Melittin

4.4.1.1 Melittin depletes membrane material on open silicon nitride support

The addition of the AMP melittin to planar lipid bilayers on open silicon nitride support initially led to disappearance of the multilammellar layers at the edges of the membrane patches, while the general fluorescence intensity of the membrane material decreased. A possible explanation is that the membrane deposits at the rims of patches were more accessible to melittin action because they were structurally less stable due to high membrane curvature and possible exposure of hydrophobic regions. Over time, individual pores abruptly lost their fluorescence. This can be interpreted as pore-by-pore rupture of the membrane covering individual pores. Indeed, SICM micrographs of treated pore-spanning membranes confirmed that the pores in question were no longer covered by membrane [107].

The observations can be interpreted as follows: The lipid material may have been removed by melittin. The remaining lipid material may have at some point been too little to maintain a lipid bilayer, leading to membrane rupture with the remaining material adhering to the inside of the pore. There, it would no longer have been detectable since the membrane fluorescence is quenched by the gold surface.

91 4.4.1.2 Melittin causes dye release on closed silica pores

Many AMPs are thought to act by perforating the biomembrane through introduction of local membrane disturbance, leading to an increased permeability of the membrane. These disturbances may be followed by complete membrane rupture or lysis, depending on the peptide. However, almost all peptides are observed to lyse the membrane at a certain peptide concentration [17].

This behavior could be visualized experimentally for melittin using a dye release assay on closed silica pores (3.2.3). Addition of melittin led to a heavy decrease in pyranine fluorescence in the pores, with only a minimal decrease in membrane fluorescence. This suggests that, overall, the membrane stayed intact. Thus, the used assay was able to provide evidence that melittin appears to work not through lysis, but through permeabilization of the membrane.

Higher melittin concentrations (600 nM) led to a pronounced continuous decrease in membrane fluorescence, suggesting material removal from the lipid bilayer. Curiously, it was possible to exploit the previously discussed artifact of membrane bulging (4.1.2) to study membrane changes: It could be observed that melittin addition led to a decrease of the membrane protrusions, as the membrane appeared to “sink in”. This observation provided further evidence for an increased material flow across the membrane after melittin addition.

By the time the experiment was terminated, all membranes were planar, and some had ruptured.

These observations suggest the succession of several processes: The decrease in membrane fluorescence suggests a removal of membrane material from the lipid bilayer, supporting the findings on open silicon nitride pores (4.4.1.1). The decrease in membrane bulging and the loss of pyranine fluorescence below the membrane indicate that melittin permeabilized the membrane to an extent that allowed the outflow of water or buffer components from the inflated cavities.

The apparent late stage membrane rupturing events upon melittin treatment were observed on closed silica pores similarly to open silicon nitride pores. However, the details and the precise cause of the rupture could not be determined using gold-functionalized substrates because of the quenching of the membrane dye directly on the substrate. Due to this, it could not be determined if lipid material was also removed from the substrate. This issue was addressed on AAO pores.

4.4.1.3 Melittin removes lipid from the membrane at a concentration-dependent rate on AAO support

AAO support provided a substrate with optical transparency that allowed the more detailed investigation of melittin action. Experiments on AAO support confirmed the observations

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made on silicon nitride and silica pores (see previous chapters). Additionally, it was observed that the rate of material depletion from the membrane was dependent on melittin concentration. The membrane fluorescence under influence from melittin decreased in a linear fashion at lower peptide concentrations (0.13 µM, 0.35 µM), and exponentially at a higher melittin level (0.7 µM). Assuming that loss of Texas Red DHPE fluorescence was proportional to loss of lipid material, it appears that melittin removed about half the lipid material and then ceased to exercise its function. This indicates that melittin removed lipid from the membrane at a concentration-dependent rate without destroying the membrane altogether.

4.4.1.4 Melittin concentration influences the dye inflow rate on AAO support

Interestingly, the size of the used dye did not affect translocation kinetics. Both the small dye pyranine and the bulky dye Dex70 showed comparable membrane translocation kinetics, indicating that the membrane defects by melittin cause enough large pores to allow passing of bulky molecules and small molecules alike.

Similarly to 4.4.1.3, a concentration dependence on dye translocation kinetics was observed.

While the lowest used concentration (0.13 µM) of melittin led to very little dye translocation, increasing the peptide concentration drastically increased the ability of pyranine to pass through the membrane. At 0.5 µM melittin, the maximum pyranine translocation rate was observed. The half time as well as the slope at half time decreased with increasing melittin concentrations. In contrast to magainin-2 (4.4.2.2), the decrease appeared exponential rather than linear. This indicates that the more melittin was used, the faster the dye translocation through a permeabilized membrane was possible. It also showed a cooperativity effect since a certain minimum concentration appeared to be necessary for effective permeabilization of the membrane.

4.4.1.5 Summary of melittin effects on pore-spanning membranes

The data collected from experiments with pore-spanning lipid bilayers on three different substrates (open silicon nitride pores, closed silica pores, closed aniodic aluminium pores) indicate that the antimicrobial peptide melittin is able to permeabilize the membrane and extract material from the lipid bilayer.

The observed threshold concentration for dye translocation suggests a cooperative mechanism of melittin action. It points towards the formation of large pores either as toroidal pores or barrel staves. Additionally, the translocation kinetics were not dependent on the hydrodynamic radii of the aqueous dyes, which implies that the formed pores have a certain minimum size that let all dyes pass. This is in line with reports that melittin acts on lipid bilayers by toroidal pore formation [61].

93 It was also reported that melittin may act like a detergent through the carpet mechanism [36][40][62][106][108]. Additionally, it was reported that the secondary structure of melittin seems to be responsible for its hemolytic, but not antimicrobial activity [109]. Melittin diastereomers that lost their helical structure could no longer lyse human erythrocytes but maintained their activity against Gram-positive and Gram-negative bacteria [110]. This has some implications in the context of this work. The membranes used in this work consisted of POPC, a lipid with neutral net charge. In that, they resembled eukaryotic host cell membranes and may also be susceptible to material removal through melittin. This may lead to the point where too little material is left to maintain the integrity of the membrane, so that individual pore-spanning membranes rupture. Possibly, the AMP gets depleted at low melittin concentrations as the melittin molecules stay attached to the extracted lipid material. This way, the dye translocation rate stays linear and this would explain why membrane rupture is only observed at high concentrations of melittin. To summarize, the experiments on different porous substrates using dye translocation assays showed successfully the versatility of the method. They evidence suggests that the activity of melittin may originate from two different modes of action- that of pore formation, and that of material removal.