Protein folding: independent unrelated pathways or predetermined pathway with optional errors

Abstract

The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway-optional error (PPOE) model but contrary to the properties implied in IUP models.

Fig. 1.

SNase folding and unfolding kinetics. (A) Chevron plot for folding and unfolding rates against final GdmCl concentration, measured as the change in fluorescence between the unfolded state and the native state (intermediate fluorescence is equal to the U state). Folding rates were obtained by multiphase fitting of kinetic data for native-state formation like that shown in B. (Inset) Unfolding rate at zero denaturant measured by kinetic native-state hydrogen exchange (24). (B) Kinetic folding data for native-state formation at zero denaturant, shown at the earliest folding times to exhibit the lag phase. The dashed curve is the two-exponential fit. The plot of residuals shows the reality of the lag phase, which is confirmed by similar results at other conditions and in other laboratories (see Table 1).

Scheme 1.

Fig. 2.

Global fit of the measured SNase kinetic folding and unfolding data by an IUP model (A–C) and a PPOE model (D–F). Shown are the chevron display of denaturant-dependent data (from Fig. 1), measured data for native-state acquisition at zero denaturant, and reaction schemes for the two different models with best global fit parameters (denaturant-dependent m values in parentheses). The curves drawn are the curves predicted by the best fit parameters for the denaturant-dependent chevron and for the time-dependent populations (zero denaturant) of the unfolded and native states and the two intermediates that significantly populate in the different models. A minor folding phase (amplitude ≈6%) on the 1-sec time scale (see Fig. 1A) was ignored.

Scheme 2.