Analysis of Membrane-Binding Properties of Dermaseptin Analogues:  Relationships between Binding and Cytotoxicity

To understand relationships between membrane-binding properties of cytolytic peptides and resulting cytotoxicity, we investigated interactions of dermaseptin analogues with model bilayers by means of surface plasmon resonance. First, we tested the system by comparing two native dermaseptins, S1 and S4, whose binding properties were previously characterized in different experimental systems. Validation experiments revealed deviations from the one-to-one interaction model and indicated the binding to proceed by a two-stage mechanism. By calculation of apparent affinity constants and individual affinities for both steps of the interaction, the biosensor technology was able to distinguish between surface-bound peptides that subsequently penetrated into the bilayer and peptides that remained essentially superficially bound. This data interpretation was sustained after analysis of a series of dermaseptin S4 derivatives whose binding data were compared with cytotoxicity, revealing cytolytic activity to correlate mainly with insertion affinity. The data indicate that the potency of highly cytolytic peptides such as K(4)K(20)-S4 is not due to the highest membrane adhesion affinity but to the highest propensity for the inserted state. Similarly, truncated derivatives of 16, 13, and 10 residues showed a progressive reduction in cytotoxicity that best correlated with progressive reduction in insertion affinity. Support for the adhesion versus inserted states was provided by proteolytic experiments with RBC-bound peptides that demonstrated K(4)K(20)-S4 to be protected from enzymatic cleavage, unlike its 13-mer derivative. Overall, using the two-stage model proved instrumental in investigating membrane-binding properties of antimicrobial peptides and capable of explaining the cytolytic properties of closely related analogues.