PROTON NMR-STUDIES OF THE STRUCTURAL AND DYNAMICAL EFFECT OF CHEMICALMODIFICATION OF A SINGLE AROMATIC SIDE-CHAIN IN A SNAKE CARDIOTOXIN -RELATION TO THE STRUCTURE OF THE PUTATIVE BINDING-SITE AND THE CYTOLYTIC ACTIVITY OF THE TOXIN

This paper presents the comparative comprehensive analysis of NMR stru ctural parameters (NOEs, scalar coupling, chemical shifts) of toxin ga mma, a cardiotoxin isolated from the venom of Naja nigricollis, and th ree chemical derivatives, i.e. the 2-nitrophenylsulphonyl (NPS)-Trp11, 3-nitro-Tyr22 and 3-nitro-Tyr51 derivatives. In previous work, the ch emical modifications of single side chains have suggested that these a romatic residues, in association with several lysine residues, contrib uted to the cytotoxicity of toxin gamma. Analysis of these results bas ed on the refined solution structure of the toxin has resulted in the proposal of a conserved phospholipid binding site through which cardio toxins are likely to interact with the membrane of target cells. The p resent work shows that modifications of either the tryptophan residue or the tyrosine residues, which are within or near the proposed bindin g site, have no influence on the three-dimensional structure of the pr otein. On the other hand: the proton exchange study of the backbone am ides indicates that the structural core of the protein is destabilized in the three derivatives. This corresponds to a decrease of the overa ll stability of the protein as indicated by the comparative solvent de naturation study of the unmodified toxin gamma and the Trp11 derivativ e. More specifically the dynamics of the three-stranded beta sheet, a part of the structural core, are highly perturbed by the chemical modi fications. This sheet was previously proposed as a part of the phospho lipid binding site of cardiotoxins. The dynamical perturbation of this site appears to be correlated with the decrease in toxicity of the ch emical derivatives.