Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 µs of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea–protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.
To perform their physiological function, proteins have to fold into their characteristic three-dimensional structure. While the folded state is stable under physiological conditions, changes in the solvent can destabilize the folded state and even induce denaturation. One of the most commonly used denaturants is urea. Despite its widespread use to study protein folding and stability, however, the molecular mechanism and particularly the driving forces of urea-induced protein denaturation are not yet understood. Two mechanisms have been suggested, according to which denaturation is driven either by polar interactions via hydrogen bonds or by hydrophobic interactions with apolar amino acids. By systematically varying urea polarity and quantifying the interactions of the solvent molecules with all amino acids of the protein, the present simulation study reveals that it is mainly the apolar interactions that drive denaturation. Our results suggest a coherent microscopic picture for urea-induced denaturation and bear more general implications for protein stability in other environments, e.g., in chaperone-assisted folding.