Chemical Synthesis of a Functional Fluorescent-Tagged α-Bungarotoxin

alpha-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of alpha-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant alpha-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged alpha-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of alpha-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged alpha-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic alpha-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent alpha-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the alpha-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged alpha-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.

Automated summary

This study describes the chemical synthesis of a fluorescent-tagged alpha-bungarotoxin and demonstrates its binding properties on muscle nicotinic receptors using TE671 cells. The synthetic fluorescent alpha-bungarotoxin showed expected nanomolar affinity for the receptors and was able to label the cells.