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: 7957402
• 负责人: ALMA L BURLINGAME
• 金额: $ 1.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957402
• 关键词: Amination; Biochemical; Bioinformatics; Buffers; Cell physiology; Chemicals; Chemistry; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Cross-Linking Reagents; Crystallography; Development; Electron Microscopy; Funding; Goals; Grant; Hydrolysis; Institution; Maps; Mass Spectrum Analysis; Messenger RNA; Methodology; Methods; Peptides; Proteins; RNA Splicing; Reagent; Research; Research Personnel; Resolution; Resources; Solutions; Source; Structure; Synaptic Vesicles; Techniques; United States National Institutes of Health; aqueous; crosslink; density; design; mass analyzer; protein complex; protein crosslink; 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 随着高质量精度/分辨率质量分析仪的出现，适合分析共价修改的肽和蛋白质，化学交联成已经蓬勃发展，作为绘制相互作用结构域并提供蛋白质复合物低分辨率结构的技术。 交联补充了其他结构方法，例如晶体学或电子显微镜，因为它可以在多种生化条件下研究溶液中的结构，并适合检查动态复合物。 但是，现有的交联试剂不适合分析大型多蛋白复合物，因为它们在水性缓冲液中水解了水解。 这导致交联肽的产量较低和“死末端”改性肽的相应高收率，其中一半的试剂发生了水解。 该项目的目的是扩展化学交联的实用性和规模，这通常仅限于对简单的二元蛋白质相互作用的研究，到较大的细胞机械集合，从而催化许多基本的细胞过程，例如突触囊泡或mRNA拼接的融合。 该项目包括：1）新的交联试剂的设计和合成，2）方法论的开发以丰富特定的交联肽； 3）生物信息学工具的开发，以帮助识别由大量相互作用的蛋白质很多很多相互作用的蛋白质引起的交联的MS/MS光谱。 新方法将还原性胺化的化学方法应用于增加交联产量，并可以从未修饰和死端修饰的肽中选择性地富集真正的交联肽。