CHEMICAL CROSSLINKING AND PROTEIN INTERACTIONS OF THE POST SYNAPTIC DENSITY

突触后密度的化学交联和蛋白质相互作用

• 批准号: 7724213
• 负责人: ALMA L BURLINGAME
• 金额: $ 1.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724213
• 关键词: Amination; Biochemical; Bioinformatics; Buffers; Cell physiology; Chemicals; Chemistry; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; Cross-Linking Reagents; Crystallography; Development; Electron Microscopy; Funding; Goals; Grant; Hydrolysis; Institution; Maps; Messenger RNA; Methodology; Methods; Peptides; Proteins; RNA Splicing; Reagent; Research; Research Personnel; Resolution; Resources; Solutions; Source; Structure; Synapses; Synaptic Vesicles; Techniques; United States National Institutes of Health; aqueous; crosslink; density; design; mass analyzer; protein crosslink; protein structure; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. With the advent of high mass accuracy/resolution mass analyzers suitable for the analysis of covalently modified peptides and proteins, chemical crosslinking has flourished as a technique for mapping interacting domains and providing low resolution structures of protein complexes. Crosslinking complements other structural methods such as crystallography or electron microscopy because it can study structure in solution under a variety of biochemical conditions, and is suited to examining dynamic complexes. However, existing crosslinking reagents are not suitable for the analysis of large, multi-protein complexes because of their proclivity to hydrolyze in aqueous buffers. This leads to low yields of crosslinked peptides and a corresponding high yield of "dead-end" modified peptides, in which hydrolysis has occurred on one half of the reagent. The goal of this project is to extend the utility and scale of chemical crosslinking, which has generally been limited to the study of simple, binary protein interactions, to larger ensembles of cellular machinery that catalyze many fundamental cellular processes such as the fusion of synaptic vesicles or mRNA splicing. This project encompasses: 1) the design and synthesis of new crosslinking reagents, 2) the development of methodology to enrich specifically crosslinked peptides and 3) the development of bioinformatic tools to assist in identifying crosslinked MS/MS spectra resulting from a large pool of interacting proteins. The new methodology applies the chemistry of reductive amination to increase crosslinking yields and allow selective enrichment of true crosslinked peptides from unmodified and dead-end modified peptides.

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