Chaperone Dependency during Primary Protein Biogenesis Does Not Correlate with Chaperone Dependency during 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 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 can differ from the functions they play during refolding experiments. Here, we used LiP-MS to probe structural changes incurred by the proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble proteome, trigger factor deletion only impacts a small number of proteins' structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold ) are more likely to be dependent on chaperones to fold . For instance, the glycolytic enzyme, phosphoglycerate kinase (PGK), cannot refold to its native form following denaturation (even with chaperones), but by LiP-MS we find that its structure is unperturbed upon DnaKJ or Tig deletion, which is further supported 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 chaperone-nonrefolders are obligate co-translational folders. Hence, for some proteins, the vectorial nature of co-translational folding is the most important "chaperone."