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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

基本信息

批准号：
9277594
负责人：
TAMARA L KINZER-URSEM
金额：
$ 22.11万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9277594
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 Hydration status Image Impaired cognition Impairment 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 Translating Transmission Electron Microscopy biological preparation calmodulin-dependent protein kinase II chemical group density high resolution imaging improved in vivo insight interest knock-down monolayer mutant nanometer nervous system disorder neurotransmitter release novel overexpression particle protein activation protein complex protein structure reconstruction response spatiotemporal surface coating synaptic function therapy design tool

项目摘要

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/文摘