β-Sheets are one of the fundamental three-dimensional building blocks for protein structures. Oppositely charged amino acids are frequently observed directly across one another in antiparallel sheet structures, suggesting the importance of cross-strand ion pairing interactions. Despite the apparent electrostatic nature of ion pairing interactions, the charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. Accordingly, the effect of charged amino acid side chain length on cross-strand ion pairing interactions at lateral non-hydrogen bonded positions was investigated in a β-hairpin motif. The negatively charged residues with a carboxylate (Asp, Glu, Aad in increasing length) were incorporated at position 4, and the positively charged residues with an ammonium (Dap, Dab, Orn, Lys in increasing length) were incorporated at position 9. The fraction folded population and folding free energy were derived from the chemical shift deviation data. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Only the Asp/Glu-Dap interactions with shorter side chains and the Aad-Orn/Lys interactions with longer side chains exhibited stabilizing energetics, mostly relying on electrostatics and hydrophobics, respectively. This suggested the need for length matching of the interacting residues to stabilize the β-hairpin motif. A survey of a nonredundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Lys < Glu-Lys, also implying the need for length matching of the oppositely charged residues.

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.