Center for Structure of Membrane Proteins

完整的膜蛋白占蛋白质组的30%左右，在代谢过程中起着关键作用。 调节和细胞间过程。人类MPS是所有治疗药物中约40%的靶点，但 MP结构数不到可溶性蛋白质结构数的0.5%。拟建的中心 将来自五个美国机构的11名调查人员聚集在一起，共同关注 确定具有高生物医学影响的整体MP结构。 具体目标平衡了多个优先事项。目标1-3是广泛的，寻求通过以下方式获得结构 提供来自(1)大肠杆菌、(2)极端嗜酸菌和(3)人类的许多靶点。对广泛的目标基础进行了分类 通过动态生物信息学筛选，在第一年年底之前将重点放在最容易处理的集合上。目标4和 5个是密集的，目标是结构具有最高生物医学相关性和影响的家庭 一般尚未获得；目标4涉及特定的原核MPS；目标5涉及最多 挑战真核MPS，包括人类治疗靶点和核孔成分 很复杂。AIM 6通过专门为MPS开发的比较建模来利用MP结构。 十个核心能力实施支持目标的方法，并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射线结晶学、核磁共振波谱或电子显微镜进行测定，并进行建模。这些核心 为支持高吞吐量的结构生物学伙伴关系提供多点入口。表达核心 介绍原核生物和真核生物(包括HEKS)活体系统，其中一个使用绿色荧光 表达的蛋白质检测，以及优化MP表达的基于大肠杆菌的体外无细胞系统。 蛋白质纯化核心，辅以几种表征方法，提供纯均一的和 稳定的蛋白质，没有多余的洗涤剂。电子显微镜核心提供纤维表征和 2D结晶。结构测定方法包括X射线衍射法和核磁共振法，其中 无细胞表达已被利用于组合标记策略，以快速确定 主干结构。X-射线晶体学核心提供机器人晶体试验和衍射 先进的光源光束线8.3.1，世界上生产率最高的蛋白质结晶学设备之一。 总体而言，主要调查人员的综合专业知识提供了一个独特的环境，以实现 建议的目标。

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.