Conformational stability of lysozyme in choline chloride-polyol aqueous deep eutectic solvents (ADES).
Naturally occurring deep eutectic solvents (NADES) offer a tunable microenvironment for biomolecular stabilization. Yet, the atomistic mechanisms governing the protein-solvent interactions remain poorly understood. Here, we present microsecond-long atomistic molecular dynamics simulations investigating lysozyme (Lys) stability in Choline Chloride (ChCl)-polyol (erythritol, xylitol, and sorbitol) aqueous deep eutectic solvents as a function of water wt. % (fw=0,25,50,75). Lys maintains its native structure across all conditions, with stable Rg, RMSD, RMSF, and secondary structure populations. The stabilization mechanism involves three coupled effects. First, solvent-solvent radial distribution functions (RDFs) and inter-component hydrogen bond (H-bond) analysis demonstrate that each NADES component preserves its characteristic H-bond network even in the presence of Lys, confirming that the bulk solvent structure is not disrupted. Second, the poorly structured protein-NADES RDFs are consistent with the preferential exclusion of NADES components from the protein surface with increasing hydration. Third, protein-polyol H-bonds remain persistently high and largely independent of water content, revealing a stable surface association layer that is not displaced by water. Among the polyols, xylitol exhibits significantly higher protein-alcohol H-bonds at low and intermediate hydration levels, owing to its intermediate flexible geometry and hydroxyl density. These findings demonstrate that the Lys stabilization in ChCl-polyol NADES arises from a polyol-dependent interplay among the preserved solvent network structure, inferential evidence for preferential exclusion, and persistent interfacial interactions. Overall, the study provides a molecular framework for rational NADES design for biomolecular stabilization applications.