Two‐dimensional ¹H‐nmr study of synthetic peptides containing the main immunogenic region of the Torpedo acetylcholine receptor

Abstract

A comparative 1H-NMR spectral study of a synthetic decapeptide containing the main immunogenic region of the Torpedo acetylcholine receptor (AChR; WNPADYGGIK, representing the alpha 67-76 fragment of Torpedo AChR) with four analogous peptides (WNP3-D5YGGIK, WNPAA5YGGIK, WNPADYGGA9K, and WNPD4DYGGV9K) has been carried out in dimethyl sulfoxide. One- and two-dimensional nmr experiments [correlated spectroscopy (COSY), relayed COSY, and phase-sensitive nuclear Overhauser enhancement spectroscopy (NOESY)] were performed to obtain complete assignments of the proton resonances. The presence of strong and multiple short- and long-range NOEs, and especially a strong long-range NOE between the two Asn2-C alpha H and Gly7-C alpha H protons, argues in favor of a rigid folded structure in all five cases. Temperature dependence measurements indicate the existence of three intramolecular interactions involving the Asp3, Gly8, and Lys10 amide protons.

Anatomy of the antigenic structure of a large memberane autoantigen, the muscle‐type nicotinic acetylcholine receptor

“…The NMR experiments did not give any evidence of conformational preferences in water which strongly solvated such short peptides. In dimethylsulfoxide (DMSO), various NOEs indicated the intrinsic conformationalpreferences depending on the location of the Ala substitution in the peptide, but no correlation between the structure and the antigen-antibody hinding capacity was seen(154,155). However, usiug MIR analogs and anti-MIR mAb6, TR-NOE studies in water showed that, on binding mAb6, some ofthe analogs adopted a rigid structure, the extent of which depended on their mAb6-biuding capacity (156), The free and mAb6bound conformations of Torpedo MIR and the [A76JMIR analog have been extensively studied by NOE and TR-NOE measurements and by restrained molecular dynamics simulations based on the interproton distances deduced from the NOEs(157,158).In DMSO.…”

Triphosgene: an efficient carbonylating agent for liquid and solid-phase aza-peptide synthesis. Application to the synthesis of two aza-analogues of the AChR MIR decapeptide

“…Different Pro-AzAsx and AzAsx-Pro model compounds, designed for structural analysis purposes (Xaa Ala and Asx Asp or Asn) were synthesized in the liquid-phase. In order to evaluate the impact of the AzXaaaXaa substitution in a longer peptide chain, we have also prepared in the liquid-phase Boc-Trp-AzAsn-Pro-NH i Pr, an analogue of the Nterminal tripeptide sequence in the main immunogenic region (MIR Trp-Asn-Pro-Ala-Asp-Tyr-Gly-Gly-Ile-Lys) of the Torpedo californica electric organ acetylcholine receptor (AChR) which is the target in an experimental autoimmune response related to myasthenia gravis [15,16]. Triphosgene was also used for the solid-phase synthesis of two azaanalogues of the MIR decapeptide containing either AzAsn 2 or AzAla 4 .…”

High‐resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor

“…Anti-MIR mAb binding has been gradually localized between amino acid residues 1-151 (Barkas et al, 1986), 46-120 (Ratnam et al, 1986), 37-85 (Barkas et al, 1987), 61-76 (Barkas et al, 1988) 67-78 (Wood et al, 1989 and finally 67-76 of the AChR ol-subunit . The conformation of the MIR decapeptide, either free (in dimethyl sulfoxide) or bound on the corresponding anti-MIR mAb, has been elucidated by the use of two-dimensional NMR experiments (Cung et al, 1989(Cung et al, , 1992. Interestingly, Sano et al (1991), by mapping antibodies raised against a recombinant human muscle AChR a-subunit domain (a1 -210), using human a-subunit peptides, showed that the sequence around a65 -72 (i. e. that strongly overlaps with the MIR decapeptide a67 -76) is very antigenic in rats, mice and rabbits.…”