Swapping the Positions in a Cross-Strand Lateral Ion-Pairing Interaction between Ammonium- and Carboxylate-Containing Residues in a β-Hairpin

Cross-strand lateral ion-pairing interactions are important for antiparallel β-sheet stability. Statistical studies suggested that swapping the position of cross-strand lateral residues should not significantly affect the interaction. Herein, we swapped the position of ammonium- and carboxylate-containing residues with different side-chain lengths in a cross-strand lateral ion-pairing interaction in a β-hairpin. The peptides were analyzed by 2D-NMR. The fraction folded population and folding free energy were derived from the chemical shift data. The ion-pairing interaction energy was derived using double mutant cycle analysis. The general trends for the fraction folded population and interaction energetics remained similar upon swapping the position of the interacting charged residues. The most stabilizing cross-strand interactions were between short residues, similar to the unswapped study. However, the fraction folded populations for most of the swapped peptides were higher compared to the corresponding unswapped peptides. Furthermore, subtle differences in the ion-pairing interaction energy upon swapping were observed, most likely due to the “unleveled” relative positioning of the interacting residues created by the inherent right-handed twist of the structure. These results should be useful for developing functional peptides that rely on lateral ion-pairing interactions across antiparallel β-strands.


Tables
Tables S1~S36. The 1 H Chemical Shift Assignments for the Peptides S2~S19 Tables S37~S45. The 3 J NH α Values of the Peptides S20~S24 Figure S1. The Hα chemical shift deviations for the residues in the experimental HPTXaaZbb peptides.

Figures
S25 Figure S2. The Hα chemical shift deviations for the residues in the fully folded reference HPTFXaaZbb peptides.                                  S27 Figure S3. The NOEs in the ROESY spectra of HPTDapAsp involving side chain protons. Figure S4. The NOEs in the ROESY spectra of HPTFDapAsp involving side chain protons. Figure S5. The NOEs in the ROESY spectra of HPTUDapAsp involving side chain protons.
S28 Figure S6. The NOEs in the ROESY spectra of HPTDabAsp involving side chain protons. Figure S7. The NOEs in the ROESY spectra of HPTFDabAsp involving side chain protons. Figure S8. The NOEs in the ROESY spectra of HPTUDabAsp involving side chain protons.

S29
. Figure S9. The NOEs in the ROESY spectra of HPTOrnAsp involving side chain protons. Figure S10. The NOEs in the ROESY spectra of HPTFOrnAsp involving side chain protons. Figure S11. The NOEs in the ROESY spectra of HPTUOrnAsp involving side chain protons.

S30
. Figure S12. The NOEs in the ROESY spectra of HPTLysAsp involving side chain protons. Figure S13. The NOEs in the ROESY spectra of HPTFLysAsp involving side chain protons. Figure S14. The NOEs in the ROESY spectra of HPTULysAsp involving side chain protons.
S31 Figure S15. The NOEs in the ROESY spectra of HPTDapGlu involving side chain protons. Figure S16. The NOEs in the ROESY spectra of HPTFDapGlu involving side chain protons. Figure S17. The NOEs in the ROESY spectra of HPTUDapGlu involving side chain protons.
S32 Figure S18. The NOEs in the ROESY spectra of HPTDabGlu involving side chain protons. Figure S19. The NOEs in the ROESY spectra of HPTFDabGlu involving side chain protons. Figure S20. The NOEs in the ROESY spectra of HPTDabGlu involving side chain protons.
S33 Figure S21. The NOEs in the ROESY spectra of HPTOrnGlu involving side chain protons. Figure S22. The NOEs in the ROESY spectra of HPTFOrnGlu involving side chain protons. Figure S23. The NOEs in the ROESY spectra of HPTUOrnGlu involving side chain protons.
S34 Figure S24. The NOEs in the ROESY spectra of HPTLysGlu involving side chain protons. Figure S25. The NOEs in the ROESY spectra of HPTFLysGlu involving side chain protons. Figure S26. The NOEs in the ROESY spectra of HPTULysGlu involving side chain protons.
Diisopropylethylamine (DIEA,8 equivalents) was then added to the solution and mixed thoroughly. The solution was then applied to the resin. The vial that contained the solution was rinsed with DMF (2x1 mL) and added to the reaction. The first coupling was carried out for 8 hours. The 8th to 14th residues were coupled for 1.5 hours. Other residues were coupled for 45 minutes. The residue with β-branching and the residue after it were coupled with double the time. After each coupling, the resin was washed with DMF (5 mL, 5x1 min). The Fmoc-group was then removed by 20% piperidine/DMF (5 mL, 3x8 min). After the final residue was coupled, a solution of acetic anhydride (20 equivalents), DIEA (20 equivalents), and DMF (3 mL) was added to resin for capping. The reaction was shaken for 2 hours.
Peptides were deprotected and cleaved off the resin by treating the resin with 5 mL 95:5 trifluoroacetic acid (TFA)/triisopropylsilane and shaken for 2 hours. For Cys-containing peptides, 5 mL 90:5:5 trifluoroacetic acid (TFA)/triisopropylsilane/ethanedithiol was used instead. The solution was then filtered through glass wool and the resin was washed with TFA (3x1.5 mL). The combined filtrate was evaporated gently by an air pump (nitrogen gas was used for the Cys-containing peptides). The resulting material was washed with hexanes S56 (3x3 mL), dissolved in water, and lyophilized. The peptide (1 mg/ mL, aqueous solution) was analyzed using analytical RP-HPLC on a 25 cm C 18 column (dia 4.6 mm) with flow rate 1 mL/min, temperature 25°C, linear 1 %/ min gradient from 100% A to 0% A (solvent A: 99.9% water, 0.1% TFA; solvent B: 90% acetonitrile, 10% water, 0.1% TFA). The disulfide bond of the Cys-containing HPTFXaaZbb peptides were formed via charcoal mediated air oxidation [3]. Peptides were purified to higher than 95% purity by Sep-Pak® Plus Short tC18 cartridges using an appropriate percentage of B solvent and by reverse phase HPLC using a preparative C 4 and C 18 columns with flow rate 10 mL·min -1 , temperature 25°C, linear 0.5 %·min -1 gradient. Appropriate linear gradients of solvent A and solvent B were used for each peptide to place the retention time for the desired peptide between 20 and 30 minutes. These gradients are listed individually for each peptide (vide infra); for example, PLG15_25 was used to purify HPTDapAsp using a C 18 column, representing the linear gradient from 15 % B to 25 % B (flow rate 10 mL·min -1 , temperature 25°C, linear 0.5 %·min -1 gradient). The identity of the peptide was confirmed by MALDI-TOF.
HPTDapAsp (Ac-Arg Thr Val Dap Val D Pro Gly Orn Asp Ile Leu Gln-NH 2 ) The peptide was synthesized using 200.2 mg (0.050 mmol) of NovaSyn ® TGR resin. The synthesis gave 286.6 mg of resin (99.2% yield). The cleavage yielded 52.6 mg of crude peptide (87.0% yield). The peptide was purified by preparative RP-HPLC using a C4 (PLG8_18) and a C18 column (PLG15_25) to give a 10.6 mg of pure peptide (96.9% purity). Retention time on analytical RP-HPLC was 27.4 minutes. The identity of the peptide was confirmed by MALDI-TOF mass spectrometry. Calculated for C 58 H 103 N 19 O 17 [MH]+: 1338.785;observed: 1338.776. The concentration of the peptide for NMR analysis was 10.5 mM.
HPTDabAsp (Ac-Arg Thr Val Dab Val D Pro Gly Orn Asp Ile Leu Gln-NH 2 ) The peptide was synthesized using 203.9 mg (0.051 mmol) of NovaSyn ® TGR resin. The synthesis gave 293.0 mg of resin (99.2% yield). The cleavage yielded 55.3 mg of crude peptide (88.0% yield). The peptide was purified by preparative RP-HPLC using a C4 (PLG7_17) and a C18 column (PLG15_25) to give a 12.3 mg of pure peptide (96.1% purity). Retention time on analytical RP-HPLC was 27.4 minutes. The identity of the peptide was confirmed by MALDI-TOF mass spectrometry. Calculated for C 59 H 105 N 19 O 17 [MH]+: 1352.801;observed: 1352.822. The concentration of the peptide for NMR analysis was 10.5 mM.
HPTOrnAsp (Ac-Arg Thr Val Orn Val D Pro Gly Orn Asp Ile Leu Gln-NH 2 ) The peptide was synthesized using 204.8 mg (0.051 mmol) of NovaSyn ® TGR resin. The synthesis gave 291.8 mg of resin (87.8% yield). The cleavage yielded 57.3 mg of crude peptide (75.8% yield). The peptide was purified by preparative RP-HPLC using a C4 column