title: In vivo application of photocleavable protein interaction reporter technology.
abstract: 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 Escherichia coli cells, and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating that many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo intercross-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.
authors.affiliation: Department of Chemistry, Washington State University, Pullman, Washington, 99164, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Chunxiang
authors.initials: C
authors.lastname: Zheng
authors.forename: Chad R
authors.initials: CR
authors.lastname: Weisbrod
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Tang
authors.forename: Gerhard R
authors.initials: GR
authors.lastname: Munske
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hoopmann
authors.forename: Jimmy K
authors.initials: JK
authors.lastname: Eng
authors.forename: James E
authors.initials: JE
authors.lastname: Bruce
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D010782:Photolysis; D025941:Protein Interaction Mapping / Q000379:methods*; D015203:Reproducibility of Results; D016415:Sequence Alignment; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D010446:Peptide Fragments