Fig 1.
Simplified zigzag representation of utilised β-hairpin peptides.
The pictograms show the relative orientation of the side chains on the upper (red) and lower side (blue) of the secondary structure in the all-l bi-disulfide 1 (Cit: l-citrulline). A pair of d-configurated amino acids flips the side chains to the other side as shown for peptide 3.
Fig 2.
Sequences of the bi-disulfide peptides 1–4 and the mono-disulfide peptides 5–10.
They contain one or a pair of d-configurated amino acids and are depicted as simplified zigzag structures in accordance with Fig 1. d-amino acids are denoted by lowercase letters. The directed bending and tethering of peptide backbones results from the combination of the hydrophobic cluster, disulfides, and d-amino acids.
Fig 3.
HPLC chromatograms of the oxidative folding process of tetra-d bi-disulfide 4 (c1-c14, c5-c10; cpeptide = 1 mg L-1).
The time periods after which the oxidation process was stopped by addition of TFA is denoted at the right end of the respective chromatograms.
Fig 4.
HSQC spectrum excerpt of bi-disulfide peptide 2md (c1/c14,C5-C10).
The 1JC,H correlations between the β-carbons of all cysteines and their corresponding protons are shown. They prove a reduced state for c1 and c14 (~25 ppm, low 1H signal dispersion) and a disulfide bridge between C5 and C10 (over 40 ppm, high 1H signal dispersion; 600 MHz, 280 K, 50 mm potassium phosphate buffer (pH = 3.0)/D2O 9:1).
Fig 5.
Amide and alkyl regions of the 1H NMR spectra of peptides 3 and 5–10.
Their different content of pairwise installed d-amino acids corresponds to the respective zigzag shapes depicted in Figs 1 and 2. The peptides are listed according to the characteristic highfield shift of L12-Hδ (right blue ribbon) which identify the completely folded hydrophobic cluster and correlates with the downfield shift of E4-NH (left blue ribbon). R9-NH is in all cases the most shielded amide proton (red ribbon; 600 MHz, 280 K, 50 mm potassium phosphate buffer (pH = 7.0)/D2O 9:1).
Fig 6.
NMR-based computer models of peptides 5 (all-l), 8 (t6,r9), 9 (s5,s10) and 10 (t6,r9,s5,s10) as overlay.
The hydrophobic clusters differ only minimally, while the citrulline (shown as thin lines) containing turn exhibits differently kinked geometries, based on the position and amount of d-amino acid pairs. Simplified zigzag structures are depicted at the respective turn.
Fig 7.
Absolute absorbance values obtained in ELISA for the CCP-positive RA patients.
The numbers of the sera have been randomly assigned during their collection. Every patient shows a distinct set of measured absorbances in regard to the utilised epitopes. The order of the peptides is chosen based on the conformational design: first the all-l reference peptide 5, then the two mono-d peptides 7 (r9) and 6 (t6), afterwards the double-d motifs 8 (t6,r9) and 9 (s5,s10), finally the tetra-d-sequence 10 (t6,r9,s5,s10) as well as the only bi-disulfide 3 (C1-C14,c5-c10).