**1. Introduction**

The β-sheet is an important protein secondary structure. About one-fourth of protein residues adopt a β-sheet conformation in protein structures [1–3]. Furthermore, β-sheets are also formed in amyloid fibrils involved in various diseases, including Alzheimer's disease [4,5], Huntington's disease [6], and Parkinson's disease [7,8]. Therefore, understanding the folding energetics of β-sheets is scientifically important with potential therapeutic applications [9,10].

The side-chains of the closest residues on adjacent strands are on the same face of a β-sheet. This would enable cross strand lateral side-chain-side-chain interactions. Statistical analysis showed that oppositely charged residues are frequently observed across antiparallel β-sheets [11–13], suggesting that cross-strand interactions between oppositely charged residues may be important for β-sheet stability. Accordingly, the energetics of cross strand ion pairs have been measured in sheet-containing host systems, including the protein G B1 domain [14,15], the zinc finger domain [16], and β-hairpins [11,17–23]. For the protein G B1 domain, a cross strand lateral Glu44-Lys53 ion-pairing interaction increased the protein stability by 1.0 kcal/mol based on thermal denaturation studies [14].

**Citation:** Huang, C.-H.; Wong, T.W.; Yu, C.-H.; Chang, J.-Y.; Huang, S.-J.; Huang, S.-L.; Cheng, R.P. Swapping the Positions in a Cross-Strand Lateral Ion-Pairing Interaction between Ammonium- and Carboxylate-Containing Residues in a β-Hairpin. *Molecules* **2021**, *26*, 1346. https://doi.org/10.3390/ molecules26051346

Academic Editor: Marilisa Leone

Received: 25 December 2020 Accepted: 11 February 2021 Published: 3 March 2021

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For the zinc finger domain, cross strand ion-pairing interactions involving Asp were more stabilizing compared to those involving Glu based on competitive metal ion binding studies [16]. In particular, cross strand Lys3-Asp10 and Arg3-Asp10 interactions stabilized the system by 0.48 and 0.26 kcal/mol, respectively [16].

The effect of charged amino acid side-chain length on cross strand lateral ion-pairing interaction was investigated in hairpin peptides [22,23]. The negatively charged carboxylatecontaining amino acids with different side-chain lengths were incorporated at the Nterminal strand guest site (position 4), whereas the ammonium-containing amino acids with different side-chain lengths were incorporated at the C-terminal strand guest site (position 9) [22]. The results showed that length matching was necessary to form a stabilizing interaction, i.e., the side-chain length of the carboxylate- and ammonium-containing residues were either both long or both short [22]. The long side-chains provided large hydrophobic surfaces to interact with one another. Alternatively, the short side-chains paid less side-chain entropic penalties to interact with one another.

Statistical analysis showed that cross strand lateral residue pairs in antiparallel βsheets are symmetric [24], meaning that swapping the position of a pair of cross strand lateral residues (i.e., orientation) should not significantly affect the interaction. However, two different experimental studies showed that swapping the positions of an amino acid pair in antiparallel β-sheets changed the stability of the system [14,19]. For the protein G B1 domain, the cross strand Phe44-Thr53 interaction stabilized the protein by 0.19 kcal/mol, but the Thr44-Phe53 interaction destabilized the protein by 0.36 kcal/mol based on thermal denaturation studies [14]. In addition, the Ile44-Phe53 and Ile44-Thr53 interactions were non-identical compared to the corresponding swapped interactions, with a change in overall thermal stability of the system [14]. Similarly, swapping the oppositely charged residues in the cross strand Lys3-Glu12 ion pair in a hairpin peptide altered the fraction folded population of the system based on NMR data [19]. As such, it appears that the statistical studies and the experimental studies contradict one another. Herein, we report the effect of lateral ion-pair interactions in a β-hairpin with the positively charged ammonium-containing residue at the N-terminal strand guest site (position 4) and negatively charged carboxylate-containing residue at the C-terminal strand guest site (position 9), effectively swapping the positions of the oppositely charged residues in a previous study [22].
