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Three phospholipase-like myotoxins, Bothrops asper myotoxin II, Agkistrodon piscivorus piscivorus K49 and Vipera ammodytes ammodytin L, can bind to long-chain fatty acids in a spontaneous autocatalytic reaction without the presence of calcium ions as cofactors. A phospholipase A2, with lysine or serine at position 49 in the active site instead of the expected aspartate residue, is found in several snake venoms. Membrane leakage is caused by these myotoxins through a unique cytolitic mechanism despite their lack of catalytic activity or calcium binding.These myotoxins are the first protein group that undergo acylation spontaneously with free fatty acids.

The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.

The capacity of the snake venom neurotoxin beta-bungarotoxin to induce fusion of small unilamellar liposomes was demonstrated. The fusion process was studied spectroscopically using three different methods: (i) by resonance energy transfer, using fluorescent lipid analogues; (ii) by the Tb/DPA assay; (iii) by electron spin resonance, using spin-labeled phospholipids. For the latter technique, a new method of analysis based on Fourier-transform component separation was developed. The fusogenic activity was found to be strongly correlated with the known phospholipase A2 activity of the toxin: both functions were shown to have a specific requirement of Ca2+ at almost stoichiometrical concentrations, much below the threshold values found for unspecific divalent cation induced vesicle fusion. Similarly, the presence of phosphatidic acid in the target membrane was essential for both fusogenic and enzymatic activities. The results suggest a molecular mechanism of fusion involving protein binding to negatively charged groups on the membrane surface, followed by local formation of lysophospholipids and as a consequence hereof the creation of point defects in the lipid structure. On the basis of these findings, a model is put forward to explain the specific mode of action of beta-bugarotoxin in vivo.