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 biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl 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 (mol 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 both 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, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 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.
Zitzewitz
J A
JA
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Bilsel
O
O
Luo
J
J
Jones
B E
BE
Matthews
C R
CR
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D016350:Leucine Zippers; D046911:Macromolecular Substances; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D006146:Guanidines; D046911:Macromolecular Substances; D015335:Molecular Probes; D019791:Guanidine