Ja. Zitzewitz et al., PROBING THE FOLDING MECHANISM OF A LEUCINE-ZIPPER PEPTIDE BY STOPPED-FLOW CIRCULAR-DICHROISM SPECTROSCOPY, Biochemistry, 34(39), 1995, pp. 12812-12819
Leucine zipper peptides provide simple model systems for studying both
the intramolecular and intermolecular interactions that govern protei
n folding. The synthetic 33-residue peptide GCN4-p1, derived from the
yeast transcriptional activator GCN4, forms a stable bimolecular coile
d-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T.
(1991) Science 254, 539-544]. The guanidineHC1 induced equilibrium un
folding of this peptide at 5 degrees C and pH 7.0 yields a standard st
ate free energy of 10.49 +/- 0.23 kcal (mel dimer)(-1) when fit to a t
wo-state model involving the native dimer and the unfolded monomer. Th
e unfolding and refolding kinetics of GCN4-p1 were monitored by stoppe
d-flow circular dichroism spectroscopy as a function of both peptide c
oncentration and final denaturant concentration. The unfolding kinetic
s displayed single-exponential behavior, consistent with a unimolecula
r reaction. The refolding kinetics, which are dependent on bath peptid
e and guanidine concentration, are well described by a simple bimolecu
lar association reaction. A simultaneous fit of all of the unfolding a
nd refolding kinetic data to the model, N-2 reversible arrow(ku) 2U, y
ields refolding and unfolding rate constants in the absence of denatur
ant of 4.2 x 10(5) M(-1) s(-1) and(kf) 3.3 x 10(-3) s(-1), respectivel
y. The equilibrium unfolding curve is accurately predicted from these
rate constants, providing further support for the validity of the two-
state kinetic model.