*2.1. Peptide Design and Synthesis*

The experimental HPTXaaZbb peptides were designed based on Gellman's YKL peptide [11,25,26] and hairpin peptides in our previous studies [3,22,23] (Figure 1a). The Tyr2 (in peptide YKL) was replaced with Thr [22] because the aromatic side-chain of Tyr may interact diagonally with the residue at position 9 due to the right-handed twist [11,27,28]. An acetyl group and a carboxamide group was incorporated at the N- and C-termini, respectively, to remove the terminal charges, preventing unintended electrostatic interactions [17]. Non-hydrogen-bonded sites at positions 4 and 9 were chosen as guest sites [3,22,23], which were near the center of the strands to avoid end fraying near the termini and excessive folding near the turn [20,21]. Our previous study placed the negatively charged carboxylatecontaining residues at position 4 and the positively charged ammonium- (or guanidinium-) containing residues at position 9 [22,23]. To investigate the effect of charged amino acid sidechain length upon swapping the position of the charged residues in the lateral cross strand ion-pairing interaction, the positively charged ammonium-containing residues (Xaa = Lys, Orn, Dab, Dap) and negatively charged carboxylate-containing residues (Zbb = Aad, Glu, Asp) were incorporated at positions 4 and 9, respectively, to give the experimental HPTXaaZbb peptides (Figure 1b). The peptides were named with an "HPT" prefix, representing hairpin peptide with Thr at position 2, followed by the positively charged Xaa residue at position 4 and the negatively charged Zbb residue at position 9.

**Figure 1.** Design of peptides to study the effect of charged amino acid side-chain length upon swapping the charged amino acid positions in lateral ion-pairing interactions. (**a**) The chemical structure of the experimental HPTXaaZbb peptides; (**b**) The sequences of the experimental HPTXaaZbb peptides, the unfolded reference HPTUXaaZbb peptides, and the folded reference HPTFXaaZbb peptides.

β The fully folded reference peptides and the fully unfolded reference peptides were necessary to determine the fraction folded population of the experimental HPTXaaZbb peptides [26]. For the fully folded reference peptides, cysteine residues were added to both termini of the experimental HPTXaaZbb peptides to form intramolecular disulfide bonds to give macrocyclic peptides to serve as the fully folded reference peptides HPT-FXaaZbb [3,11,22,23,25,26]. For the fully unfolded reference peptides, the DPro6 in the experimental HPTXaaZbb peptides was replaced with Pro to give the fully unfolded reference peptides HPTUXaaZbb [3,11,22,23,25,26], because Pro does not favor β-hairpin structures [11,26].

μ − The peptides were synthesized by solid-phase peptide synthesis using Fmoc-based chemistry [29,30]. The disulfide bond in the folded reference HPTFXaaZbb peptides was formed via charcoal mediated air oxidation [31]. All peptides were purified by reversephase high-performance liquid chromatography (RP-HPLC) to higher than 95% purity and confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). Since the nuclear magnetic resonance (NMR) spectra (chemical shift and line width) of analogous hairpin peptides did not change with concentration (20 µM to 10 mM) [3,11,12,32], the peptides in this study (2.0–15.4 mM) should not aggregate in solution. Accordingly, the experimental data should reflect the intramolecular interactions with minimal interference from intermolecular interactions.
