cobra-cardiotoxin-proteins and dimyristoylphosphatidic-acid

Membrane-damaging activity of Naja naja atra cardiotoxin 3 (CTX3) on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/1,2-dimyristoyl-phosphatidic acid (DMPA) vesicles was approximately 3-fold that of N. naja atra cardiotoxin 4 (CTX4), while CTX3 and CTX4 displayed insignificantly permeabilizing activity in 1,2-dipalmitoyl-phosphatidylcholine (DPPC)/DMPA vesicles. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles did not closely correlate with membrane-damaging potency of CTX3 and CTX4. Geometrical arrangement of CTX3 in contact with POPC/DMPA vesicles was different from that noted with CTX4, and binding forces between CTX3 and POPC/DMPA were stronger than those between CTX4 and POPC/DMPA. Unlike POPC/DMPA, the interaction between CTXs and DPPC/DMPA was drastically reduced by increasing salt concentration. Color transformation of phospholipid/polydiacetylene membrane assay and FTIR spectra analyses revealed that CTX3 and CTX4 adopted different conformationsand modes upon absorption on POPC/DMPA and DPPC/DMPA vesicles. Taken together, our data show that, in addition to membrane packing density and phospholipid-binding capability, membrane-bound conformation of CTXs plays a vital role in displaying membrane-damaging activity.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Animals; Cobra Cardiotoxin Proteins; Colorimetry; Elapid Venoms; Elapidae; Glycerophospholipids; Liposomes; Membranes, Artificial; Molecular Sequence Data; Permeability; Phospholipids; Sequence Alignment; Taiwan

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.

Topics: Cobra Cardiotoxin Proteins; Dimyristoylphosphatidylcholine; Fourier Analysis; Glycerophospholipids; Phosphatidic Acids; Spectrophotometry, Infrared; Temperature