*Article* **Swapping the Positions in a Cross-Strand Lateral Ion-Pairing Interaction between Ammonium- and Carboxylate-Containing Residues in a** β**-Hairpin**

**Cheng-Hsin Huang 1,†, Tong Wai Wong 1,†, Chen-Hsu Yu <sup>1</sup> , Jing-Yuan Chang <sup>1</sup> , Shing-Jong Huang <sup>2</sup> , Shou-Ling Huang <sup>2</sup> and Richard P. Cheng 1,\***


**Abstract:** Cross-strand lateral ion-pairing interactions are important for antiparallel β-sheet stability. Statistical studies suggested that swapping the position of cross-strand lateral residues should not significantly affect the interaction. Herein, we swapped the position of ammonium- and carboxylatecontaining residues with different side-chain lengths in a cross-strand lateral ion-pairing interaction in a β-hairpin. The peptides were analyzed by 2D-NMR. The fraction folded population and folding free energy were derived from the chemical shift data. The ion-pairing interaction energy was derived using double mutant cycle analysis. The general trends for the fraction folded population and interaction energetics remained similar upon swapping the position of the interacting charged residues. The most stabilizing cross-strand interactions were between short residues, similar to the unswapped study. However, the fraction folded populations for most of the swapped peptides were higher compared to the corresponding unswapped peptides. Furthermore, subtle differences in the ion-pairing interaction energy upon swapping were observed, most likely due to the "unleveled" relative positioning of the interacting residues created by the inherent right-handed twist of the structure. These results should be useful for developing functional peptides that rely on lateral ion-pairing interactions across antiparallel β-strands.

**Keywords:** ion-pairing interaction; side-chain length; charged amino acids; β-hairpin; peptide
