Local Conformational Transition of Toxin B from Naja naja as Studied by Nuclear Magnetic Resonance and Circular Dichroism

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Abstract
The 270‐MHz proton NMR spectra of toxin B, a long neurotoxin from Naja naja, in 2H2O solution were analyzed. From the analysis of the discontinuous pH dependence of His‐21 C‐2 proton chemical shift at 23 C. toxin B is found to exhibit a slow‐exchange conformational transition between the states A and B with the transition midpoint at pH 5.75. Separate peaks due to the states A and B were also observed for His‐21 C‐4 proton, Tyr‐25 ring protons, phenylalanine ring protons and two C‐α protons. This local conformational transition as induced by the protonation of His‐21 residue is further confirmed by the measurement of the pH dependence of circular dichroism. In state A, the pKa, value of His‐21 is 6.6–6.9, indicating that His‐71 residue is not significantly perturbed. The His‐21 residue in state B is accessible to solvent, as found by the analysis of hydrogen → deuterium exchange rate. The low pKa value (4.6–4.9) of His‐21 in state B is probably due to the formation of a hydrogen bond of the iinidazole ring. The conformational transition rate at 23°C is obtained as 2.5 s−l at the transition midpoint (pH 5.75), by the saturation transfer method. The conformationnl transition at 60°C corresponds to a fast excliiiiige case for thc Tyr‐25 ring proton resonances and to a n intermediate exchangc case Tor the His‐21 (2‐2 and C‐4 proton resoiiiinces. The transition rate at the midpoint is about 300 s −1 at 60°C.