Kinetic Studies of the Monellin: Evidence for Switching Between Alternative Parallel Pathways

Abstract

To determine whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding, to have an upward curvature. Upward curvatures are normally not observable, but may become evident upon mutation if the mutation differentially destabilizes the transition states on the parallel pathways. Folding and unfolding kinetic studies performed with heterodimeric monellin (dcMN) and monomeric monellin (scMN), respectively, using the intrinsic tryptophan fluorescence of the protein as the probe, show chevron arms with upward curvatures. In this study, the folding mechanism of dcMN has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations. Folding is shown to occur in multiple kinetic phases. In the first stage of folding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes, and a productive folding intermediate is identified on one but not on both of these routes. The formation of the intermediate occurs in a fast kinetic phase, and its folding to native dcMN occurs in a slow kinetic phase. The folding chevron arms for both the fast and slow phases of folding are shown to have upward curvatures, suggesting that at least two pathways are operational during these kinetic phases of structure formation, and that folding switches from one pathway to the other as the GdnHCl concentration is increased.