*4.7. Double Mutant Cycle Analysis*

Double mutant cycle analysis [44] was performed to determine the interaction free energy (∆Gint) between charged residues Xaa4 and Zbb9 in the HPTXaaZbb peptides using Equation (7) [44,45]. This analysis accounted for the effect of each charged residue (individually) on strand stability using data from the corresponding Ala-containing peptides HPTXaaAla [3] and HPTAlaZbb [3] to determine the Xaa4-Zbb9 ion-pairing interaction exclusively (Table 3). The peptide with Ala incorporated at positions 4 and 9, HPTAlaAla [22], was used as the reference peptide.

$$
\Delta \mathbf{G}\_{\text{int}} = (\Delta \mathbf{G}\_{\text{HFT}\mathbf{\hat{a}a}\mathbf{Z}\mathbf{b}} - \Delta \mathbf{G}\_{\text{HFT}\mathbf{\hat{a}a}\mathbf{A}\mathbf{A}}) - (\Delta \mathbf{G}\_{\text{HFT}\mathbf{\hat{a}a}\mathbf{A}\mathbf{A}} - \Delta \mathbf{G}\_{\text{HFT}\mathbf{\hat{a}a}\mathbf{Z}\mathbf{b}}) - (\Delta \mathbf{G}\_{\text{HFT}\mathbf{\hat{a}a}\mathbf{Z}\mathbf{b}} - \Delta \mathbf{G}\_{\text{HFT}\mathbf{\hat{a}a}\mathbf{A}\mathbf{A}}) \tag{7}
$$
