Kinetics of dye efflux and lipid flip-flop induced by delta-lysin in phosphatidylcholine vesicles and the mechanism of graded release by amphipathic, alpha-helical peptides.
Delta-lysin is a 26-residue, amphipathic, alpha-helical peptide of bacterial origin. Its specificity is to some extent complementary to that of antimicrobial peptides. Therefore, understanding its mechanism is important for the more general goal of understanding the interaction of amphipathic peptides with membranes. In this article, we show that delta-lysin induces graded efflux of the contents of phosphatidylcholine vesicles. In view of this finding, carboxyfluorescein efflux kinetics were re-examined. In addition, peptide-induced lipid flip-flop was directly measured using fluorescence energy transfer between two lipid fluorophores initially placed on opposite leaflets of the bilayer. Carboxyfluorescein efflux and lipid flip-flop occur with essentially identical rate constants. On the basis of a detailed, quantitative analysis of the kinetics of peptide-vesicle interactions, we conclude that the peptide translocates across the bilayer as a small, transient aggregate, most likely a trimer. Dye efflux and lipid flip-flop occur concomitantly with the transient peptide-induced perturbation of the membrane. The experimental data are interpreted by comparing the predictions of the available models for the mechanism of action of amphipathic alpha-helical peptides. We demonstrate how the combination of the quantitative kinetic analysis, graded efflux, and reversibility of the peptide-vesicle interaction can be used to reject several models for this particular peptide. Two models are compatible with the data, the toroidal pore model and the sinking raft model. On the basis of the small aggregate size, a trimer, the latter appears to be more plausible. Some significant modifications are introduced in the sinking raft model to take into account the new finding of graded dye release. Furthermore, we present an explanation for the phenomenon of graded release in general, which, contrary to all-or-none efflux, has not been well-understood.
Pokorny
Antje
A
Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, North Carolina 28403, USA.
Almeida
Paulo F F
PF
D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains; D008954:Models, Biological; D009088:Mucoproteins / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D009088:Mucoproteins; D010455:Peptides; D010713:Phosphatidylcholines; C010393:lysin, gastropoda; C024098:6-carboxyfluorescein; C028694:1-palmitoyl-2-oleoylphosphatidylcholine