Dispersive interactions determine Urea and TMAO thermodynamic effects across the protein folding landscape

The molecular mechanisms by which osmolytes promote protein denaturation or protection are under permanent debate, because the characterization of the structure and solvation of denatured states is a major challenge. Here we show that similar qualitative interactions occur between urea and TMAO and the β-sheet SH3 domain and the helical B domain of protein A (BdpA). The strength of non-specific interactions is the determining feature for cosolvent preferential accumulation or exclusion and dictate the free energy differences of native and denatured states. Urea and TMAO hydrogen-bonds with the proteins, on the other side, are disfavored in the denatured states relative to water. Notably, urea destabilizes partially denatured states of BdpA, which are only marginally affected by TMAO, demonstrating that osmolytes can also selectively interfere with denaturation pathways. These analyses are possible by the study of the solvation structures throughout complete folding landscapes using coarse-grained and atomistic simulations, the use of minimum-distance distribution functions, and the Kirkwood-Buff solvation theory. Cosolvent effects on folding free energies match experimental data within 1 kcal mol ^-1 , validating force-fields and analysis methods. These findings provide a novel perspective on osmolyte-protein interplay, with implications for solvent and biomolecular design.