Lipid specific penetration of melittin into phospholipid model membranes

Abstract

The relative depth of penetration of melittin into egg phosphatidylcholine and bovine heart cardiolipin model membranes was investigated using fluorescence spectroscopy techniques. The tryptophan intrinsic fluorescence shift suggests a more hydrophobic surrounding of this residue in cardiolipin, while the accessibility for charged and uncharged aqueous quenchers is decreased in the cardiolipin system when compared with the phosphatidylcholine-bound situation. A lipid incorporated hydrophobic, collisional quencher and a resonance energy transfer acceptor on the other hand are more effective in quenching the tryptophan fluorescence of cardiolipin bound melittin. The combination of these results is interpreted as prove of a deeper positioning of the tryptophan containing part of the peptide molecule in the cardiolipin system in comparison with the situation in phosphatidylcholine. Models that take this difference into account are presented, which try to explain the opposite effect of melittin binding to the two lipid systems with respect to supramolecular structure, as reported in the preceding article (Batenburg, A.M., Hibbeln, J.C.L., Verkleij, A

Interaction of melittin with negatively charged phospholipids: Consequences for lipid organization

“…The blue-shift, which is slightly more pronounced than the average 15 nm shift reported for PC systems [3,7,22], and the quantum yield increase ( fig. 1) indicate a transfer to a more hydrophobic surrounding.…”

“…Binding of melittin to negatively charged phospholipids can be accurately studied by following the fluorescence of tryptophan-19 [7,21]. The blue-shift, which is slightly more pronounced than the average 15 nm shift reported for PC systems [3,7,22], and the quantum yield increase ( fig.…”

“…The conversion of egg PC and DOPC to egg PG, DOPS and DOPA by phospholipase D catalysed transesterification was also described before [13]; lipid purity was confirmed by thin layer layer chromatography. Melittin from commercial sources (Serva, Sigma) appeared to be severely contaminated with phospholipase and the peptide was therefore isolated from whole bee venom (Serva) by Sephadex G-50 gel filtration [14], followed by covalent chromatography with thiopropyl-Sepharose (Pharmacia) as described elsewhere [7]. Incubation of the egg-yolk PC liposomes with melittin thus obtained (Ri = 4) in a 10 mM Ca 2 ÷ containing buffer for 20 h at 37°C, did not result in detectable amounts of degradation products; the absence of phospholipase activity was also checked after most of the experiments.…”

“…Binding experiments were performed by following the effect of titration with SUV on the fluorescence spectrum of a 10 #M melittin solution [7] with a Perkin Elmer MPF3 fluorimeter. The data on the shift of the fluorescence maximum were analysed with a nonlinear regression program using the equation Ko [18].…”

“…Binding to the negatively charged cardiolipin however resulted in large structures composed of the hexagonal Hn phase [6]. Furthermore a correlation was demonstrated between the type of structures formed in PC versus cardiolipin systems and the depth of penetration of the peptide [7].…”