CONFORMATION OF COBROTOXIN IN AQUEOUS-SOLUTION AS STUDIES BY NUCLEAR MAGNETIC-RESONANCE

The 270‐MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His‐32 is slightly lower than the pKa value (6.65) of the reference model of N‐acetylhistidine methylamide, because of the electrostatic interaction with Arg‐33 and Asp‐31. The pKa value (5.3—5.4) of His‐4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg‐59. The hydrogen‐deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole‐bearing models. The second‐order rate constants of N‐acetylhistidine methylamide, N‐acetylhistidine and imidazole acetic acid satisfy the Brønsted relation. With reference to this Brønsted relation, the imidazole ring of His‐32 is confirmed to be exposed. The imidazole ring of His‐4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg‐59 in the proximity. All the methyl proton resonances are assigned to amino‐acid types, by conventional double‐resonance method and more effectively by the spin‐echo double‐resonance method. Eight methyl proton resonances are identified as due to the γ and/or δ‐methyl groups of Val‐46, Leu‐1, Ile‐50 and Ile‐52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nuclear Overhauser effect enhance‐ ments. The aromatic proton resonances of Trp‐29 are affected by the ionizable groups of Asp‐31, His‐32 and Tyr‐35. The methyl groups of Ile‐50 are in the proximity to the aromatic ring of Trp‐29 and the methyl groups of Ile‐52 are in the proximity to Tyr‐25. The highest‐field methyl proton reso‐ nance is due to a threonine residue in the proximity to His‐4. The appreciable temperature‐dependent chemical shift of this methyl proton resonance suggests a temperature‐dependent local conformational equilibrium around the His‐4 residue of the first loop of the cobrotoxin molecule. Copyright © 1979, Wiley Blackwell. All rights reserved