Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL−GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL−GroES complexes. The results show clear evidence for destabilization of both GroEL 14 and GroES 7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (T m ∼ 70 °C). This destabilization is accompanied by temperaturedependent reaction products that have previously unreported stoichiometries, viz. GroEL 14 −GroES y −ATP n , where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg 2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg 2+ and ATP concentrations on the stoichiometry of the GroEL−GroES complex, with Mg 2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL−GroES complex (GroEL 14 −GroES 7 vs GroEL 14 −GroES 8 ) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL−gp31 complex.

The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery.

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Polymers of septin protein complexes play cytoskeletal roles in eukaryotic cells. The specific subunit composition within complexes controls functions and higher-order structural properties. All septins have globular GTPase domains. The other eukaryotic cytoskeletal NTPases strictly require assistance from molecular chaperones of the cytosol, particularly the cage-like chaperonins, to fold into oligomerization-competent conformations. We previously identified cytosolic chaperones that bind septins and influence the oligomerization ability of septins carrying mutations linked to human disease, but it was unknown to what extent wild-type septins require chaperone assistance for their native folding. Here we use a combination of in vivo and in vitro approaches to demonstrate chaperone requirements for de novo folding and complex assembly by budding yeast septins. Individually purified septins adopted non-native conformations and formed non-native homodimers. In chaperonin- or Hsp70-deficient cells, septins folded slower and were unable to assemble post-translationally into native complexes. One septin, Cdc12, was so dependent on co-translational chaperonin assistance that translation failed without it. Our findings point to distinct translation elongation rates for different septins as a possible mechanism to direct a stepwise, co-translational assembly pathway in which general cytosolic chaperones act as key intermediaries.