Biochemical characterization and nuclear magnetic resonance structure of novel α-conotoxins isolated from the venom of Conus consors

Abstract

Two novel α-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named α-CnIA and α-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The α- CnIA represents the main α-conotoxin contained in the venom, whereas α- CnIB is present in a relatively small amount. Chemical synthesis of α-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The α-CnIA inhibited the fixation of iodinated α-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 μM which can be compared to the IC50 of 0.31 μM found for the previously characterized α-MI isolated from the piscivorous Conus magus. The synthetic α-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for α-GI and α-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 °C.