The force-dependent mechanism of DnaK-mediated mechanical folding

It is well established that chaperones modulate the protein folding free-energy landscape. However, the molecular
determinants underlying chaperone-mediated mechanical folding remain largely elusive, primarily because the
force-extended unfolded conformation fundamentally differs from that characterized in biochemistry experiments.
We use single-molecule force-clamp spectroscopy, combined with molecular dynamics simulations, to study the effect
that the Hsp70 system has on the mechanical folding of three mechanically stiff model proteins. Our results demonstrate
that, when working independently, DnaJ (Hsp40) and DnaK (Hsp70) work as holdases, blocking refolding by binding to
distinct substrate conformations. Whereas DnaK binds to molten globule–like forms, DnaJ recognizes a cryptic sequence
in the extended state in an unanticipated force-dependent manner. By contrast, the synergetic coupling of the Hsp70
system exhibits a marked foldase behavior. Our results offer unprecedented molecular and kinetic insights into the
mechanisms by which mechanical force finely regulates chaperone binding, directly affecting protein elasticity.