Polypeptide collapse modulation and folding stimulation by GroEL-ES

Unfolded proteins ubiquitously collapse into a compact yet dynamic state. While this compaction is pivotal to protein folding, aggregation, intrinsic disorder, and phase separation, its role in protein quality control mechanisms remains obscure. Collapse has been characterized mainly for polypeptides that are free in solution, in terms of kinetics, chain expansion, and effect on folding. Yet, theory suggests that the solvent-mediated forces driving collapse can be altered near hydrophobic and charged surfaces, which are observed for many proteins including GroEL-ES. Notably, while GroEL-ES is the archetypal protein-folding chaperone, its folding mechanism remains unresolved. GroEL-ES is proposed to sterically confine polypeptides within its closed chamber, unfold misfolded states, or promote folding indirectly by suppressing aggregation. Here, using integrated protein manipulation and imaging, we show that GroEL-ES can strengthen the collapse of polypeptide substrates, and hence stimulate folding directly. Strikingly, attractive forces pull substrate chains into the open GroEL cavity -unclosed by GroES-, and hence trigger a gradual 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. Peptides corresponding to these C-termini alone are sufficient to strengthen the collapse. The results show a mechanism that allows folding to be stimulated: by strengthening the collapse, residues are brought together that must contact to fold. The notion that one protein can modulate the collapse of another may be generally important in protein conformation and coacervation control, for systems ranging from the GroEL-ES homologue TRiC/CCT, to the oncogenic c-Myc/Max complex, and the nuclear pore transporter transportin.