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.

项目总结/摘要 在正常的学习和记忆过程中，突触连接（称为突触连接）强度的动态变化， 可塑性）是通过神经递质释放，蛋白质合成， 蛋白定位和细胞骨架重组。这些过程的时间、强度和位置 由蛋白质信号网络内的蛋白质结合和酶活化事件决定。在许多 这些蛋白质相互作用的空间和时间调节被破坏。因此在 为了有效地设计治疗这些复杂的疾病，详细的信息，空间 蛋白质信号分子的组织是绝对必要的。目前的实验范式 在突变动物或动物中敲低、过表达或突变特定蛋白质的定性研究 细胞系本身不足以将我们的知识提高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）一种蛋白质标记技术， 和2）一种新的不结垢的点击化学， 功能化透射电子显微镜（TEM）网格涂层。网格涂层将能够选择性地 将标记的蛋白质单独和与其相互作用的蛋白质复合物共价捕获到TEM网格上， 冷冻电磁成像这允许精细控制反应、洗涤和孵育条件， 因此允许控制i）感兴趣的蛋白质的活化状态，ii）感兴趣的蛋白质的活化状态， 蛋白质的表面沉积，和iii）蛋白质与其相关蛋白质的结合。此外 TEM网格涂层是无污染的，因此最大限度地减少了非特异性结合相互作用， 蛋白质复合物鉴定模糊。复合物的直接成像将使用cryo-EM进行;这 保持蛋白质在其自然水合状态，并允许大的蛋白质复合物在高 分辨率将进行单粒子分析以将复合物重建到亚纳米分辨率。