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Toxin alpha from Naja nigricollis (61 amino acids, four disulfide bridges) belongs to the "three finger" fold family, which contains snake toxins with various biological activities and nontoxic proteins from different origins. In this paper, we report an extensive 1H and 15N NMR study of the dynamics of toxin alpha in solution. 15N relaxation, 1H off-resonance ROESY, and H-D exchange experiments allowed us to probe picosecond to hour motions in the protein. Analysis of these NMR measurements demonstrates that toxin alpha exhibits various time scale motions, i.e., particularly large amplitude picosecond to nanosecond motions at the tips of the loops, observable microsecond to millisecond motions around two disulfide bridges, second time scale motions around the C-N bonds of asparagine and glutamine side chains which are more or less rapid depending on their amino acid solvent accessibility, and minute to hour motions in the beta-sheet structure. The less well-defined regions of toxin alpha solution structures are subject to important picosecond to nanosecond motions. The toxic site is organized around residues belonging to the rigid core of the molecule but also comprises residues exhibiting dynamics on various time scales. The Malpha1 epitope is subject to large picosecond to millisecond motions, which are probably modified by the interaction with the antibody. This phenomenon could be linked to the neutralizing properties of the antibody.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

We investigated the interaction of toxin gamma, a cardiotoxin from the venom of the elapid Naja nigricollis, with perdeuterated dodecylphosphocholine (DodPCho) micelles using standard two-dimensional proton NMR spectroscopy. The proton spectrum resonances of the micelle-bound toxin gamma were assigned, and the chemical shifts of the backbone and side-chain protons were compared with those determined in the absence of DodPCho. We observed that DodPCho induced large chemical shift changes on residues localized on the hydrophobic face of the toxin. These changes are not associated with conformational changes of the toxin. However, the micellar environment may induce some stabilization of the triple-stranded beta sheet, the major component of the protein structural core. Since the proton NMR spectrum of toxin alpha, a structurally related neurotoxin extracted from the same venom, was unaffected by the presence of the micelles, we came to the conclusion that the observed effects are specific to cardiotoxins. The present results give direct evidence of the contribution of the hydrophobic face of the toxin to the toxic site and further suggest a possible mechanism of action of cardiotoxin on biological bilayers.