Polar or Apolar—The Role of Polarity for Urea-Induced Protein Denaturation

Abstract

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.

Toward an atomistic description of the urea-denatured state of proteins

“…The differential solvation effect of urea is also evident in the contact coefficient (CC UW ) (9), which reaches a value of 14.0 for apolar and only 10.6 for polar residues. As anticipated by others (8)(9)(10)14), hydrophobic residues are the main differential target for the preferential interaction of urea with unfolded conformations of the protein. Clearly, our simulations suggest that at least for this protein and in the context of a fully unfolded ensemble, the preferential solvation of apolar solutes by urea is the main stabilizing factor of the open state.…”

Interconnection of Salt-induced Hydrophobic Compaction and Secondary Structure Formation Depends on Solution Conditions

“…Although urea has been widely used as a protein denaturant, the mechanism of its action has been debated extensively (32)(33)(34)(35)(36). Although an "indirect" mechanism has been proposed, it is now generally believed that urea unfolding occurs through "direct" binding to protein backbone.…”

Mechanisms of amphipathic helical peptide denaturation by guanidinium chloride and urea: a molecular dynamics simulation study

“…The contact coefficient of C DW = 1.0 shows that the residue X has no preferential interaction with either denaturant or water. Values below 1.0 indicate preferential interaction with water; values above 1.0 show preferential interaction with denaturant [18,35].…”

“…In the third model, there is a combination of direct and indirect effects [10][11][12]. Overall, previous molecular dynamics simulations support the role of both of these effects in urea and GdmCl denaturation [13][14][15][16][17][18][19][20]. To study the interactions of osmolytes with the surface of a folded peptide, we have performed all-atom molecular dynamics simulations of helical peptide, magainin2 (PDB ID: 2mag), in different concentrations of GdmCl and urea.…”