Intrinsic fluorescence study of lipid-protein interactions in membrane models. Binding of melittin, an amphipathic peptide, to phospholipid vesicles

Abstract

Melittin is a small peptide extracted from bee venom, which has a direct lytic activity on living cells, and equally disrupts the liposome structure. In agreement with a previous work of Mollay, C. and Kreil, G. ((1973) Biochim. Biophys. Acta 316, 196–203), it is shown that intrinsic fluorescence of the only tryptophan of melittin is very sensitive to the binding to phospholipids. The observed blue shifts, from 352 to 333 nm, upon addition of lipid vesicles, indicate that the Trp residue is going from a polar to a non-polar environment, and clearly show that melittin displays hydrophobic interaction with zwitter-ionic or negative phospholipids, whatever the ionic strength or pH. The hydrophobic nature of the interactions is confirmed by the sensitivity of the fluorescence intensity of the Trp residue to the phase transitions of phosphatidylserine, dimyristoyl and dipalmitoyl phosphatidylcholine, which implies a close contact between this residue and aliphatic chains. It is also shown that the length of the aliphatic chains has no significant effect on binding, but that their fluidity is a critical parameter. Binding is indeed much less efficient when aliphatic chains are in their crystalline state, below the phase transition temperature. Binding is strongly dependent on the net electrical charge borne by the vesicles. The effect of a pH increase, or of an addition of dicetylphosphate to phosphatidylcholine vesicles leads to the conclusion that binding is enhanced by an increase of the net negative charge of the lipid bilayer. This result is illustrated by the fact that melittin is totally bound when the lipid to protein molar ratio is equal to 3 or 4 in the case of phosphatidylserine, and about 25 for phosphatidylcholines. Phosphatidylserine vesicles can then bind up to 8 times more melittin than do phosphatidylcholines. In conclusion, both electrostatic and hydrophobic forces have to be considered as important binding parameters: the first step could be an ionic interaction between Lys and Arg residues of melittin and negative groups of phospholipids, either phosphate or carboxylic, the second step being the insertion of hydrophobic residues within the bilayer, this involving at least the Trp residue, but probably all the hydrophobic part of the peptide.