Synthesis and biological activity of six monosubstituted analogs of a sea anemone polypeptide neurotoxin.

Monosubstituted analogs of Stichodactyla helianthus neurotoxin I (ShI), a sea anemone polypeptide, were synthesized by solid-phase methods. The analogs were selected to probe the importance of charged residues in the amino-terminal region; ionizable side chains were replaced with a corresponding neutral isosteric amino acid side chain. Following oxidation of the three disulfide bonds and refolding, the analogs were purified by gel, ion-exchange and reversed-phase chromatography. Circular dichroism and fluorescence spectral analyses indicate that the analogs have folded structures very similar to the native toxin. Crustacean paralytic bioassays and neuronal receptor binding assays of ShI analogs show that the anionic triad Asp 6-Asp 7-Glu 8 is essential for activity, as replacement of any of these side chains with the corresponding amide reduces toxicity and binding at least 1000-fold. Substitution at Lys 4 or Asp 11 reduces biological activity at least 10-fold; these residues are apparently not as critical as the polyanionic region. Replacing the N-terminal Ala residue with a Tyr residue has only a slight effect upon crab toxicity and axolemma binding. Our results suggest that several amino acid side chains near the N-terminus of this sea anemone neurotoxin are important for binding to crab neuronal sodium channels.