Context‐Dependence of the Contribution of Disulfide Bonds to β‐Hairpin Stability

Abstract Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to β‐hairpin stability, a previously reported β‐hairpin‐forming peptide was taken as a template to design a series of Cys‐containing peptides. The conformational behavior of these peptides in their oxidized, disulfide‐cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1 H and 13 C chemical shifts. We found that the effect of disulfide bonds on β‐hairpin stability depends on its location within the β‐hairpin structure, being very small or even destabilizing when connecting two hydrogen‐bonded facing residues. When the disulfide bond is linking non‐hydrogen‐bonded facing residues, we estimated that its contribution to the free‐energy change of β‐hairpin folding is approximately −1.0 kcal mol −1 . This value is larger than those reported for most β‐hairpin‐stabilizing cross‐strand side‐chain–side‐chain interactions, except for some aromatic–aromatic interactions, in particular the Trp–Trp one, and the cation–π interaction between Trp and the non‐natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross‐links.