A novel fluorescent multi-domain protein construct reveals the individual steps of the unfoldase action of Hsp70

Abstract

A detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the detailed characterization of the aggregates, which are typically heterogeneous. To tackle this problem, we designed here a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase core, flanked by stress-resistant fluorescent mTFP and Venus domains, which upon denaturation formed a discrete stable population of small aggregates. Combining Förster Resonance Energy Transfer and enzymatic activity measurements provided unprecedent details on MLucV states, including native, aggregated, unfolded and chaperone-bound conformations. Using MLucV, we probed the various steps undertaken by bacterial Hsp70 to convert stable discrete aggregates into native proteins. The mechanism first involved an ATP-fuelled disaggregation and unfolding step of the stable pre-aggregated substrate, with a consequent stretching of MLucV beyond simply-unfolded conformations, followed, upon release, by native refolding. Furthermore, the ATP-fuelled unfolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures, highly adverse to the native state. These results unambiguously excluded binding and preventing aggregation from the non-equilibirum mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

Real-time single-molecule observation of chaperone-assisted protein folding

“…By directly observing the conformational dynamics of individual clients upon interaction with molecular chaperones over time, which has typically been inaccessible using other ensemble or single-molecule confocal-based approaches ( 18 , 19 , 33 ), we have been able to validate the mechanisms by which these different chaperone classes interact with their clients. The findings of this study, as well as others ( 19 , 21 , 49 ), show that the entropic pulling of client proteins by DnaK helps to resolve folding-incompetent, misfolded structures and that chaperones can conformationally remodel the client for subsequent refolding attempts.…”