Mapping the energy surface for the folding reaction of the coiled-coil peptide GCN4-p1.

The energy surface for the folding/unfolding reactions of the homodimeric coiled-coil peptide M2V GCN4-p1, a 33-residue segment comprising the leucine zipper domain of the transcriptional activator GCN4, was mapped by equilibrium and kinetic methods. Circular dichroism (CD) spectroscopy was used to monitor the urea-induced unfolding reaction at a series of temperatures and temperature-induced unfolding at a series of urea concentrations. A global analysis of the urea- and temperature-induced equilibrium unfolding data provides strong support for a two-state mechanism. The absence of a detectable population of intermediate states is also consistent with differential scanning calorimetry and thermal CD melts as a function of peptide concentration. Furthermore, a global analysis of stopped-flow CD kinetic data is consistent with a kinetic two-state mechanism as well. The urea dependence of the apparent folding and unfolding rate constants at a series of temperatures reveals that the activation enthalpy and entropy for unfolding in the absence of denaturant are both significantly greater than those for the refolding reaction. Although the unfolding barrier is dominated by the activation enthalpy, the activation entropy dominates the refolding barrier. The relative magnitudes of the urea dependence of the unfolding and refolding rate constants indicate that 55-65% of the surface area is buried in the transition state. The activation parameters imply a partially organized transition state and are consistent with a previous model in which the pair of C-terminal heptad repeats are docked in a coiled-coil-like motif [Zitzewitz et al. (2000) J. Mol. Biol. 296, 1105-1116].