Pore-Forming Peptides Induce Rapid Phospholipid Flip-Flop in Membranes

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Abstract
A kinetic model for pore-mediated and perturbation-mediated flip-flop is presented and used to characterize the mechanism of peptide-induced phospholipid flip-flop in bilayers. The model assumes that certain peptides can bind to and aggregate within the membrane. When the aggregate attains a critical size (M peptides), a channel is created that results in a fast flip-flop of phospholipids. In addition, certain peptides induce flip-flop through perturbation of the membrane without forming a pore. Donor phospholipid vesicles with an asymmetrical distribution of the fluorescent phospholipid 1-oleoyl-2-[12-[(7-nitro-1,2,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) were used to measure the extent of flip-flop by quantitating the decrease in fluorescence as the NBD-PC exchanged from the donor vesicles to acceptor vesicles that contained a quencher of the NBD fluorescence. Flip-flop curves generated at lipid/peptide ratios ranging from 30/1 to 300000/1 could be well-simulated by the model. Pore-forming peptides, such as melittin or the synthetic peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA), induce rapid phospholipid flip-flop with half-times for flip-flop of seconds at low peptide/vesicle ratios. The deduced pore sizes are M = 10 +/- 2 for GALA and M = 2 - 4 for melittin. The synthetic peptide LAGA (WEAALAEAEALALAEHEALALAEAELALAA) can catalyze flip-flop via bilayer perturbation. In contrast, hydrophobic peptides such as gramicidin A and valinomycin intercalate into the membrane, but induce little flip-flop. Modeling of the kinetics of phospholipid translocation supports pore formation as the key factor in accelerating phospholipid flip-flop. Thus, amphipathic segments from membrane proteins may account for non-energy-dependent phospholipid flip-flop in biological membranes.

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