A buried polar residue in the hydrophobic interface of the coiled-coil peptide, GCN4-p1, plays a thermodynamic, not a kinetic role in folding.

The hydrophobic interfaces of coiled-coil proteins and peptides are typically interspersed with buried polar residues. These polar residues are known to be important for defining oligomeric specificity and chain orientation in coiled-coil formation; however, their effects on the folding/assembly reaction have not been investigated. The commonly studied 33-residue dimeric leucine zipper peptide, GCN4-p1, contains a single polar Asn in the center of the hydrophobic interface at position 16. Peptides containing either a valine or an alanine replacement at this position, N16V and N16A, respectively, were studied in order to investigate both the thermodynamic and kinetic roles of the buried polar side-chain on the folding of GCN4-p1. Equilibrium sedimentation confirmed that both the N16V and N16A mutations reduce the dimeric specificity of GCN4-p1, leading to the population of both dimers and trimers in the absence of denaturant. Guanidine hydrochloride-induced equilibrium unfolding of the mutant peptides demonstrated that N16V is more stable than the wild-type sequence, while the N16A peptide is moderately destabilized. Comparison of the refolding reactions indicate that Asn16 is not involved in the rate-limiting association step leading to the native dimer; only the unfolding reaction is sensitive to the mutations. More complex unfolding kinetics for both peptides at high peptide concentrations can be attributed to the presence of trimers in the absence of denaturant. These results show that the role of buried polar residues in leucine zipper peptides can be primarily thermodynamic; subunit exchange reactions can be controlled by the stability of the native coiled coil and its influence on the unfolding/dissociation process.