Role of aromatic residues in the structure-function relationship of alpha-bungarotoxin.

The conformation of alpha-bungarotoxin and its cyanogen bromide cleaved and nitrated derivatives was studied by circular dichroism (CD). Native toxin contains no helices but some beta forms and possibly beta turns. Its ordered conformation is little affected when the peptide bond between Met-27 and Trp-28 is cleaved; however, the CD due to Trp-28 is abolished. The CNBr-cleaved derivative retains its immunoaffinity toward anti-toxin sera but loses its neurotoxicity toward the acetylcholine receptor. On the basis of both CD and fluorescence spectra, Trp-28 is probably stabilized by a short-range interaction with the carboxylate group of Asp-30. The ordered conformation of the toxin is also unaltered when one of the two tyrosine residues, identified as Tyr-54, is nitrated with tetranitromethane. This Tyr(NO2)-54 derivative possesses both immunoaffinity and neurotoxicity. However, the toxin is denatured and loses its activities when the other tyrosine residue, Tyr-24, is also nitrated in 6 M guanidine hydrochloride, even after the denaturant is removed. Spectrophotometric titration of the toxin indicates that Tyr-54 has a normal apparent dissociation constant (pKa = 9.7) and Tyr-24 ionizes at pH above 11.2. Both tyrosine residues are in a polar environment, but Tyr-24 is not readily accessible to reagents and is stabilized by long-range interactions, probably involving Glu-41.