A Quantitative Model for the All-or-None Permeabilization of Phospholipid Vesicles by the Antimicrobial Peptide Cecropin A

Abstract

The mechanism of the all-or-none release of the contents of phospholipid vesicles induced by the antimicrobial peptide cecropin A was investigated. A detailed experimental study of the kinetics of dye release showed that the rate of release increases with the ratio of peptide bound per vesicle and, at constant concentration, with the fraction of the anionic lipid phosphatidylglycerol in neutral, phosphatidylcholine membranes. Direct measurement of the kinetics of peptide binding and dissociation from vesicles revealed that the on-rate is almost independent of vesicle composition, whereas the off-rate decreases by orders of magnitude with increasing content of anionic lipid. A simple, exact model fits all experimental kinetic data quantitatively. This is the first time that an exact kinetic model is implemented for a peptide with an all-or-none mechanism. In this model, cecropin A binds reversibly to vesicles, which at a certain point enter an unstable state. In this state, a pore probably opens and all vesicle contents are released, consistent with the all-or-none mechanism. This pore state is a state of the whole vesicle, but does not necessarily involve all peptides on that vesicle. No peptide oligomerization on the vesicle is involved; alternative models that assume oligomerization are inconsistent with the experimental data. Thus, formation of well-defined, peptide-lined pores is unlikely.

Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity

“…These proteins, magainin and Bax, respectively, have been shown to follow a kinetic profile similar to our report here in which stochastic nucleation is followed by a subsequent slowdown to a nonzero equilibrium leakage rate. We also note that leakage in the antimicrobial, Cecropin A, has been suggested to be rate limited by changes in state and not pore size (24). Further parallels can be seen in Aβ from Alzheimer's disease (34), where, for example, the size distribution of oligomers is reported to have an effect on overall leakage rates (35).…”

“…Other groups (23), including our own (8) have suggested persistent and porous mat-like proteolipid structure on the membrane. Such states have also been characterized as less structurally defined chaotic pores (24,25), an appealing view given the nonspecific and pathological nature of aggregation.…”

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

“…Permeabilization of Model Membranes Induced by Cyclotides-The ability of peptides to induce permeabilization of phospholipids vesicles has been correlated with their ability to disrupt the cell membranes of target organisms (48,49). Therefore, the ability of cyclotides to disrupt model membranes was evaluated with lipid systems of varying composition.…”

Resolving the kinetics of lipid, protein and peptide diffusion in membranes

“…The sample then comprises a mixture of completely empty vesicles and completely filled vesicles. Furthermore, the addition of a new marker to the solution outside the vesicles leads to marker ingress into porated vesicles only (Gregory et al, 2009;Gregory et al, 2008). (Fuertes et al, 2010).…”

“…The distinction between stable protein ensembles and efflux mediated by transient non-equilibrium structures can be made by assessing whether marker release is graded or all-or-none using fluorescence- (Pokorny and Almeida, 2004;Ladokhin et al, 1997). Recently the kinetics of marker release have been used to probe the poration mechanism, with the data modeled as an initial peptide binding step followed by a transformation to a permeative state (Gregory et al, 2009;Gregory et al, 2008). The kinetics of peptide association and dissociation were determined independently of marker release, and the rate of marker permeation (k efflux ) was accounted for, bringing this method close to model in Fig.…”