Extraction and Refolding Determinants of Chaperone-Driven Aggregated Protein Reactivation

Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and biotechnological applications. The molecular details of the chaperone systems involved are known to a great extent but how the overall reactivation process is achieved has remained unclear. Here, we quantified reactivation over time through a predictive mechanistic model and identified the key parameters that control the overall dynamics. We performed new targeted experiments and analyzed classical data, covering multiple types of non-ordered aggregates, chaperone combinations, and experimental conditions. We found that, irrespective of the behavior observed, the balance of surface disaggregation and refolding in solution universally determines the reactivation dynamics, which is broadly described by two characteristic times. This characterization makes it possible to use activity measurements to accurately infer the underlying loss of aggregated protein and to quantify, for the first time, the refolding rates of the soluble intermediates.