A more compact structural conformation, higher active-site flexibility, lower viscosity, and higher solvent medium polarity are found to probably facilitate enzymatic activity in a hydrated deep eutectic solvent (DES).

The mechanism of protein stabilization by osmolytes remains one of the most important and long-standing puzzles. The traditional explanation of osmolyte-induced stability through the preferential exclusion of osmolytes from the protein surface has been seriously challenged by the observations like the concentration-dependent reversal of osmolyte-induced stabilization/destabilization. The more modern explanation of protein stabilization/destabilization by osmolytes considers an indirect effect due to osmolyte-induced distortion of the water structure. It provides a general mechanism, but there are numerous examples of protein-specific effects, i.e., a particular osmolyte might stabilize one protein, but destabilize the other, that could not be rationalized through such an explanation. Herein, we hypothesized that osmolyte-induced modulation of associated water might be a critical factor in controlling protein stability in such a medium. Taking different osmolytes and papain as a protein, we proved that our proposal could explain protein stability in osmolyte media. Stabilizing osmolytes rigidify associated water structures around the protein, whereas destabilizing osmolytes make them flexible. The strong correlation between the stability and the associated water dynamics, and the fact that such dynamics are very much protein specific, established the importance of considering the modulation of associated water structures in explaining the osmolyte-induced stabilization/destabilization of proteins. More interestingly, we took another protein, bromelain, for which a traditionally stabilizing osmolyte, sucrose, acts as a stabilizer at higher concentrations but as a destabilizer at lower concentrations. Our proposal successfully explains such observations, which is probably impossible by any known mechanisms. We believe this report will trigger much research in this area.