Excitingly, these efforts resulted in identification of interacting regions on proteins that are resistant to crystallization 13. coli experiments revealed novel in vivo topological information on interacting proteins. Recently, a few applications on complex biological systems, including cells and cell lysates have been reported 11– 13. In the past, cross-linking strategies were used on limited applications to study topologies of one or a few proteins. Cross-linking strategies, on the other hand, can provide unique information including the identities of native interacting partners and topological features of the interacting regions in vivo. However, in very few cases are any topological features of the interacting regions known or revealed from these studies. Exceptionally large numbers of potential protein interactions can be identified with YTH or IP-based methods 8– 10. Cross-linker application can result in creation of new covalent bonds between proteins that allow visualization of interacting regions. Chemical technologies such as chemical cross-linking have been applied for many years, but have gained increasing interest in recent years 4– 7. Traditional methods to study protein-protein interactions from cells include yeast two-hybrid (YTH) 1, immunoprecipitation (IP) 2 and tagged immunoprecipitation 3, eg. Mapping protein interaction networks is a critical goal of proteomics research to better understand the biological function of proteins in cells. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. From those labeled sites, 53 in vivo inter-cross-linked peptide pairs were identified and manually validated. coli were identified, indicating many protein sites react with pcPIR in vivo. coli cells and in vivo protein interactions and topologies are measured. In the present report, the pcPIR strategy is applied to E. 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. 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. In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems.
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