Translocation of Cationic Amphipathic Peptides across the Membranes of Pure Phospholipid Giant Vesicles

Abstract

The ability of amphipathic polypeptides with substantial net positive charges to translocate across lipid membranes is a fundamental problem in physical biochemistry. These peptides should not passively cross the bilayer nonpolar region, but they do. Here we present a method to measure peptide translocation, and test it on three representative membrane-active peptides. In samples of giant unilamellar vesicles (GUVs) prepared by electroformation, some GUVs enclose inner vesicles. When these GUVs are added to a peptide solution containing a membrane-impermeant fluorescent dye (carboxyfluorescein), the peptide permeabilizes the outer membrane, and dye enters the outer GUV, which then exhibits green fluorescence. The inner vesicles remain dark if the peptide does not cross the outer membrane. But if the peptide translocates, it permeabilizes the inner vesicles as well, which then show fluorescence. We also measure translocation, simultaneously on the same GUV, by the appearance of fluorescently-labeled peptides on the inner vesicle membranes. All three peptides examined are able to translocate, but to different extents. Peptides with smaller Gibbs energy of insertion into the membrane translocate more easily. Further, translocation and influx occur broadly over the same period but with very different kinetics. Translocation across the outer membrane follows approximately an exponential rise, with a characteristic time of 10 minutes. Influx occurs more abruptly. In the outer vesicle, influx happens before most of the translocation. But some peptides cross the membrane before any influx is observed. In the inner vesicles, influx occurs abruptly sometime during peptide translocation across the membrane of the outer vesicle.

Optical Microscopy of Giant Vesicles as a Tool to Reveal the Mechanism of Action of Antimicrobial Peptides and the Specific Case of Gomesin

“…GUVs were also used by the same group to reveal the ability of peptides to translocate the bilayer [55]. GUVs encapsulating smaller vesicles were imaged and the fluorescent probe carboxyfluorescein (CF) was added to the external medium of the GUVs.…”

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

“…wall may have a "sieving" effect with some larger peptides that would be lost in spheroplasts, requiring additional controls comparing spheroplasts and "normal" cells in other assays (18). However, even with these caveats we believe that spheroplasts provide an excellent model system compared to other alternatives to overcome size and shape limitations, such as giant unilamellar vesicles (19)(20)(21), as spheroplasts preserve a physiological bacterial membrane composition and are viable if returned to growth conditions (13,22). Moreover, although spheroplasts have generally been produced from E. coli, protocols can be adjusted to make them from strains of other species (23).…”

GUVs Melt Like LUVs: The Large Heat Capacity of MLVs Is Not Due to Large Size or Small Curvature

“…DPPC GUVs were prepared by electroformation in a 0.1 M sucrose solution, at 50 C, as previously described in Wheaten et al (5) and Svetlovics et al (6). The effect of 0.1 M sucrose on the phase transition of DPPC is negligible (7).…”

“…Alternatively, it is possible that the apparent higher cooperativity of the phase transition in MLVs may be due to inhibition of curvature fluctuations by adjacent bilayers in each vesicle (13). Understanding the effect of vesicle size and interbilayer coupling on the phase transition of phospholipids is significant for investigations that use model membranes in protein-lipid interactions, membrane protein function, mechanisms of antimicrobial and cell-penetrating peptides (5), and the lipid lateral organization in membranes. GUVs are good models of plasma membranes, because of their large size and low curvature.…”