Hydrophobic Association in Urea–Water Solutions: A Structural and Thermodynamic Perspective

The formation of a hydrophobic core is fundamental to protein folding and the biological function of most cellular proteins, yet the molecular basis of hydrophobic association in osmolyte-rich environments remains unclear. Using all-atom molecular dynamics simulations, we investigated how urea concentration and hydrophobe isomerism influence the solvation and association of neopentane (NEO), isopentane (ISO), and normal pentane (PEN) in urea-water solutions. Radial distribution functions, running coordination numbers, potentials of mean force, preferential binding coefficients, and conditional solvation free energies were employed to characterize hydrophobic associations between the hydrophobes. Saturation of hydrophobic association for neopentane-neopentane, isopentane-isopentane, and n-pentane-n-pentane is observed in the 2 M urea-water solution, with the association strength following the order NEO-NEO > ISO-ISO > PEN-PEN, highlighting the influence of molecular branching. Urea becomes increasingly enriched in the first solvation shell, accompanied by a reduction in local water density. Conditional solvation free energies of hydrophobes become less positive with increasing urea concentration, showing the salting-in effect of urea on hydrophobes.