IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. Cell (0092-8674) 2009 imex curation Kadokura H., Beckwith J. The proteins were first immunoprecipitated with anti-phoA, then complexes under-went a second round of IP using anti-DsbA. hkadokura@bs.naist.jp 23-Feb-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 2 ecoli Escherichia coli (strain K12) anti bait coip anti bait coimmunoprecipitation anti bait coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Kadokura H., Beckwith J. The proteins were first immunoprecipitated with anti-phoA, then complexes under-went a second round of IP using anti-DsbA. hkadokura@bs.naist.jp Accepted 2010-FEB-12 by JYOTI. 23-Feb-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 2 imex curation Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. Detecting folding intermediates of a protein as it passes through the bacterial translocation channel. Cell (0092-8674) 2009 imex curation Kadokura H., Beckwith J. The proteins were first immunoprecipitated with anti-phoA, then complexes under-went a second round of IP using anti-DsbA. hkadokura@bs.naist.jp 23-Feb-2010: Contacted by JYOTI. jyoti Only protein-protein interactions 2 ecoli Escherichia coli (strain K12) anti tag coip anti tag coimmunoprecipitation anti tag coip ms participant Identification by mass spectrometry ms participant Kadokura H., Beckwith J. The proteins were first immunoprecipitated with anti-phoA, then complexes under-went a second round of IP using anti-DsbA. hkadokura@bs.naist.jp Accepted 2010-FEB-12 by JYOTI. 23-Feb-2010: Contacted by JYOTI. Cell (0092-8674) 2009 jyoti Only protein-protein interactions 2 imex curation ppb_ecoli Alkaline phosphatase phoA b0383 JW0374 protein protein ecoli Escherichia coli (strain K12) MKQSTIALALLPLLFTPVTKARTPEMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEITAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAASATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQDATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQLVSDAASLNSVTEANQQKPLLGLFADGNMPVRWLGPKATYHGNIDKPAVTCTPNPQRNDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQDHAANPCGQIGETVDLDEAVQRALEFAKKEGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLK DIP protein P00634 original sequence version: 122 DIP protein P00634 original sequence version: 123 DIP protein P00634 original sequence version: null 8A8DE1F29D9D9253 dsba_ecoli Thiol:disulfide interchange protein DsbA dsbA dsf ppfA b3860 JW3832 protein protein ecoli Escherichia coli (strain K12) MKKIWLALAGLVLAFSASAAQYEDGKQYTTLEKPVAGAPQVLEFFSFFCPHCYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTIRSASDIRDVFINAGIKGEEYDAAWNSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMDVFVQQYADTVKYLSEKK DIP protein P0AEG4 original sequence version: 53 DIP protein P0AEG4 original sequence version: 48 10527E876FCAEE55 phoa-dspa-1 1 4 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 3 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association We suggest that the five higher-molecular-weight bands (Figure 1B, lane 7, asterisks) represent disulfide-linked intermediates containing both DsbA and PhoA since these bands disappear when the samples are treated with reductant before electrophoresis (not shown, see also Figures 2A and 2B and related discussion on it), and both anti-DsbA and anti-PhoA antibodies precipitate them (Figure 1C). Fig. 1C Molecular weight of bands suggest multiple copies of one or both proteins is contained in the complex. phoa-dspa-2 2 3 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range 4 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association To analyze the structure of these high-molecular-weight complexes, we purified them using a hexa-histidine tag fused to DsbA and a FLAG tag fused to PhoA. Upon analysis of the purified complexes by mass spectrometry, we detected tryptic fragments from DsbA and PhoA Fig. 4A