TOWARD AN OPTIMIZED PROTEIN-PROTEIN CROSS-LINKING STRATEGY FOR PROTEIN COMPLEXES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 随着适用于共价修饰的肽和蛋白质的分析的高质量准确度/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。 该项目的目标是将化学交联的实用性和规模扩展到更大的细胞机器集合，化学交联通常仅限于简单的二元蛋白质相互作用的研究。 在这种规模上应用结构交联的最大障碍是由与传统活化酯型交联试剂的反应产生的分子内交联的极小产率。 这种类型的试剂经受水解，并且因此未修饰的和“死端”修饰的肽倾向于在交联反应的反应产物中占主导地位。 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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. With the advent of high mass accuracy/resolution mass analyzers suitable for the analysis of covalently modified peptides and proteins, chemical crosslinking has blossomed as a technique for mapping interacting domains and providing low resolution structures of protein complexes. 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. The largest barrier to applying structural crosslinking on this scale is the miniscule yield of intramolecular crosslinking resulting from reactions with traditional activated ester type crosslinking reagents. Reagents of this type are subject to hydrolysis, and as a result unmodified and "dead-end" modified peptides tend to predominate the reaction products of crosslinking reactions. 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.