Urea-Mediated Solvent Reorganization of Bovine Serum Albumin in an Acid-Induced Expanded Conformation at pH 3.7

Abstract

Understanding the molecular mechanisms by which denaturants modulate protein structure remains a central challenge in protein biophysics. In this study, molecular dynamics (MD) simulations were employed to investigate the effects of urea on the structural stability of bovine serum albumin (BSA) in its F isoform at pH 3.7 across a broad range of urea concentrations, from pure aqueous solution (0 M) to a fully urea-solvated environment. The simulations reveal a concentration-dependent remodeling of the protein hydration shell. At low urea concentrations, backbone-water hydrogen bonds decrease by approximately 40 %t, accompanied by an approximately 45 % increase in protein-urea hydrogen bonds between 1 and 7 M urea, consistent with a competitive solvation process in which urea progressively replaces water molecules at the protein surface. As urea concentration increases, urea-urea self-association becomes increasingly significant, reducing the number of direct protein-urea contacts; concurrently, the remaining water molecules form protein-water hydrogen bonds more efficiently on a per-water-molecule basis, without implying a net increase in the absolute number of hydration water molecules. Despite these pronounced solvent rearrangements, the secondary structure of BSA remains largely preserved throughout the simulations. In contrast, local structural organization and global conformational features, particularly within Domain III, exhibit increased solvent exposure and enhanced conformational flexibility.