biostudies-literatureYadav DHHS | National Institutes of Health (NIH)Amgen (Amgen Inc.)Research Corporation for Science Advancement (RCSA)Camille and Henry Dreyfus Foundation (Dreyfus Foundation)National Science Foundation (NSF)139-164https://www.ebi.ac.uk/biostudies/studies/S-EPMC12759075biostudies-literatureUnknown22(1)Many proteins require molecular chaperones to fold into their functional native forms. However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments. Here, we use limited proteolysis mass spectrometry (LiP-MS) to probe structural changes incurred by the E. coli proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble E. coli proteome, trigger factor deletion only impacts a small number of proteins' structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold in vitro) are not more likely to be dependent on chaperones to fold in vivo. We find that chaperone-nonrefolders (pMolecular systems biologyChaperone dependency during biogenesis does not correlate with chaperone dependency during refolding.PMC12759075Amgen ScholarsCamille Dreyfus Teacher-Scholar AwardDP2-GM140926MCB-2045844Cottrell Scholars AwardFakler MFried SDDemiralp IIYadav DfalseChaperone dependency during biogenesis does not correlate with chaperone dependency during refolding.Many proteins require molecular chaperones to fold into their functional native forms. However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments. Here, we use limited proteolysis mass spectrometry (LiP-MS) to probe structural changes incurred by the E. coli proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble E. coli proteome, trigger factor deletion only impacts a small number of proteins' structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold in vitro) are not more likely to be dependent on chaperones to fold in vivo. We find that chaperone-nonrefolders (p2026-01-01T00:00:00Z2026 Jan2026-06-06T10:44:21.36Z2026-05-29T03:12:59.875ZS-EPMC127590754115253210.1038/s44320-025-00166-6