Characterization by infrared spectroscopy of the interaction of a cardiotoxin with phosphatidic acid and with binary mixtures of phosphatidic acid and phosphatidylcholine

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Abstract
The effect of cardiotoxin IIa from Naja mossambica mossambica, a small basic protein extracted from snake venom, on dimyristoylphosphatidic acid (DMPA) and on equimolar mixtures of DMPA and dimyristoylphosphatidylcholine (DMPC) has been studied by Fourier transform infrared spectroscopy. The interaction of cardiotoxin with DMPA dispersions decreases both the cooperativity of the phase transition of the lipid and the molecular order of the lipid acyl chains in the gel phase. This effect increases with the proportion of the toxin in the complexes and leads to the total abolition of the phase transition of DMPA at a lipid-to-protein molar ratio of 5. Small-angle X-ray results demonstrate that the structure of the lipid-protein complexes is poorly ordered and gives rise to broad diffusion peaks rather than to well-resolved diffraction patterns. Infrared spectra of oriented cardiotoxin-DMPA films show that the protein is not homogeneously oriented with respect to the bilayer surface. The destabilization of the gel-phase structure of DMPA by cardiotoxin also results in a deeper water penetration in the interfacial region of the lipid since more carbonyl ester groups appear to be hydrogen bonded in the presence of the toxin. The infrared results on the phosphate group vibrations also indicate clearly that the basic residues of cardiotoxin interact strongly with the phosphate group of DMPA that becomes partly ionized at a pH as low as 6.5. The results obtained on the interaction of cardiotoxin with an equimolar mixture of DMPA and DMPC clearly demonstrate the ability of this toxin to induce lateral phase separation in this mixture with one phase containing DMPA-rich domains perturbed by cardiotoxin while the second phase is composed of regions enriched in DMPC. Comparison of the results of the current study with those obtained on other basic proteins and polypeptides suggests that charge-induced phase separation occurs only when the charge density on certain regions of the protein structure is high enough to lead to efficient electrostatic interactions with anionic phospholipids. This condition occurs only when the conformation of the protein or polypeptide is well-ordered at the lipid interface.