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.

The β-sheet is one of the common protein secondary structures, and the aberrant aggregation of β-sheets is implicated in various neurodegenerative diseases. Cross-strand interactions are an important determinant of β-sheet stability. Accordingly, both diagonal and lateral cross-strand interactions have been studied. Surprisingly, diagonal cross-strand ion-pairing interactions have yet to be investigated. Herein, we present a systematic study on the effects of charged amino acid side-chain length on a diagonal ion-pairing interaction between carboxylate- and ammonium-containing residues in a β-hairpin. To this end, 2D-NMR was used to investigate the conformation of the peptides. The fraction folded population and the folding free energy were derived from the chemical shift data. The fraction folded population for these peptides with potential diagonal ion pairs was mostly lower compared to the corresponding peptide with a potential lateral ion pair. The diagonal ion-pairing interaction energy was derived using double mutant cycle analysis. The Asp2-Dab9 (Asp: one methylene; Dab: two methylenes) interaction was the most stabilizing (−0.79 ± 0.14 kcal/mol), most likely representing an optimal balance between the entropic penalty to enable the ion-pairing interaction and the number of side-chain conformations that can accommodate the interaction. These results should be useful for designing β-sheet containing molecular entities for various applications.

Interactions between charged amino acids significantly influence the structure and function of proteins. The encoded charged amino acids Asp, Glu, Arg, and Lys have different number of hydrophobic methylenes linking the backbone to the charged functionality. It remains to be fully understood how does this difference in the number of methylenes affect protein structure stability. Protein secondary structures are the fundamental three‐dimensional building blocks of protein structures. β‐Sheet structures are particularly interesting, because these structures have been associated with a number of protein misfolding diseases. Herein, we report the effect of charged amino acid side chain length at two β‐strand positions individually on the stability of a β‐hairpin. The charged amino acids include side chains with a carboxylate, an ammonium, or a guanidinium group. The experimental peptides, fully folded reference peptides, and fully unfolded reference peptides were synthesized by solid phase peptide synthesis and analyzed by 2D NMR methods including TOCSY, DQF‐COSY, and ROESY. Sequence specific assignments were performed for all peptides. The chemical shift data were used to derive the fraction folded population and the folding free energy for the experimental peptides. Results showed that the fraction folded population increased with increasing charged amino acid side chain length. These results should be useful for developing functional peptides that adopt the β‐conformation.

Collagen is a major structural component of the extracellular matrix and connective tissue. The key structural feature of collagen is the collagen triple helix, with a Xaa-Yaa-Gly (glycine) repeating pattern....

Cross-strand interactions are important for the stability of β-sheet structures. Accordingly, cross-strand diagonal interactions between glutamate and arginine analogs with varying side-chain lengths were studied in a series of β-hairpin peptides. The peptides were analyzed by homonuclear two-dimensional nuclear magnetic resonance methods. The fraction folded population and folding free energy of the peptides were derived from the chemical shift data. The fraction folded population trends could be rationalized using the strand propensity of the constituting residues, which was not the case for the peptides with lysine analogs, highlighting the difference between the arginine analogs and lysine analogs. Double-mutant cycle analysis was used to derive the diagonal ion-pairing interaction energetics. The most stabilizing diagonal cross-strand interaction was between the shortest residues (i.e., Asp2–Agp9), most likely due to the least side-chain conformational penalty for ion-pair formation. The diagonal interaction energetics in this study involving the arginine analogs appears to be consistent with and extend beyond our understanding of diagonal ion-pairing interactions involving lysine analogs. The results should be useful for designing β-strand-containing molecules to affect biological processes such as amyloid formation and protein-protein interactions.