*2.4. Lateral Cross Strand Xaa-Zbb Interactions*

Double mutant cycle analysis was performed to derive the interaction free energy (∆Gint) for each lateral Xaa4-Zbb9 interaction (Table 3) [44,45]. For the reference peptides with minimal cross strand interaction, Ala was incorporated at position 4, position 9, or both positions 4 and 9 simultaneously because of the small side-chain of Ala [22,23,44]. The difference in folding energetics between peptides HPTXaaZbb and HPTAlaAla [22] would reflect the effect of simultaneously incorporating Xaa at position 4 and Zbb at position 9. This energy difference would include the effect of incorporating the Xaa residue and Zbb residue individually at positions 4 and 9, respectively, and the interaction between Xaa4 and Zbb9. Therefore, the effect of individually incorporating Xaa and Zbb would need to be considered to derive the Xaa4-Zbb9 interaction energy. The difference in folding energetics between peptides HPTXaaAla [3] and HPTAlaAla [22] would represent the effect of only incorporating Xaa at position 4. Similarly, the difference in folding energetics between peptides HPTAlaZbb [3] and HPTAlaAla [22] would represent the effect of only incorporating Zbb at position 9. The Xaa4-Zbb9 interaction energy (∆Gint) was determined from the folding energetics for the peptides HPTXaaZbb, HPTXaaAla [3], HPTAlaZbb [3], and HPTAlaAla [22] using Equation (7) (Table 3).


**Table 3.** The Xaa4-Zbb9 ion-pairing interaction energy (∆Gint, kcal/mol) <sup>1</sup> .

<sup>1</sup> Average value for residues 2, 3, 9, and 10. <sup>2</sup> Average value for residues 2, 3, and 10.

All of the cross strand lateral Xaa4-Zbb9 ion-pairing interactions were apparently stabilizing (Table 3). For the HPTXaaAsp peptides, the Xaa4-Asp9 interaction energy followed the trend Dap < Dab < Orn < Lys. Similarly, for the HPTXaaGlu peptides, the Xaa4- Glu9 interaction energy followed the trend Dap < Dab ~ Orn ~ Lys. For the HPTXaaAad peptides, the Xaa4-Aad9 interaction energy followed the trend Dap > Dab ~ Orn < Lys. If one disregards the Dap4-Aad9 interaction, the Xaa4-Zbb9 interaction generally becomes more stabilizing with decreasing Xaa4 side-chain length for a given Zbb9. Interestingly, the Dap4-Asp9 and Dap4-Glu9 interactions were the most stabilizing, providing more than 1 kcal/mol stabilization (Table 3). In contrast, the Dap4-Aad9 interaction provided the least stabilization, being essentially nonexistent. This showed that interaction between

oppositely charged residues with short side-chains form stabilizing lateral cross strand ion-pairing interactions.
