[1H-NMR study of the Naja naja oxiana neurotoxin II and its spin-labeled derivatives. Conformation of "short" neurotoxins]. / 1H-IaMR-issledovanie neirotoksina II Naja naja oxiana i ego spin-mechenykh proizvodnykh. Konformatsiia "korotkikh" neirotoksinov.

ABSTRACT

In 1H NMR spectra of neurotoxin II N. n. oxiana the chemical shift pH-dependences in H2O and 2H2O solutions were studied, and also the deuterium exchange rates and chemical shift temperature gradients were measured for the amide protons. The spin probe method was applied to assess the degree of exposure into solvent of the amide and side chain protons. With the purpose of establishing mutual disposition of certain neurotoxin II groupings, nuclear Overhauser effect was studied in the 1H NMR spectra, along with the broadening of proton resonances induced by spin labels selectively attached to epsilon-amino groups of Lys26, Lys27, Lys45 or Lys47. The mobility of these labels was determined from the EPR spectra. The methyl resonances of Val and Leu residues were assigned to a definite position in the amino acid sequence. The following pKa were determined alpha-NH2 Leu1 (9,2), gamma-COOH Glu2 (3,7), alpha-COOH Asn62 (1,3). The protonation of a carboxyl group(s) in neurotoxin II (alpha-COOH Asn62 seems to be involved) decreases the temperature stability of the neurotoxin II conformation. On the basis of studies on neurotoxin II and some other homologous neurotoxins, the model for the "short" neurotoxin folding in solution was proposed. Comparison of experimental data for the disposition of equivalent groups in homologous neurotoxins and in the X-ray structure of erabutoxin b Laticauda semifasciata revealed that the Val46 side chain in solution might change its orientation by 180 degrees with respect to polypeptide backbone. Binding of spin labeled neurotoxin II derivatives to the acetylcholine receptor was discussed in light of the obtained data.