[NMR study of the spatial structure of the Naja naja siamensis toxin 3]

The 300 and 500 MHz 1H NMR spectra of a long-chain neurotoxin, toxin 3 Naja naja siamensis (S-T3), its derivatives and close homolog, toxin 3 Naja naja naja (N-T3), have been analyzed. A combination of double resonance, difference spectroscopy and usage of a series of toxin S-T3 derivatives acetylated or trifluoroacetylated at lysine residues allowed to assign the signals from all aromatic protons and a number of signals in the aliphatic region, and to elucidate the microenvironment of certain functionally important residues. Analysis of chemical shifts pH-dependences in the 1H and 19F NMR spectra of toxin S-T3 and its trifluoroacetylated congeners delineated a considerable region of the neurotoxin molecule affected by the conformational transition, with the midpoint at pH 5,4, between two states. This conformational transition is induced by protonation of His22 and is accompanied by a change in the accessibility of its imidazole ring for the solvent, along with the alteration of the distance between the side chains of functionally important Lys27 and Lys53 residues. According to EPR data (-196 degrees) for the respective S-T3 derivative having spin labels at Lys27 and Lys53 epsilon-amino groups, the inter-label distance increases from 18 A at pH 7,5 to 23 A at pH 3,5. Notwithstanding this conformational change, the totality of the obtained results evidences in favor of considerable similarity of the toxin S-T3 spatial structure in the solution and crystalline state.