An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution

Recent studies in the field of de novo protein design have focused on the c onstruction of native-like structures. Here we describe the design and char acterization of an isoleucine zipper peptide intended to form a parallel tr iple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of beta-branched Ile res idues for complementary side chain packing. The peptide forms a stable trip le-stranded coiled coil, as determined by circular dichroism and sedimentat ion equilibrium analyses. A fluorescence quenching assay after the incorpor ation of acridine revealed a parallel orientation of the peptides. The stru ctural uniqueness of the coiled coil was confirmed by proton-deuterium amid e hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of ma gnitude slower than those expected if the exchange occurred via global unfo lding. A hydrophobic dye does not bind to the peptide. These results strong ly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in th e hydrophobic interface can improve the side chain packing, which can impar t native-like structural uniqueness to the designed coiled coil.