The unusual conformation of cross‐strand disulfide bonds is critical to the stability of β‐hairpin peptides

The cross‐strand disulfides (CSDs) found in β‐hairpin antimicrobial peptides (β‐AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles and a short Cα‐Cα distance. While the sequence and disulfide bond connectivity of disulfide‐rich peptides is well studied, much less is known about the disulfide geometry found in CSDs and their role in the stability of β‐AMPs. To address this, we solved the nuclear magnetic resonance (NMR) structure of the β‐AMP gomesin (Gm) at 278, 298, and 310 K, examined the disulfide bond geometry of over 800 disulfide‐rich peptides, and carried out extensive molecular dynamics (MD) simulation of the peptides Gm and protegrin. The NMR data suggests Cα‐Cα distances characteristic for CSDs are independent of temperature. Analysis of disulfide‐rich peptides from the Protein Data Bank revealed that right‐handed and left‐handed rotamers are equally likely in CSDs. The previously reported preference for right‐handed rotamers was likely biased by restricting the analysis to peptides and proteins solved using X‐ray crystallography. Furthermore, data from MD simulations showed that the short Cα‐Cα distance is critical for the stability of these peptides. The unique disulfide geometry of CSDs poses a challenge to biomolecular force fields and to retain the stability of β‐hairpin fold over long simulation times, restraints on the torsion angles might be required.