Comparison of C−H···π and Hydrophobic Interactions in a β-Hairpin Peptide:  Impact on Stability and Specificity

We have examined the impact of C−H···π and hydrophobic interactions in the diagonal position of a β-hairpin peptide through comparison of the interaction of Phe, Trp, or Cha (cyclohexylalanine) with Lys or Nle (norleucine). NMR studies, including NOESY and chemical shift perturbation studies, of the Lys side chain indicates that Lys interacts in a specific geometry with Phe or Trp through the polarized Cε. In contrast, Nle does not interact in a specific manner with the diagonal aromatic residue. Thermal denaturation provides additional support that Lys and Nle interact in fundamentally different manners. Folding of the peptide with a diagonal Trp···Lys interaction was found to be enthalpically driven, whereas the peptide with a diagonal Trp···Nle interaction displayed cold denaturation, as did the control peptide with a diagonal Cha···Nle interaction, indicating different driving forces for interaction of Lys and Nle with Trp. These findings have significant implications for specificity in protein folding and de novo protein design.