Structural studies of .alpha.-bungarotoxin. 3. Corrections in the primary sequence and x-ray structure and characterization of an isotoxic .alpha.-bungarotoxin

Abstract

The most plausible set of chemical shift assignments for .alpha.-bungarotoxin as deduced from the combined use of two-dimensional J-correlated and two-dimensional nuclear Overhauser effect 1H nuclear magnetic resonance (NMR) spectroscopy was in conflict with the accepted amino acid sequence between residues 8 and 12 and residues 66 and 70 [Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M., and Kuntz, I. D. (1988) Biochemistry (first paper of three in this issue)]. Furthermore, NMR spectra of .alpha.-bungarotoxin, purified by conventional methods, evidenced a second species at the level of approximately 10% total protein. The minor component was separated from .alpha.-bungarotoxin by Mono-S (cationic) chromatography. Sequencing of Mono-S-purified .alpha.-bungarotoxin and one of its tryptic peptides showed that the correct sequence for .alpha.-bungarotoxin is Ser-Pro-Ile at positions 9-11 and Pro-His-Pro at positions 67-69. The electron density map of .alpha.-bungarotoxin [Love, R. A., and Stroud, R. M., (1986) Protein Eng. 1, 37] was refined with the new sequence data. Improvements in the structure were found primarily for residues 9-11. Sequence analysis of two overlapping tryptic peptides proved that the minor species differed from .alpha.-bungarotoxin by replacement of a valine for an alanine at position 31. This new toxin, .alpha.-bungarotoxin(Val-31), binds to the acetylcholine receptor with an affinity that is comparable to that of .alpha.-bungarotoxin.