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Molecular Mechanisms of Urea Interactions with Bovine Serum Albumin in an Acid-Expanded Conformation (pH 3.7)

Understanding the molecular mechanism by which denaturants modulate protein structure remains a central challenge in protein biophysics. In this work, molecular dynamics simulations were employed to investigate the effects of urea on the structural stability of bovine serum albumin, its F isoform at pH 3.7, over a broad range of urea concentrations (0 M to a fully urea/solvated system). The results reveal that urea induces a concentration/dependent dehydration/rehydration mechanism within the protein hydration shell. At low urea concentrations, a marked reduction in protein/water hydrogen bonds is observed, accompanied by a corresponding increase in protein/urea interactions, consistent with a competitive solvation process. At higher concentrations, urea/urea self-association becomes significant, limiting direct protein/urea interactions and promoting partial rehydration of the protein surface. Despite these solvent rearrangements, the secondary structure of BSA remains largely preserved, whereas local and tertiary structural features, particularly in Domain III, exhibit increased solvent exposure and conformational flexibility. These findings support a dynamic compensation mechanism in which urea partially replaces water in the solvation shell without fully disrupting the hydrogen-bonding network. Overall, this study provides molecular-level insight into the interplay between preferential interactions, solvation dynamics, and protein stability under denaturing conditions.