Center for Structure of Membrane Proteins

完整的膜蛋白约占蛋白质组的30%，在代谢、调节和细胞间过程中起着关键作用。人的MPS是~40%的治疗药物的靶标，但MPS的结构数不到可溶性蛋白质结构数的0.5%。拟议的中心汇集了五个美国机构的11名研究人员，共同专注于确定具有高生物医学影响的完整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是广泛的，寻求通过提供来自(1)大肠杆菌、(2)极端细菌和(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靶基进行分类，以在第一年结束时将重点放在最容易处理的集合上。目标4和5是密集的，目标是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MPS；目标5涉及最具挑战性的真核MPS，包括人类治疗靶点和核孔复合体的成分。AIM 6通过专门为MPS开发的比较建模来利用MP结构。十项核心能力实现了支持目标的方法，涵盖了结构确定的方方面面，包括目标选择、克隆、表达、纯化、结晶、通过X射线结晶学、核磁共振光谱或电子显微镜确定结构，以及建模。这些核心为高吞吐量结构生物学伙伴关系提供了多点入口。表达核心包括原核和真核(包括HEKS)体内系统，一个使用绿色荧光蛋白检测表达的系统，以及一个针对MP表达而优化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，提供纯净、均匀和稳定的蛋白质，没有多余的洗涤剂。电子显微镜核心提供了进一步的表征和2D结晶。结构确定方法包括X射线衍射和核磁共振波谱，其中无细胞表达已被利用于组合标记策略，以快速确定主干结构。X射线结晶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，这是世界上生产率最高的蛋白质结晶学设施之一。总体而言，首席调查员的综合专业知识为实现拟议目标提供了独特的环境。

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.