We synthesized a 18 residues cyclic ppcicle correqonding to a loop involved in tk cumremimetic action of a snake toxic pmtein. This peptide. whii ccnn~les with Ihe native toxin for the binding lo the nicolinic ucetylcholii mzeptor, inclti the Bturn inducing Pro-AW moiety. Gibe peptide wax pqxued and cyclixed on solid phase, starting from Boc-Asp-OFm linked 1o Ibe nu%hylbenxbydrylamine resin through cbe ikarboxylic function. Fii treatment by HF converts this C-krmiud Asp inlo Asn.
Curatemimetic proteins (60-74 amino acids) from snake venoms (Elupiduc and Hyrlrophi&e) bind specifically and with high affinity (K,J IO-9 to 10-11 M) to the nicotinic acetylcholine receptors (AchR) thus inducing flaccid paralysis. The three-dimensional structure of some toxins has been previously determined, revealing that the polypeptide chain is fold into three loops projecting from a core region. Sequence comparisons** 2, chemical modilications 1-3 and mutagenesis studid have been used to determine the residues involved in the toxin/receptor complex formation. Most of residues presently identified (Lys27, Trpm. Aspsl, Phe/His/Trp32. Arg33, Ly#) are located mostly in the three-stranded p-sheet structure (LYs~~. TIPSY, Asp31, PhelHisfZ, A@) on loop 2, and only Ly&' helongs to loop 3. During tbe past few years. a number of synthetic peptides corresponding to portions of curaremimetic toxins were described. either to study their interactions with AchR5*6,7 or to generate toxin-specific neutralizing antibodiesg. The role of the secondary and tertiary structures in the biological properties of synthetic loop 2 peptides is now currently addressed in several lahoratories7v 9. In particular, it was shown that after cyclisation, the immunogenic property of the synthetic loop 2 of Nuju nigricolfis better mimicked those of the native loop 2 than the corresponding linear peptide8. Neverless. a recent NMR study revealed that the cyclic peptide possessed in solution a native-like p-turn. but not the expected p-sheet-like structure 9. In this case, cyclisation was made hy introducing a disulfide bridge hetween the first and the last amino acid positions (Cysu and Cy&).
In an attempt to further reduce the conformational space of the synthetic peptide and with the aim of bet&r initiating the &sheet structuw that the loop 2 adopts in the toxin molecule, we have linked N to C-terminal ends of the selected sequence by the Pro-Asn moiety, which is known to induce p-turns (Pro and Asn present at positions i+l and i+2 of p-turn, respectively) to. In order to respect the alternation and the orientation of potential hydrogen bonds as they exist in the native protein, Cysz4 and Cys4t were replaced by Asn and Pro, respectively.