Associated Water Stabilization Mechanism Reconciles the Enigmatic Differential Response of Proteins to a Single Osmolyte

The complexity of the protein-osmolyte interaction stems from the protein-specific stabilizing effects, challenges that traditional theories fail to address. In the same context, this study uses the associated water stabilization mechanism (AWSM) to investigate sorbitol's effect on papain and bromelain. Through a combination of melting, solvation, and conformational dynamics studies, we investigated the effect of sorbitol on the thermal stability, hydration shell, and internal structure of both proteins. Our findings show that sorbitol retards the dynamics of the protein's associated water, which is caused by the strengthening of water-water and water-protein hydrogen bonds. This, in turn, leads to a retardation in the protein's internal conformational dynamics, suggesting a stronger, more rigid interior and ultimately resulting in thermal stabilization. While both proteins individually follow the AWSM framework, the superior thermal stability of papain over bromelain cannot be explained simply by comparing their absolute dynamics in sorbitol. Instead, a more insightful approach is to consider the relative change in dynamics compared to that of the buffer. Our results demonstrate that papain exhibits a greater extent of retardation in its associated water dynamics, which directly correlates with a higher degree of retardation in its internal conformational dynamics. The greater strengthening of the internal structure leads to its superior thermal stabilization. These findings validate the AWSM framework as a more nuanced approach to understanding protein-osmolyte interactions. Our results highlight the importance of the hydration shell in modulating protein stability and provide a deeper, more mechanistic view of these complex relationships.