Phase separation induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry.
Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical rate of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C14 lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10-20 degrees C. This second group of lipids is constituted by monoacid C16 lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C16 aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C14 chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.
D000838:Anions; D001514:Bee Venoms / Q000494:pharmacology; D002151:Calorimetry; D005454:Fluorescence Polarization; D008051:Lipid Bilayers; D008555:Melitten / Q000494:pharmacology; D008560:Membrane Fluidity / Q000187:drug effects*; D010712:Phosphatidic Acids; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D010743:Phospholipids; D013329:Structure-Activity Relationship
D000838:Anions; D001514:Bee Venoms; D008051:Lipid Bilayers; D010712:Phosphatidic Acids; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D010743:Phospholipids; D008555:Melitten