β-Sheets have been implicated in various neurological disorders, and ∼20% of protein residues adopt a sheet conformation. Therefore, studies on the structural origin of sheet stability can provide fundamental knowledge with potential biomedical applications. Oppositely charged amino acids are frequently observed across one another in antiparallel β-sheets. Interestingly, the side chains of natural charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the backbone to the hydrophilic charged functionalities. To explore the inherent effect of charged amino acid side chain length on antiparallel sheets, the stability of a designed hairpin motif containing charged amino acids with varying side chain lengths at non-hydrogen bonded positions was studied. Peptides with the guest position on the N-terminal strand and the C-terminal strand were investigated by NMR methods. The charged amino acids (Xaa) included negatively charged residues with a carboxylate group (Asp, Glu, Aad in increasing length), positively charged residues with an ammonium group (Dap, Dab, Orn, Lys in increasing length), and positively charged residues with a guanidinium group (Agp, Agb, Arg, Agh in increasing length). The fraction folded and folding free energy for each peptide were derived from the chemical shift deviation data. The stability of the peptides with the charged residues at the N-terminal guest position followed the trends: Asp > Glu > Aad, Dap < Dab < Orn ∼ Lys, and Agb < Arg < Agh < Agp. The stability of the peptides with the charged residues at the C-terminal guest position followed the trends: Asp < Glu < Aad, Dap ∼ Dab < Orn ∼ Lys, and Agb < Arg ∼ Agp < Agh. These trends were rationalized by thermodynamic sheet propensity and cross-strand interactions.

β-Sheet is one of the major protein secondary structures. Oppositely charged residues are frequently observed across neighboring strands in antiparallel sheets, suggesting the importance of cross-strand ion pairing interactions. The charged amino acids Asp, Glu, Arg, and Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on lateral cross-strand ion pairing interactions at non-hydrogen-bonded positions, β-hairpin peptides containing Zbb-Agx (Zbb = Asp, Glu, Aad in increasing length; Agx = Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by NMR methods. The fraction folded population and folding energy were derived from the chemical shift deviation data. Peptides with high fraction folded populations involved charged residue side chain lengths that supported high strand propensity. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Minimal interaction was observed between residues with short side chains, most likely due to the diffused positive charge on the guanidinium group, which weakened cross-strand electrostatic interactions with the carboxylate side chain. Only the Aad-Arg/Agh interactions with long side chains clearly exhibited stabilizing energetics, possibly relying on hydrophobics. A survey of a non-redundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Arg < Glu-Arg, implying the need for matching long side chains. This suggested the need for long side chains on both guanidinium-bearing and carboxylate-bearing residues to stabilize the β-hairpin motif.

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