Choline Chloride as a Protective Osmolyte: Comparative Stabilization of HP36, Trp-Cage, and GB1 Against Urea Denaturation

Proteins undergo structural perturbations in the presence of denaturants such as urea, which destabilizes the native fold of the proteins. While protecting osmolytes stabilize proteins against denaturants like urea, the molecular mechanism by which they preserve the folded state has limited understanding. Competing hypotheses suggest stabilization arises either from preferential exclusion or direct interactions of protecting osmolytes with proteins. To address this, we performed atomistic simulations of three mini proteins: villin headpiece subdomain (HP36), tryptophan-Cage (Trp-Cage), and β-hairpin domain (residue 41-56) of the immunoglobulin-binding domain of streptococcal protein G (GB1) in different aqueous solutions containing choline chloride (ChCl) and urea. Comparison of global structural metrics, secondary structure, dynamics, hydrogen bonding, and microscopic solvation structure reveals a consistent pattern: urea unfolds proteins through preferential binding and hydrogen bonding, ChCl enhances stability by strengthening hydration shells, and in mixtures, ChCl counteracts urea-induced denaturation by reducing protein-urea contacts and enhancing hydration while being preferentially excluded from the protein surface. These findings establish ChCl as a molecular protectant that preserves folded protein states against urea and provide molecular-level insight into osmolyte-driven stabilization mechanisms helping to resolve long-standing uncertainties of the topic.