Protein Tagging for High Resolution Structural Analysis of Synaptic Protein Complexes Using Clickable CryoEM Grids

使用可点击 CryoEM 网格进行蛋白质标记，用于突触蛋白复合物的高分辨率结构分析

• 批准号: 9182565
• 负责人: TAMARA L KINZER-URSEM
• 金额: $ 18.25万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182565
• 关键词: Address; Adsorption; Affinity; Alkynes; Animals; Azides; Binding; Binding Proteins; Brain; Cell Line; Chemistry; Complex; Cryoelectron Microscopy; Cytoskeletal Modeling; Data; Deposition; Disease; Drug Design; Engineering; Enzyme Activation; Etiology; Event; Film; Functional disorder; Future; Goals; Image; Impaired cognition; Intervention; Investigation; Knowledge; Label; Learning; Learning Disorders; Lipids; Location; Mediating; Memory; Memory Disorders; Mental disorders; Methodology; Molecular; Mutation; Neurons; Pharmacologic Substance; Phosphotransferases; Polymers; Process; Protein Biosynthesis; Protein-Protein Interaction Map; Proteins; Reaction; Regulation; Resolution; Signaling Molecule; Signaling Protein; Site; Structure; Surface; Synapses; Synaptic plasticity; Techniques; Time; Translating; Transmission Electron Microscopy; abstracting; base; biological preparation; calmodulin-dependent protein kinase II; chemical group; density; improved; in vivo; insight; interest; knock-down; monolayer; mutant; nanometer; nervous system disorder; neurotransmitter release; novel; overexpression; particle; protein activation; protein complex; reconstruction; response; surface coating; synaptic function; therapy design; tool

Project Summary/Abstract In normal learning and memory, dynamic changes in the strength of synaptic connections (called synaptic plasticity) are brought about through exquisite coordination of neurotransmitter release, protein synthesis, protein localization and cytoskeletal reorganization. The timing, magnitude, and location of these processes are determined by protein binding and enzyme activation events within protein signaling networks. In many neurological disorders the spatial and temporal regulations of these protein interactions are disrupted. Thus, in order to effectively design treatments for these complex disorders, detailed information about the spatial organization of protein signaling molecules is absolutely necessary. Current experimental paradigms of qualitative studies with knock-down, overexpression or mutation of particular proteins in mutant animals or in cell lines alone are not adequate to advance our knowledge to the necessary level of mechanistic detail. To address this gap, we are simultaneously developing: 1) a protein labeling technique that is site-specifically and covalently tags a protein with click chemistry functionality and 2) a novel non-fouling, click chemistry- functionalized transmission electron microscopy (TEM) grid coating. The grid coating will enable selective covalent capture of the tagged protein alone and in complex with its interacting proteins onto TEM grids for cryo-EM imaging. This allows to fine control over the reaction, wash, and incubation conditions that the proteins are subjected to, thus allowing control over i) the state of activation of the protein of interest, ii) the surface deposition of the protein, and iii) the binding of the protein with its associated proteins. In addition the TEM grid coatings are non-fouling and thus minimize non-specific binding interactions that would otherwise obscure protein complex identification. Direct imaging of the complexes will be performed using cryo-EM; this maintains proteins in their naturally hydrated state and allows for large protein complexes to be imaged at high resolution. Single particle analysis will be performed to reconstruct the complexes to sub-nanometer resolution.

项目摘要/摘要 在正常的学习和记忆中，突触连接强度的动态变化（称为突触 可塑性是通过精致的神经递质释放，蛋白质合成的协调来实现的， 蛋白质定位和细胞骨架重组。这些过程的时间，大小和位置 由蛋白质信号网络中的蛋白质结合和酶激活事件确定。许多人 神经疾病会破坏这些蛋白质相互作用的空间和时间法规。因此，在 为了有效地设计这些复杂疾病，有关空间的详细信息 蛋白质信号分子的组织是绝对必要的。当前的实验范例 在突变动物中的特定蛋白质或在 单独的细胞系不足以使我们的知识达到必要的机械细节水平。 为了解决这一差距，我们正在同时开发：1）一种特定于站点的蛋白质标记技术 并共价标记具有点击化学功能的蛋白质和2）新型的非污染，请点击化学 - 官能化的透射电子显微镜（TEM）网格涂料。网格涂料将使选择性 单独捕获标记蛋白的共价捕获，并具有复杂的相互作用蛋白 冷冻EM成像。这样可以智能控制反应，洗涤和孵育条件 蛋白质受到允许控制i）感兴趣蛋白质的激活状态，ii） 蛋白质的表面沉积，iii）蛋白质与其相关蛋白的结合。另外 TEM网格涂层是非污染的，因此可以最大程度地减少非特异性结合相互作用，否则 模糊的蛋白质复合物鉴定。将使用冷冻EM进行配合物的直接成像；这 将蛋白质保持在自然水分状态，并允许在高成像的大蛋白质复合物中成像 解决。将进行单个粒子分析以重建复合物为亚纳米分辨率。