来利用 MP 结构。十大核心能力落实支撑目标的方法，涵盖结构的各个方面测定，包括靶标选择、克隆、表达、纯化、结晶、结构通过 X 射线晶体学、核磁共振波谱或电子显微镜和建模进行测定。核心提供高通量结构生物学合作伙伴关系的多点入口。表达核心涵盖原核和真核（包括 HEK）体内系统，其中一种使用绿色荧光蛋白质表达检测，以及针对 MP 表达进行优化的基于大肠杆菌的无细胞体外系统。蛋白质纯化核心在多种表征方法的帮助下，提供纯均质和稳定的蛋白质，不含过量的洗涤剂。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NMR光谱，其中无细胞表达已被用于组合标记策略，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光源光束线 8.3.1，世界上生产力最高的蛋白质晶体学设施之一。总体而言，主要研究人员的综合专业知识为实现拟议的目标。