Molecular mechanisms for protein-denaturation in urea and guanidinium chloride mixtures

Using replica-exchange molecular dynamics simulations, we have studied the thermodynamic stability and the conformational changes of two synthetic mini-proteins, Trp-cage and Trp-zip1, in pure and mixed denaturant solutions of guanidinium chloride (GdmCl) and urea. We have found that urea, which denatures proteins through favorable preferential interactions with protein sidechains and backbone, is more effective in destabilizing and elongating alpha-helical secondary structures of proteins than beta-hairpin structures. Contrarily, GdmCl, which denatures proteins by inhibiting salt-bridge formations between charged amino acids, is more effective in destabilizing beta-hairpin structures than alpha-helical structures. The extent of GdmCl-induced protein-denaturation is not determined by protein-GdmCl preferential interactions. In mixed denaturant conditions, our results indicate that GdmCl may however enhance the overall denaturing effects of urea by promoting protein-urea preferential interactions when full-length proteins are considered, but it may also potentially lead to local compaction of smaller segments of proteins, partially counteracting urea-induced extension of the protein segments.