biorxiv.org
Osmolyte-Induced Structural and Thermodynamic Effects on the Protein Folding Landscape
Understanding how osmolytes influence protein folding is fundamental in biology and biotechnology. We developed a computational pipeline to investigate the structural and thermodynamic effects of osmolytes across the protein folding landscape: Using coarse-grained simulations to generate folding ensembles, we then employed atomistic simulations to explore each conformation in aqueous solutions of urea and TMAO. Minimum-distance distribution functions and the Kirkwood-Buff solvation theory provide unprecedented details on the solvation structures and thermodynamics. We studied two model proteins: the β-sheet SH3 domain and the helical B domain of protein A (BdpA). Urea stabilizes most denatured states, while TMAO destabilizes them. However, urea destabilizes partially denatured states of BdpA, which are only marginally affected by TMAO, demonstrating that osmolytes can selectively interfere with denaturation pathways. We show that urea-induced denaturation arises from strengthened non-specific interactions in unfolded states, leading to increased protein dehydration. Cosolvent effects on folding free energies match experimental data within 1 kcal/mol, validating force-fields and analysis methods. These findings provide a novel perspective on osmolyte-protein interplay, with implications for solvent and biomolecular design. ### Competing Interest Statement The authors have declared no competing interest.
