Melittin induces HII phase formation in cardiolipin model membranes

Abstract

The interaction of melittin with bovine heart cardiolipin model membranes was investigated via binding assays, 3t P-NMR, freeze-fracture electron microscopy, small angle X-ray diffraction and fluorescence based fusion assays. A strong binding (K d < 10-7 M) appeared to be accompanied by the formation of large structures, resulting from a fusion process of extremely fast initial rate. As the melittin content is increased, bilayer structure is gradually lost and from a cardiolipin to melittin ratio of about 6 the lipid starts to organize itself in an hexagonal H ii phase. At lower temperatures (T < 40 ° C) the coexistence of another structure is observed, characterized by a broad isotropic 31p-NMR signal and giving rise to sharp X-ray reflections, most probably a cubic phase, as suggested also by freeze-fracture images, showing orderly stacked particles. The results are discussed in relation to contrasting observations on the structural changes induced by melittin in the zwitterionic phospholipid system of dipalmitoylphosphatidylcholine (Dufourcq, J. et al. (1986) Biochim. Biophys. Acta 859, 33-48). The biological relevance of the observations with respect to the process of protein insertion into membranes is indicated.

Lipid specific penetration of melittin into phospholipid model membranes

“…This difference is a first indication suggestive of a deeper penetration in the cardiolipin system, though it should be kept in mind that depth of penetration and blue shift of fluorescence are not directly related. The fluorescence changes reach a plateau value at a phospholipid to melittin ratio of 2.03 + 0.07 : 1, which is in good agreement with the stoichiometry established with a centrifugation based binding assay [1]. The K a was calculated to be (1.6 + 0.7).…”

“…The vast majority of these reports were limited to the insertion of melittin into detergent micelles or phosphati- dylcholine bilayers. Our 3ap-NMR, freeze-fracture and X-ray diffraction studies however, as reported in the preceding article [1] show a remarkable difference between the structural alterations induced by melittin in the negatively charged cardiolipin system (formation of inverted non-bilayer structures) and those reported for phosphatidylcholines (formation of disc and micellar type of structures [16,19]). The hypothesis that this lipid specific structural reorganization is correlated with a different mode of insertion is addressed in this report, using fluorescence techniques.…”

“…With this thoroughly purified material the penetration of melittin into PC and cardiolipin was investigated in a comparative way, in order to explain the completely opposite effect of melittin 161 on the macroscopic organization of these two phospholipids [1]. The mode of insertion of melittin into zwitterionic phospholipids has been extensively investigated already with many techniques.…”

“…In order to explain the different structures of PC-melittin and cardiolipin-mefittin complexes [1], we will now propose models which take into account the different position of the tryptophan residue in these two lipid systems. For these models arbitrarily a monomeric and lipid independent structure of the peptide molecule is assumed and in the analysis different proposed orientations of the melittin helices will be considered.…”

“…7) to situation B1 and thus to translocation of the site were the mature sequence of the protein is attached, the mentioned situations being sequential steps in the export process. Many export-models are based on signal peptide-phospholipid interaction [44][45][46] and it would be worthwile to investigate further the role of these orientations and their consequences for phospholipid structure [1] using melittin as a model peptide or, better, synthetically prepared signal sequences.…”