19F NMR determination of intramolecular distances in spin- and fluorine-labelled proteins: neurotoxin II Naja naja oxiana.

selected groupings gives crucial information on the conformation of proteins in solution and characterizes the topography of the sites responsible for biological activity, It also enables a comparison of the protein’s spatial structure in solution and in the crystal to be made on a quantitative basis, which is essential for understanding the dynamic features and conformational potentialities of the molecule. Evaluation of the intramolecular distances in proteins in solution can be done by optical spectroscopy, EPR and NMR techniques. For instance, detailed information can be obtained through the analysis of shift and relaxation effects of paramagnetic metal ions [l] or covalent spin labels (stable nitroxyl radicals) [2] in the NMR spectra of proteins. However, there arises a serious problem of signal assignment in overlapping proton spectra of proteins. In this respect it appears promising to use line-broadening evoked by spin labels in 19F NMR spectra of fluorine-containing protein derivatives. Such derivatives are obtained via chemical modification or biosynthetically, whereas their spectra usually consist of a limited number of signals [3,4 1. Feasibility of such an approach is demonstrated here with neurotoxin II (NT-II) isolated from the venom of Central Asian cobra (Naja naja oxiuna). A spin label was incorporated at the e-amino group of the Lys 27* residue. The amino groups of Leu 1, Lys 15, Lys 26, Lys 45 and Lys 47 residues were trifluoroacetylated. The distances between the spin label and the trifluoroacetyl groups were evaluated by r9F NMR. Comparison of the results on the spatial structure of neurotoxins in solution [2,6] and in the crystal [7] points to adequacy of the utilized approach.