Chaperone 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 (proteins that cannot refold even with chaperone assistance) do not generally require chaperones to fold in vivo, strengthening the view that chaperone-nonrefolders are obligate co-translational folders. Hence, for some E. coli proteins, the vectorial nature of co-translational folding is the most important "chaperone".