Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride

Significance Guanidinium chloride (GdmCl) has been used to modulate the stability of proteins for more than 50 years; surprisingly, however, the molecular mechanism of its action is still poorly understood. Here, we provide direct kinetic evidence for the hypothesis that GdmCl unfolds proteins by a two-step mechanism. In the first step, it binds to the protein surface, resulting in the formation of a “dry molten globule,” an expanded form of the native protein with a dry core. Core solvation and global structural disruption occur in the second step. The observation of a dry molten globule during unfolding indicates that dispersion forces, and not only the hydrophobic effect, also play an important role in stabilizing proteins. Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).