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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 随着适合于分析共价修饰多肽和蛋白质的高质量精度/分辨率质谱仪的出现，化学交联作为一种绘制相互作用结构域和提供蛋白质复合体低分辨率结构的技术得到了蓬勃发展。该项目的目标是将化学交联剂的用途和规模扩大到更大的细胞机械集合，化学交联剂通常仅限于研究简单的二元蛋白质相互作用。在这种规模上应用结构交联剂的最大障碍是与传统的活化酯类交联剂反应所产生的极小的分子内交联率。这类试剂容易被水解，因此，未修饰和“死端”修饰的多肽往往占交联化反应的反应产物的大多数。该项目包括：1)设计和合成新的交联剂，2)开发丰富特定交联肽的方法学，3)开发生物信息学工具，以帮助识别由大量相互作用的蛋白质产生的交联质谱。

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.