In vivo application of photocleavable protein interaction reporter technology.
In vivo application of photocleavable protein interaction reporter technology.
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DOI：
10.1021/pr200775j
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发表时间：
2012-02-03
影响因子：
4.4
通讯作者：
Bruce, James E.
中科院分区：
文献类型：
--
作者：
Yang, Li;Zheng, Chunxiang;Weisbrod, Chad R.;Tang, Xiaoting;Munske, Gerhard R.;Hoopmann, Michael R.;Eng, Jimmy K.;Bruce, James E.
关键词：
体内蛋白质结构和蛋白质-蛋白质相互作用对蛋白质在生物系统中的功能至关重要。作为传统蛋白质相互作用识别方法的补充，交联策略开始提供关于蛋白质和蛋白质复合物拓扑特征的额外数据。以前，通过交联纯蛋白质和蛋白质复合物以及使用紫外光切割或释放交联肽以实现鉴定，证明了光可切割蛋白质相互作用报告基因（pcPIR）技术。在本报告中，pcPIR策略应用于E.大肠杆菌细胞和体内蛋白质相互作用和拓扑结构的测量。从E.结果表明，pcPIR在大肠杆菌中有多个蛋白位点与pcPIR发生反应。从这些标记的位点中，鉴定并手动验证了53个体内相互交联的肽对。大约有一半的相互作用已被报道使用其他技术，虽然详细的结构存在的很少。三个蛋白质或蛋白质复合物的详细晶体结构进行了比较，从体内应用的pcPIR技术获得的交联结果。
In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to E. coli cells and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo inter-cross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology.
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