Interactions of membrane-active peptides with thick, neutral, nonzwitterionic bilayers

Abstract

Alamethicin is a well-studied channel-forming peptide that has a prototypical amphipathic helix structure. It permeabilizes both microbial and mammalian cell membranes, causing loss of membrane polarization and leakage of endogenous contents. Antimicrobial peptide-lipid systems have been studied quite extensively and have led to significant advancements in membrane biophysics. These studies have been performed on lipid bilayers that are generally charged or zwitterionic and restricted to a thickness range of 3-5 nm. Bilayers of amphiphilic diblock copolymers are a relatively new class of membranes that can have significantly different physicochemical properties compared with those of lipid membranes. In particular, they can be made uncharged, nonzwitterionic, and much thicker than their lipid counterparts. In an effort to extend studies of membrane-protein interactions to these synthetic membranes, we have characterized the interactions of alamethicin and several other membrane-active peptides with diblock copolymer bilayers. We find that although alamethicin is too small to span the bilayer, the peptide interacts with, and ruptures, thick polymer membranes.

Figure 1. Relative size of peptide and membranes

Alamethicin forms an amphiphilic helix that is only a few angstroms shorter than the ∼3 nm typical hydrophobic core thickness of lipid membranes. The hydrophobic core thickness of the OE7 (PEO-PEE) diblock copolymer membrane is around 8 nm as determined by cryo-TEM and the corresponding thickness for OB18 (PEO-PBD) is ∼14 nm (not shown).

Figure 2. LAURDAN spectral shift

(A) Addition of alamethicin to PC, OE7 and OB18 vesicles causes LAURDAN emission maxima to blue shift. The data for PC is shown in the inset. A threshold concentration below which alamethicin did not result in a detectable blue shift was observed for PC and OE7. (B) Emission spectra of LAURDAN incorporated in OE7 vesicles with different concentrations of alamethicin. Note that the curves intersect at a common point. The arrows denote directions of increasing molar ratio of alamethicin to OE7. (C) Two-population fits for blue-shifted LAURDAN spectra. The fits were determined with the formula F_i = αF₀ + (1-α)F_∞, where α is the fitting parameter, F₀ is the spectrum in the absence of alamethicin, and F_∞ is the spectrum at a saturating concentration of alamethicin, which we took to be 2:1 alamethicin to OE7. The values for α determined from the fits are shown in the table on the right. Representative fits are shown in the graph on the left. (D) LAURDAN blue-shift for three other membrane-active peptides added to OE7 vesicles. Melittin and mastoparan are two antimicrobial peptides that are also known to permeabilize lipid vesicles. They both show weak blue-shifts, indicating at least some interaction with the OE7 bilayer. Polymyxin, an anti-bacterial agent that binds to lipopolysaccharides found in gram-negative bacterial cell walls, does not affect the spectral properties of LAURDAN in the bilayer.

Figure 3. Representative graphs of alamethicin-induced calcein leakage

Fluorescence was normalized by the fluorescence level after detergent lysis (0.6 % Triton X 100). The arrows indicate the instant at which the peptide or detergent was added. The concentration of peptide is represented as a molar ratio (alamethicin to polymer or lipid). (A) Alamethicin induces 100% leakage from 200 nm PC vesicles. (B) Alamethicin induces partial calcein leakage from 200 nm OE7 vesicles. (C) Alamethicin does not induce calcein leakage from 200 nm OB18 vesicles.

Figure 4. Calcein leakage from OE7 vesicles as a function of alamethicin concentration

Below a threshold molar ratio of about 0.25, alamethicin does not cause any leakage from OE7. Complete leakage is not observed except at very high concentrations. The amount of leakage varies between different samples of OE7 polymersomes, with a maximum variation of about 20%.

Figure 5. Circular dichroism measurements of alamethicin in the presence of OE7 polymersomes

The ellipticity at 222 nm (θ₂₂₂) was determined for various molar ratios of alamethicin to OE7 and subtracted from the corresponding ellipticity for a solution of alamethicin alone (θ_222,Alam). For all measurements, the OE7 concentration was 42 μM.

Figure 6. Micropipette delivery of alamethicin

(A) Alamethicin was allowed to diffuse freely from a micropipette placed near an OE7 vesicle. Phase contrast was achieved by loading the lumen with a sucrose solution that was equi-osmotic to the surrounding PBS buffer. The pipette contained alamethicin at 0.05 mg/ml. No phase loss was observed before disintegration of the vesicle. Instead, a dramatic collapse occurred, triggered by the loss of membrane integrity at one spot on the vesicle. (B) An OE7 vesicle was held in a micropipette and alamethicin was allowed to diffuse from a second pipette. The amount of membrane aspirated into the pipette increased with time until vesicle rupture while the radius of the vesicle remained unchanged. (The scale is the same as in A.)

Figure 7. Membrane thinning due to micropipette delivery of alamethicin

(A) Increase in the membrane projection inside the micropipette as a function of time. Zero time denotes the frame before any noticeable change was observed in the length of the membrane projection. The curve terminates at the point of rupture. (B) The increase in membrane surface area due to the increase in membrane inside the micropipette can be used to estimate the extent of bilayer thinning if we assume the hydrophobic core of the bilayer is incompressible. The thinning just prior to rupture is plotted as the percentage of initial membrane thickness vs. vesicle diameter.

Figure 8

(A) Alamethicin was allowed to diffuse locally near a PC vesicle. Loss of phase contrast was observed before vesicle disintegration. Smaller vesicles trapped inside the larger vesicle were pushed out prior to complete lysis of the large vesicle. (B) Phase loss followed by lysis of an OB18 vesicle placed in a bath of 0.8 mg/ml alamethicin. OB18 is inert to alamethicin except at very high concentrations of peptide.

Figure 9. Dynamic light scattering from 200 nm vesicles

The size distribution is plotted as mass fraction (assuming scattering from spherical particles) vs. radius. (A) The size distribution shifts to higher values after addition of alamethicin to PC vesicles. This indicates possible rupture and re-aggregation. (B) The size distribution broadens on adding alamethicin to OE7 vesicles. (C) There is very little change in the size distribution after addition of alamethicin to OB18 vesicles.

Figure 10

(A) Scattering intensity vs. wave number (Q) from neutron scattering experiments. The first minimum shifts towards larger Q (smaller length scale) with increasing alamethicin. (B) Length scale associated with the first minimum in the scattering intensity (2π/Q_min), as a function of the molar ratio of peptide to polymer.