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

Abstract

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.

The Effects of Charged Amino Acid Side-Chain Length on Diagonal Cross-Strand Interactions between Carboxylate- and Ammonium-Containing Residues in a β-Hairpin

“…In general, the fraction folded population for the HPDZbbXaa peptides with a given positively charged residue Xaa9 increased as the side-chain length of the negatively charged residues Zbb2 increased from Asp to Glu but mostly remained similar upon increasing Glu to Aad (Table 3). This trend is similar to the fraction folded population trend for the the HPDZbbAla peptides (with the negatively charged residue Zbb at position 2 and Ala at position 9) [38], suggesting that there may be small variations in the diagonal Zbb2-Xaa9 interactions in the HPDZbbXaa peptides. However, the fraction folded population for the HPDZbbXaa peptides with a given negatively charged residue Zbb2 generally decreased as the side-chain length of the positively charged residue Xaa9 increased (Table 3).…”

“…However, the fraction folded population for the HPDZbbXaa peptides with a given negatively charged residue Zbb2 generally decreased as the side-chain length of the positively charged residue Xaa9 increased (Table 3). This trend is opposite of the fraction folded population trend for the HPDAlaXaa peptides (with Ala at position 2 and the positively charged ammonium-containing residue Xaa at position 9) [38], suggesting variation in the diagonal Zbb2-Xaa9 interaction in the HPDZbbXaa peptides. Indeed, the diagonal Zbb2-Xaa9 interaction varied with side-chain length combination (Table 5).…”

“…The fully folded reference peptides HPDFZbbXaa and the fully unfolded reference peptides HPDUZbbXaa were required to determine the fraction folded population of the experimental HPDZbbXaa peptides (Figure 1b) [3,19,[25][26][27][36][37][38]. The fully folded reference HPDFZbbXaa peptides were designed by introducing cysteines at both the Nand C-termini of experimental HPDZbbXaa peptides to form intramolecular disulfide bonds [3,19,[25][26][27][36][37][38]. The fully unfolded reference HPDUZbbXaa peptides were designed by replacing DPro with LPro at position 6 of the experimental HPDZbbXaa peptides, to disrupt the β-hairpin conformation [3,19,[25][26][27][36][37][38].…”

“…The fully folded reference HPDFZbbXaa peptides were designed by introducing cysteines at both the Nand C-termini of experimental HPDZbbXaa peptides to form intramolecular disulfide bonds [3,19,[25][26][27][36][37][38]. The fully unfolded reference HPDUZbbXaa peptides were designed by replacing DPro with LPro at position 6 of the experimental HPDZbbXaa peptides, to disrupt the β-hairpin conformation [3,19,[25][26][27][36][37][38]. The peptides were synthesized by solid-phase peptide synthesis using Fmoc-based chemistry [39,40].…”

“…The energy difference between the ∆G fold of HPDZbbAla (and HPDAlaXaa) and HPDAlaAla represented the effect of individually incorporating Zbb2 (and Xaa9) on β-hairpin stability. The Zbb2-Xaa9 interaction energy (∆G int ) was derived from the folding free energy for the peptides HPDZbbXaa, HPDZbbAla [38], HPDAlaXaa [38], and HPDAlaAla (Table 5). Most of the cross-strand diagonal Zbb2-Xaa9 interactions were apparently stabilizing (Table 5).…”