Expected and unexpected results from combined β-hairpin design elements

Abstract

A model β-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II′ β-turn sequence and a Trp–zip interaction, while also incorporating a β-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and ¹H NMR spectroscopy. A peptide representing the folded conformation of the model β-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and ¹H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model β-hairpin indicates that the Trp–zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model β-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the β-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp–zip interaction in a similar position to the parent β-hairpin [EGFWVpGKWITK] results in a folded β-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting β-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.