**5. Conclusions**

We investigated the effect of swapping the cross strand interacting charged amino acid positions in a β-hairpin from the original Zbb4-Xaa9 in a previous study (HPTZbbXaa peptides) to the swapped Xaa4-Zbb9 in this study (HPTXaaZbb peptides). The general trends for the fraction folded population, and side-chain interaction energetics remained similar upon swapping the position of potentially interacting charged residues. Nonetheless, the fraction folded populations for most of the swapped HPTXaaZbb peptides were higher compared to the corresponding original HPTZbbXaa peptides, consistent with the inherent effect of the positively charged Xaa residue on hairpin formation at the two different positions. The most stabilizing cross strand interactions were between short residues (Dap4-Asp9 and Dap4-Glu9) even after swapping the position of the charged residues. However, subtle differences were present, most likely due to the unleveled relative placement of the residues at positions 4 and 9 created by the right-handed twist of the sheet structure. These results should be useful for developing functional peptides that rely on lateral ion-pairing interactions across antiparallel β-strands.

**Supplementary Materials:** The following are available online. Tables S1–S36: The 1H chemical shift assignments for the peptides. Tables S37–S45: The 3JNHα values of the peptides. Figure S1: The Hα chemical shift deviations for the residues in the experimental HPTXaaZbb peptides. Figure S2: The Hα chemical shift deviations for the residues in the fully folded reference HPTFXaaZbb peptides. Figures S3–S38: The NOEs observed involving the side-chains of the peptides. Figures S39–S50: Wüthrich diagrams of the backbone NOE connectivities involving the α-protons and amide protons for the peptides. Figure S51: The fraction folded of the residues in the peptides. Figure S52: The ∆Gfold of the residues in the peptides. Figure S53: The low-energy conformations for peptide HPTAadDab. Figure S54: The low-energy conformations for peptide HPTDabAad. Detailed peptide synthesis procedures and peptide characterization data.

**Author Contributions:** Conceptualization, R.P.C.; methodology, R.P.C.; software, C.-H.H., T.W.W., and C.-H.Y.; validation, C.-H.H. and C.-H.Y.; formal analysis, C.-H.H., T.W.W., and C.-H.Y.; investigation, C.-H.H., S.-J.H., and S.-L.H.; resources, C.-H.H., T.W.W., and C.-H.Y.; data curation, C.-H.H., T.W.W., C.-H.Y., and J.-Y.C.; writing—original draft preparation, C.-H.H. and T.W.W.; writing—review and editing, J.-Y.C. and R.P.C.; visualization, C.-H.H., T.W.W., C.-H.Y., and R.P.C.; supervision, R.P.C.; project administration, R.P.C.; funding acquisition, R.P.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science and Technology in Taiwan, MOST-101-2113-M-002-006-MY2,MOST-103-2113-M-002-018-MY3,and MOST-109-2113-M-002-011.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the Supplementary Materials. The raw data are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Hsiou-Ting Kuo, Jhe-Hao Li, Po-Yi Wu, and Chien-Hsiang Liu for their assistance in acquiring the NMR data. The authors would like to thank the Computer and Information Networking Center at National Taiwan University for the support of the high-performance computing facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.
