We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of peptide insertion into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and dye release from those vesicles were measured by stopped-flow fluorescence, and the secondary structure of the peptides on the membranes was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Dye release from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower in quantitative agreement with the prediction.

We previously proposed three hypotheses relating the mechanism of antimicrobial and cytolytic peptides in model membranes to the Gibbs free energies of binding and insertion into the membrane [Almeida, P.F., and Pokorny, A. (2009) Biochemistry 48, 8083–8093]. Two sets of peptides were designed to test those hypotheses, by mutating the sequences of δ-lysin, cecropin A, and magainin 2. Peptide binding and activity were measured on phosphatidylcholine membranes. In the first set, the peptide charge was changed by mutating basic to acidic residues or vice versa, but the amino acid sequence was not altered much otherwise. The type of dye release changed from graded to all-or-none according to prediction. However, location of charged residues in the sequence with the correct spacing to form salt bridges failed to improve binding. In the second set, the charged and other key residues were kept in the same positions, whereas most of the sequence was significantly but conservatively simplified, maintaining the same hydrophobicity and amphipathicity. This set behaved completely different from predicted. The type of release, which was expected to be maintained, changed dramatically from all-or-none to graded in the mutants of cecropin and magainin. Finally, contrary to the hypotheses, the results indicate that the Gibbs energy of binding to the membrane, not the Gibbs energy of insertion, is the primary determinant of peptide activity.

The results indicate significant differences in the membrane interactions of cationic and anionic peptides, confirming the importance of electrostatic interactions.