FIGURE 4.

Examples of analytical techniques to characterize peptide’s membranotropic properties. (A) Fusogenic properties: 1) Membrane leakage experiments. After pore formation upon peptide addition, dilution of the self-quenching dye from labeled vesicles to unlabeled vesicles results in fluorescence increase, 2) Lipid mixing with FRET. The increasing distance between two fluorophores composing a FRET system upon lipid mixing can be visualized by monitoring the energy transfer efficiency decrease, 3) Lipid mixing can also be highlighted by dynamic light scattering (DLS) monitoring the vesicle size increase, (B) Insertion or internalization propensity: 1) Peptide insertion can be monitored by following Trp fluorescence. Upon membrane insertion, the hydrophobic environment around Trp results in a blue-shift in fluorescence, 2) NBD/sodium dithionite experiments. NBD-labeled sequences are incubated with liposomes and a reducing agent, sodium dithionite, is added. After reduction, the remaining fluorescence indicate the degree of membrane insertion/internalization, 3) Imaging experiments using fluorescence microscopy, such as confocal or total internal reflection fluorescent (TIRF) microscopy, with labeled peptide and liposomes or cells (C) Structural characterization in contact with model or cellular membranes to visualize conformational changes upon membrane interaction.