Effect of Charged Amino Acid Side Chain Length on Lateral Cross-Strand Interactions between Carboxylate-Containing Residues and Lysine Analogues in a β-Hairpin

Abstract

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.

Longer charged amino acids favor β‐strand formation in hairpin peptides

“…The peptides were designed based on a β‐hairpin‐forming peptide initially developed by Gellman 25 and later modified by our group 10,27,28 (Figure 2). Instead of using positions 4 and 9 as the guest positions (as in our previous study), 10,27,28 positions 2 and 9 were used individually as guest positions in this study. Thr was introduced at position 4 for high thermodynamic sheet propensity 13,14 to maintain the hairpin structure in the experimental peptides.…”

“…The peptides were named H air P ins with D iagonal relative guest positioning (i.e., positions 2 and 9), and the corresponding three letter codes for the residues at positions 2 and 9. In order to determine the fraction folded for the experimental peptides, peptides corresponding to the fully folded state and the fully unfolded state were required 10,24,25,27–29 . Cys residues were introduced at both termini for intramolecular cyclization via disulfide bond formation, serving as the fully folded reference peptides HPDFXaaAla and HPDFAlaXaa (Figure 2), which were presumed to be fully folded 10,24,25,27–29 .…”

“…In order to determine the fraction folded for the experimental peptides, peptides corresponding to the fully folded state and the fully unfolded state were required 10,24,25,27–29 . Cys residues were introduced at both termini for intramolecular cyclization via disulfide bond formation, serving as the fully folded reference peptides HPDFXaaAla and HPDFAlaXaa (Figure 2), which were presumed to be fully folded 10,24,25,27–29 . DPro6 in the experimental peptides was replaced with Pro to give the fully unfolded reference peptides HPDUXaaAla and HPDUAlaXaa (Figure 2), which were presumed to be fully unfolded 10,24,27,28 .…”

“…Cys residues were introduced at both termini for intramolecular cyclization via disulfide bond formation, serving as the fully folded reference peptides HPDFXaaAla and HPDFAlaXaa (Figure 2), which were presumed to be fully folded 10,24,25,27–29 . DPro6 in the experimental peptides was replaced with Pro to give the fully unfolded reference peptides HPDUXaaAla and HPDUAlaXaa (Figure 2), which were presumed to be fully unfolded 10,24,27,28 . The fully folded and fully unfolded reference peptides were named by adding the letters “F” and “U” after the “HPD” prefix, respectively.…”

“…The Hα chemical shift deviation (ΔδHα) is the difference between the observed Hα chemical shift and the Hα random coil chemical shift value; the chemical shifts for the fully unfolded reference peptides were assumed to represent the random coil values 10,25,27,28 . The ΔδHα for the residues in the experimental (HPDXaaAla and HPDAlaXaa) and fully folded reference peptides (HPDFXaaAla and HPDFAlaXaa) were derived from the corresponding Hα chemical shift values.…”