Center for Structure of Membrane Proteins

膜蛋白结构中心

基本信息

批准号：
8693620
负责人：
Robert M Stroud
金额：
$ 144.76万
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 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8693620
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

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.

整合膜蛋白约占蛋白质组的 30%，在代谢、调节和细胞间过程中发挥着关键作用。人类 MP 是约 40% 的治疗药物的靶标，但 MP 结构的数量不到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，共同致力于确定具有高生物医学影响力的完整 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 范围广泛，寻求通过提供来自 (1) 大肠杆菌、(2) 极端微生物和 (3) 人类的许多目标来获得结构。通过动态生物信息学筛选对广泛的目标基础进行分类，以在第一年年底前直接关注最容易处理的群体。目标 4 和 5 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，这些家庭通常尚未获得其结构；目标 4 涉及特定的原核 MP；目标 5 涉及最具挑战性的真核 MP，包括人类治疗靶点和核孔复合体的成分。目标 6 通过专门为 MP 开发的比较模型来利用 MP 结构。十项核心能力实施支持目标的方法，涵盖结构测定的各个方面，包括靶标选择、克隆、表达、纯化、结晶、通过 X 射线晶体学、核磁共振波谱或电子显微镜进行结构测定以及建模。这些核心提供了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，一种使用绿色荧光蛋白检测表达，以及一种针对 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，可提供纯净、均质且稳定的蛋白质，不含过量的去垢剂。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用于组合标记策略，以快速测定主链结构。 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目标提供了独特的环境。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism.

DOI：
10.1126/science.1260590
发表时间：
2015-01-30
期刊：
Science (New York, N.Y.)
影响因子：
0
作者：
Li F;Liu J;Zheng Y;Garavito RM;Ferguson-Miller S
通讯作者：
Ferguson-Miller S

Patch-clamp characterization of the MscS-like mechanosensitive channel from Silicibacter pomeroyi.

波默罗氏硅杆菌 MscS 类机械敏感通道的膜片钳表征。

DOI：
10.1016/j.bpj.2013.01.055
发表时间：
2013
期刊：
Biophysical journal
影响因子：
3.4
作者：
Petrov,Evgeny;Palanivelu,Dinesh;Constantine,Maryrose;Rohde,PaulR;Cox,CharlesD;Nomura,Takeshi;MinorJr,DanielL;Martinac,Boris
通讯作者：
Martinac,Boris

Merging single-shot XFEL diffraction data from inorganic nanoparticles: a new approach to size and orientation determination.

合并无机纳米颗粒的单次 XFEL 衍射数据：确定尺寸和方向的新方法