Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation

Abstract

According to their distinct biological functions, membrane-active peptides are generally classified as antimicrobial (AMP), cell-penetrating (CPP), or fusion peptides (FP). The former two classes are known to have some structural and physicochemical similarities, but fusogenic peptides tend to have rather different features and sequences. Nevertheless, we found that many CPPs and some AMPs exhibit a pronounced fusogenic activity, as measured by a lipid mixing assay with vesicles composed of typical eukaryotic lipids. Compared to the HIV fusion peptide (FP23) as a representative standard, all designer-made peptides showed much higher lipid-mixing activities (MSI-103, MAP, transportan, penetratin, Pep1). Native sequences, on the other hand, were less fusogenic (magainin 2, PGLa, gramicidin S), and pre-aggregated ones were inactive (alamethicin, SAP). The peptide structures were characterized by circular dichroism before and after interacting with the lipid vesicles. A striking correlation between the extent of conformational change and the respective fusion activities was found for the series of peptides investigated here. At the same time, the CD data show that lipid mixing can be triggered by any type of conformation acquired upon binding, whether α-helical, β-stranded, or other. These observations suggest that lipid vesicle fusion can simply be driven by the energy released upon membrane binding, peptide folding, and possibly further aggregation. This comparative study of AMPs, CPPs, and FPs emphasizes the multifunctional aspects of membrane-active peptides, and it suggests that the origin of a peptide (native sequence or designer-made) may be more relevant to define its functional range than any given name.

Fig. 1

Fusogenic activities and structural properties of AMPs and CPPs. a Lipid-mixing activities of different AMPs (red horizontal) and CPPs (green vertical), displayed in order of decreasing activity. The amplitudes of the fluorescence resonance energy transfer (FRET) lipid-mixing signal are shown relative to the extent of fusion induced by the detergent Triton X-100 (=100%). These values can be compared to the activity of the viral fusion peptide FP23 (left column). b Alternative assay for fusion activity based on dynamic light scattering (DLS), which gives the increase in average vesicle diameter (from originally 120 nm) after peptide-induced fusion. c Circular dichroism (CD) was used to determine the extent of the conformational change experienced by the peptides upon inducing fusion. Here we show the absolute difference in mean residue ellipticity (MRE) at 195 nm before and after fusion (see Fig. 2). d Total charge of each peptide (including free termini). e Mean residue hydrophobicity of each peptide, normalized according to the Eisenberg consensus scale (Eisenberg et al. 1984). All experimental values (FRET, DLS, CD data) represent the mean value of three or more independent experiments

Fig. 2

Conformational change upon membrane binding. CD spectra of the peptides before fusion (dashed lines: 10 mM phosphate buffer, pH = 7) and 20 min after inducing vesicle fusion (thick lines: LUVs of LM3 lipid mixture). The extent of the conformational change is illustrated by the difference spectra (dotted lines) and is indicated by the arrows at 195 nm (where the loss of random coil signal is most pronounced)