The composite physical and chemical approach to the solution spatial structure of polypeptide neurotoxins

Abstract

The most fruitful approach to the solution structure of peptides and proteins is a dovetailing of physical (experimental and theoretical) and chemical (selective modification) methods so as to best attain the common purpose of assessing the intra-and intermolecular interactions of the give substance. Of the physical methods the most informative in such study is high resolution NMR spectroscopy. We have made extensive use of this technique within the framework of the above approach in an analysis of the spatial structures os some polypeptide neurotoxin components of bee and snake venoms. For signal assignment in the 1H and 13C NMR spectra of the bee venom component apamin (18 amino acid residues, two disulfide bonds) a multifrequency homo- and heteronuclear decoupling procedure was developed, aimed at identification of the spin systems of the particular amino acid residues and determination of the residue position in the amino sequence. The apamin spatial structure has been elucidated by selection of the optimal calculated conformation on the basis of the NMR parameters of the native toxin and its selectively modified analogs. In a study of snake neurotoxins, mainly neurotoxin II (61 amino acid residues, four disulfide bonds) isolated from the venom of the Central Asian cobra Naja naja oxiana, use was made of the dependence of the 1H NMR parameters on the conditions of the aqueous medium, and of selective chemical modification, in particular, insertion of spin labels. Additional information was obtained from comparison of the spectra with those of homologous toxins from other snake. The contacts revealed between the amino acid side chains provided a general picture of the holding of the backbone and detailed information on the antiparallel β-structure of its central segment. A comparison snake venom erabutoxins, as well as with NMR data for Naja naja oxiana cytotoxins I and II. An EPR study of the binding of selectivity spin labeled neurotoxin II derivatives to the Torpedo marmorata acetylcholine receptor revealed the role played by lysine residues in this specific interaction