Proton‐Nuclear‐Magnetic‐Resonance Study on Molecular Conformations of Long Neurotoxins

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Abstract
The 270‐MHz proton NMR spectra were analyzed of the long neurotoxins α‐bungarotoxin fromBungarus multicinctus and Toxin B fromNaja naja.The aromatic proton resonances were completely assigned to individual nuclei for α‐bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least‐square method, for obtaining PKavalues and protonation shifts. The PKavalues of Tyr‐25, an invariant residue of neurotoxins, are 12.1 for α‐bungarotoxin and 11.3 for toxin B, suggesting the presence of a strong hydrogen bond involving Tyr‐25 in α‐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 α‐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 α‐bungarotoxin exchange most slowly with solvent deuterons and are found by interproton nuclear Overhauser effects to be in the β‐sheet near the aromatic ring of Tyr‐25 residue. Hydrogen → deuterium exchange rates in 2H2O solution at 37 °C were measured of slowly exchanging amide protons of α‐bungarotoxin, toxin B, and two short neurotoxins, namely cobrotoxin and erabutoxin b. The two long neurotoxins 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, α‐bungarotoxin > toxin B > cobrotoxin ∼ erabutoxin b, in agreement with the order of neurotoxicity as reported previously by Chicheportiche et al. and by Lee and Chen.