Chaperone Dependency during Primary Protein Biogenesis Does Not Correlate with Chaperone Dependency during in vitro Refolding

Many proteins require molecular chaperones to fold into their functional native forms. Previously we used limited proteolysis mass-spectrometry (LiP-MS) to find that ca. 40% of the E. coli proteome do not efficiently refold spontaneously following dilution from denaturation, a frequency that drops to ca. 15% once molecular chaperones like DnaK or GroEL are provided.  However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments.  Here, we used 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.  For instance, the glycolytic enzyme, phosphoglycerate kinase (PGK), cannot refold to its native form in vitro following denaturation (even with chaperones), but by LiP-MS we find that its structure is unperturbed upon DnaKJ or Tig deletion, which we further demonstrate with biochemical and biophysical assays. Thus, PGK folds to its native structure most efficiently during co-translational folding and does so without chaperone assistance.  This behaviour is generally found among chaperone-nonrefolders (proteins that cannot refold even with chaperone assistance), strengthening the view that this class of proteins are obligate co-translational folders.  Hence, for some E. coli proteins, the vectorial nature of co-translational folding is the most important “chaperone.”