CHEMICAL CROSSLINKING AND PROTEIN INTERACTIONS OF THE POST SYNAPTIC DENSITY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 随着适用于分析共价修饰的肽和蛋白质的高质量准确度/分辨率质量分析仪的出现，化学交联作为用于映射相互作用结构域和提供蛋白质复合物的低分辨率结构的技术已经蓬勃发展。 交联补充了其他结构方法，如晶体学或电子显微镜，因为它可以在各种生化条件下研究溶液中的结构，并适合于检查动态复合物。 然而，现有的交联试剂不适合分析大的、多蛋白质复合物，因为它们倾向于在水性缓冲液中水解。 这导致交联肽的低产率和相应的“死端”修饰肽的高产率，其中在一半的试剂上发生水解。 该项目的目标是将化学交联的实用性和规模扩展到催化许多基本细胞过程（如突触囊泡融合或mRNA剪接）的细胞机制的更大集合，化学交联通常仅限于研究简单的二元蛋白质相互作用。 该项目包括：1）新交联试剂的设计和合成，2）富集特异性交联肽的方法学的开发，以及3）生物信息学工具的开发，以帮助识别由大量相互作用蛋白质产生的交联MS/MS谱。 新方法应用还原胺化的化学作用来增加交联产率，并允许从未修饰的和死端修饰的肽中选择性富集真正的交联肽。

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.