Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4.

Abstract

One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation. However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation. A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A. (1991) Science 254, 539-544]. Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form. N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7). In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length. Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities. These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.