Biophysical Characterization of Two Different Stable Misfolded Monomeric Polypeptides That Are Chaperone-Amenable Substrates

Misfolded polypeptide monomers may be regarded as the initial species of many protein aggregation pathways, which could accordingly serve as primary targets for molecular chaperones. It is therefore of paramount importance to study the cellular mechanisms that can prevent misfolded monomers from entering the toxic aggregation pathway and moreover rehabilitate them into active proteins. Here, we produced two stable misfolded monomers of luciferase and rhodanese, which we found to be differently processed by the Hsp70 chaperone machinery and whose conformational properties were investigated by biophysical approaches. In spite of their monomeric nature, they displayed enhanced thioflavin T fluorescence, non-native β-sheets, and tertiary structures with surface-accessible hydrophobic patches, but differed in their conformational stability and aggregation propensity. Interestingly, minor structural differences between the two misfolded species could account for their markedly different behavior in chaperone-mediated unfolding/refolding assays. Indeed, only a single DnaK molecule was sufficient to unfold by direct clamping a misfolded luciferase monomer, while, by contrast, several DnaK molecules were necessary to unfold the more resistant misfolded rhodanese monomer by a combination of direct clamping and cooperative entropic pulling.

Graphical AbstractFigure optionsDownload high-quality image (113 K)Download as PowerPoint slideHighlights
► Misfolded monomers are the primary targets for molecular chaperones.
► Two stable misfolded monomeric polypeptides were produced and characterized.
► Their structural differences accounted for differences in Hsp70 amenability.
► They are useful tools to investigate chaperone activity.
► They are useful to study early events in protein misfolding and aggregation pathways.