Chaperone-Mediated Mechanical Protein Folding at the Single Molecule Level

DnaK, DnaJ and GrpE heat shock proteins form the main conserved chaperone system in Escherichia coli. Such a complex chaperone system participates in the correct folding of emergent polypeptides in the ribosome to ensure that they reach their native structure, while preventing the aggregation of proteins under stress conditions in the cell. It is well known from bulk experiments that DnaK and DnaJ accomplish these functions by binding to both unfolded and partially unfolded protein substrates. However, the extended protein conformations that characterise the nascent proteins in the ribosome are rarely accessible by biochemistry denaturing experiments. Moreover, the detailed conformation of the substrates required for an optimal chaperone binding still remains controversial. Here we use single molecule force-clamp spectroscopy AFM to study the effect of molecular chaperones on the mechanical folding of individual proteins. This approach enables to monitor the individual folding trajectories from highly extended states, and to dissect the different conformational ensembles visited by the single protein during its folding pathway. Our experiments reveal that DnaJ binds to the unfolded state of Ubiquitin (which contains the consensus binding sequence GKQLEDG) with more affinity than to the molten globule, collapsed state. DnaJ binding blocks ubiquitin refolding by decreasing its folding efficiency by over 40%, thus highlighting its “holdase” functionality. By contrast, DnaK presents instead a higher affinity for the molten globule conformations. Crucially, both proteins, together with GrpE, readily increase the refolding of Ubiquitin by over 10%, within an ATP-mediated process. Altogether, our nanomechanical experiments demonstrate that the binding of different chaperones to a unique substrate is highly conformation-dependent, enabling us to postulate a kinetic scheme that underlies the synergetic functionality of a complex chaperone system, at the single molecule level.