Center for Structure of Membrane Proteins

描述（由申请人提供）：整合膜蛋白占蛋白质组的约30%，在代谢、调节和细胞间过程中发挥关键作用。人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11名研究人员在五个美国机构合作，重点放在确定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。目的1-3是广泛的，寻求通过提供来自（1）E.大肠杆菌，（2）极端微生物，和（3）人类。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1年年底前直接关注最易处理的目标。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的家族，其结构通常尚未获得;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，包括人类治疗靶标和核孔复合物的组分。目标6通过专门为MP开发的比较建模来利用MP结构。十大核心能力实现了支持目标的方法，涵盖了结构测定的各个方面，包括靶点选择，克隆，表达，纯化，结晶，通过X射线晶体学，NMR光谱或电子显微镜进行结构测定，以及建模。核心提供多点进入高吞吐量启用结构生物学伙伴关系。表达核心涵盖原核和真核（包括HEK）体内系统，一个使用绿色荧光蛋白检测表达，一个使用E.基于大肠杆菌的无细胞体外系统优化MP表达。蛋白质纯化核心，在几种表征方法的帮助下，提供纯净均匀和稳定的蛋白质，不含过量的去污剂。电子显微镜核心提供进一步的表征和2D结晶。结构测定方法包括X射线衍射和NMR光谱法，其中无细胞表达已被利用于快速测定骨架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，这是世界上最具生产力的蛋白质晶体学设施之一。总体而言，主要研究者的综合专业知识为实现拟议目标提供了独特的环境。 公共卫生相关性：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关键作用。人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11名研究人员在五个美国机构合作，重点放在确定高生物医学影响的整体MP结构的总体目标。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.