Proton-nuclear-magnetic-resonance study on molecular conformations of long neurotoxins. alpha-Bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja.

The 270-MHz proton NMR spectra were analyzed of the long neurotoxins alpha-bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja. The aromatic proton resonances were completely assigned to individual nuclei for alpha-bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts. The pKa values of Tyr-25, an invariant residue of neurotoxins, are 12.1 for alpha-bungarotoxin and 11.3 for toxin B, suggesting the presence of a strong hydrogen bond involving Tyr-25 in alpha-bungarotoxin. The Trp-29 residues of both toxins show a common titration shift due to the carboxylate group of Asp-31 and a similar structural arrangement of functionally invariant pair of Trp-29 and Asp-31 is implied. From the temperature dependences of the chemical shifts of His-68 and a methyl group of alpha-bungarotoxin, the local structure around His-68 near the tail part is shown to be more flexible than the other part. The six main-chain amide protons of alpha-bungarotoxin exchange most slowly with solvent deuterons and are found by interproton nuclear Overhauser effects to be in the beta-sheet near the aromatic ring of Tyr-25 residue. Hydrogen leads to deuterium exchange rates in 2H2O solution at 37 degrees C were measured of slowly exchanging amide protons of alpha-bungarotoxin, toxin B, and two short neurotoxins, namely cobrotoxin and erabutoxin b. The two long neurotoxins have amide protons with relatively long half-times spanning as long as 10-100 h, but the two short neurotoxins do not have amide protons with half-times longer than 3 h. The distributions of the half-times of amide proton exchange indicate the structural rigidity of neurotoxins in the order, alpha-bungarotoxin greater than toxin B greater than cobrotoxin approximately erabutoxin b, in agreement with the order of neurotoxicity as reported previously by Chicheportiche et al. and by Lee and Chen.