PROBING THE FOLDING MECHANISM OF A LEUCINE-ZIPPER PEPTIDE BY STOPPED-FLOW CIRCULAR-DICHROISM SPECTROSCOPY

Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable bimolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidineHC1 induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mel dimer)(-1) when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on bath peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N-2 reversible arrow(ku) 2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M(-1) s(-1) and(kf) 3.3 x 10(-3) s(-1), respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.