The utilization of competing unfolding pathways of monellin is dictated by enthalpic barriers.

Direct evidence of the presence of competing pathways for the folding or unfolding reactions of proteins is difficult to obtain. A direct signature for multiple pathways, seen so far only rarely in folding or unfolding studies, is an upward curvature in the dependence of the logarithm of the observed rate constant (λU) of folding on denaturant concentration. In this study, the unfolding mechanism of the wild-type (wt) and E24A variants of monellin has been investigated, and both variants are shown to display upward curvatures in plots of log λU versus denaturant concentration. Curvature is distinctly more pronounced for E24A than for the wt protein. Kinetic unfolding studies of E24A were conducted over a range of denaturant concentrations and across a range of temperatures, and the kinetic data were globally analyzed assuming two parallel pathways L and H, which proceed through transition states TS(L) and TS(H), respectively. The observation of the upward curvature in the unfolding kinetics permitted a thermodynamic analysis of how unfolding switches from one pathway to the other upon a change in unfolding conditions. The m −N and ΔCp values indicate that TSL is more compact than TS(H). The major contribution to the free energy of activation on either pathway is seen to be enthalpic and not entropic in origin.