QCM, when correlating mass changes measured by using different harmonics overtones of the sensor crystal, is able to provide information about the spatial distribution of mass change related to the sensor surface 30 — Although a few feasibility studies have been published 33 , 34 , no detailed works have used QCM to study structural aspects of membrane disruption thus far. Atomic force microscopy AFM imaging of surface-confined membranes and membrane-protein systems has been published by several authors 35 — Membrane disruption by antimicrobial peptides, however, was primarily studied on whole bacterial cells and only a few works have used model membranes 43 — 45 , which is necessary to achieve high resolution.
Furthermore, although dynamic processes such as lipid membrane formation and phase transitions have been imaged before 46 , 47 , in the case of membrane-disrupting peptides, AFM studies are limited to before-after pictures and do not attempt to follow the process in real time. Here we report results using the short peptide aurein and the longer membrane-spanning peptides that exhibit pronounced kinks associated with the proline residues maculatin and caerin.
Features exhibited by both pore-forming and membrane-disruptive carpet-like mechanisms were elucidated by correlating QCM and AFM measurements. Chloroform and methanol were purchased from Sigma-Aldrich St. Sodium chloride, potassium phosphate monobasic, and potassium phosphate dibasic were purchased from Sigma-Aldrich. Ultrapure water with a resistivity of Liposomes were prepared as previously described 42 , 48 ; in brief, lipids were resuspended in 20 mM phosphate buffered saline mM NaCl at pH 6.
The sensor crystals used were 5 MHz, AT-cut, polished quartz discs chips with evaporated gold sensor surface. Measurements at natural frequency 5 MHz were not considered due to this resonance being very sensitive to bulk solution changes and generating unreliable data. A flow-through system allowed successive application of a set of sample fluids to the sensor. Before assembling into the chamber, sensor chips were rinsed with ethanol and dried under a gentle stream of N 2 gas, after which they were placed into a 1: Subsequently, chips were thoroughly rinsed with ultrapure water and ethanol, dried, and immediately assembled into the QCM-D chamber ready to use.
One chip was used to perform at least 10 experiments. The gold surface of the chip was chemically modified in situ before liposome deposition. A clean gold crystal was treated with MPA to form a self-assembled monolayer of uniform negatively charged surface as a support for the artificial membrane This process was typically 20 min in duration, using a 1 mM solution of MPA in propanol.
Peptide-methionine (R)-S-oxide reductase
A typical experiment involved two main stages after MPA modification of the gold crystal Fig. The original data were analyzed by graphing software Origin 7. A difference in frequency between baselines 2 and 4 was used to calculate a total amount of lipid on the chip surface. The positive change in frequency between baselines 4 and 6 suggests a mass loss due to the peptide action on a phospholipid bilayer.
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QCM experiments were repeated 3—5 times for each peptide and at each concentration. Small frequency variations were observed; however, these correlated with the amount of lipid. For the same lipid mass, the peptide response was reproducible within 1 Hz, that is, within the sensitivity of the instrument. A total of 0.
Imaging was performed on regions of exposed membrane boundaries, where the underlying mica surface can be clearly distinguished. After images of the membrane have been obtained and the static nature of the membrane confirmed, peptide solution was injected without stopping imaging to maintain the timeline. The effect of peptide exposure was thus observed in real time. AFM images reveal specific morphology, which is distinguishable and recognizable for each peptide-membrane interaction.
Imaging was repeated twice for each peptide. In each case, phospholipid surfaces were prepared in situ and a stable baseline was obtained before exposure to the peptide and analysis of the effects. Data were recorded as frequency change where decrease corresponds to an increase in mass on the modified chip In each case, the dissipation parameter data not shown reflected the structural changes indicated by the frequency response, thus confirming our analysis and conclusions.
First, the effect of the three peptides on DMPC bilayers was assessed. Typical QCM traces are shown in Fig. Upon introduction into the measurement cell, the peptides interacted with the membrane immediately. Initial decrease in QCM frequency suggests mass uptake for all three peptides. For caerin, the mass uptake continued for all concentrations, proceeding to saturation. Since the final mass uptake was proportional to the concentration, this could be interpreted as depletion of peptides and the achievement of a steady state in the reacting volume.
Saturation of the membrane occurred at higher peptide concentrations. Solid trace in a , d , and g: At higher concentrations the mass removal became dominant. For aurein, the trace was somewhat different to the other two peptides: As concentration was increased, the initial mass gain moved toward net mass removal, similar to that observed for maculatin. Recording mass change using different overtones of the natural frequency of the QCM sensor chip provides a means of characterizing processes as a function of distance from the surface 30 — Although a discrete sectioning cannot be achieved, transmembrane and surface effects can be simply distinguished.
The first overtone was not used, as it senses mostly the solution and thus gives meaningless information. For caerin, the mass measured by all overtones was the same, indicating a process that has the same structural characteristics across the whole thickness of the membrane, consistent with a vertical transmembrane insertion. Maculatin initially behaved in a similar manner. However, once the insertion process changed to membrane removal, different masses were recorded.
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This suggests an initial transmembrane insertion, followed by an asymmetrical disruption mechanism once a threshold concentration was reached. Aurein, on the other hand, behaved differently: This behavior can be explained by a structural effect: Thus, even without actual material removal, the third overtone would indicate mass loss as the movement of the surface would drag a lower amount of water.
Specific and Selective Peptide-Membrane Interactions Revealed Using Quartz Crystal Microbalance
The ninth overtone, which is dissipated mostly within the membrane, senses the additional mass of the peptide on the bilayer. A summary of the overtone effects as a function of concentration is depicted as a bar chart in Fig. For caerin, identical mass changes at all four overtones indicate that transmembrane insertion was dominant, although at higher concentrations some surface adsorption might also have occurred.
Importantly, membrane removal did not occur even at high peptide concentrations. A different mechanism was observed for aurein. Concentration-independent surface association dominated the low range, with apparent material removal at lower, and mass gain at higher, overtones. Based on these results, aurein appeared to act on the surface of the membrane bilayer.
Overtone effects showing change in frequency Hz as a function of peptide concentration for DMPC bilayers. In context of the literature models of membrane disruption, we propose that caerin acts as a pore-forming peptide by inserting across the membrane. Aurein appeared to adopt the mechanism known as carpet, where sudden disruption occurs after a certain threshold is reached. Maculatin, however, seemed to switch from transmembrane insertion to lysis above a threshold concentration.
When comparing overtone effects Fig. This behavior suggests that when a threshold concentration within the membrane is reached, caerin is also able to form mixed micelles and thus incite material removal. This did not lead, however, to complete material removal at higher concentrations data not shown , confirming that the primary action remains by pore formation. Maculatin exhibited a different behavior: Importantly, large-scale membrane disruption, as observed for DMPC, did not occur.
The conclusion is that both aurein and maculatin are lipid-selective peptides. The dynamics of membrane activity insertion and disruption for caerin and maculatin were further examined using AFM imaging. As a three-dimensional imaging tool, AFM is able to visualize the morphological changes of the membrane and thus identify the differences in mechanism revealed by the QCM measurements.
Imaging was performed on DMPC membranes at the phase transition temperature, where both fluid and crystalline phases are present. The membrane appeared a featureless smooth surface, with domains of crystalline and fluid phase exhibiting different heights e. Peptide was added as follows: High-resolution imaging on the membrane surface was unable to find any pore structures. This is not unexpected due to the high mobility of the peptides and thus the pores within the membrane. This interpretation is consistent with data obtained with solid-state NMR 20 , The absence of peptides on the surface, and the lack of any visible disruption, is consistent with the proposed vertical insertion of peptides with a high likelihood of pore formation although the presence of pores could not be proven directly.
In contrast, the data obtained by addition of maculatin resulted in visible changes. A sequence of consecutive images of the membrane disruption is shown in Fig. A movie of the full-scale sequence is available as Supplementary Material. The resolution was deliberately set low, so that the large-scale disruption events could be better observed. The first two frames show the membrane before peptide injection. The domains are static; no changes can be observed.
Visible membrane disruption is apparent and starts at frame 8 and the lower, fluid phase domain is fully disrupted by the consecutive frame. This layer propagates from the membrane covered area across the surface. The time evolution of the three-dimensional morphology by AFM, together with the QCM data, provides a basis for speculation for the mechanism of antimicrobial peptide action.
The QCM results indicate maculatin incorporation into the bilayer in a transmembrane manner at lower solution phase concentrations. However, upon reaching a threshold concentration within the membrane, disruption begins. The fact that the threshold concentration has to be reached within the membrane is deduced from the curves that show significant disruption over time but indicate peptide incorporation at the beginning Fig. This is consistent with the AFM observations, where no significant membrane disruption was observed before increasing the concentration beyond the threshold value.
We anticipate the threshold concentration required for disruption to be a function of temperature, and since the QCM and AFM operate over different temperature ranges and the concentrations in the AFM liquid cell are estimates due to the uncontrollable evaporation of the liquid, it is not feasible to directly—or quantitatively—compare these threshold values. AFM revealed, in accordance with QCM results, that lysis is not instantaneous and proceeds from the edges of the membrane inward. This suggests that the mechanism proceeds from pore formation to a weak detergent effect, without an immediate loss of the entire membrane integrity.
The thin layer of debris remaining on the surface is indicative of mixed micelle formation. One can hypothesize that inhomogeneities or phase boundaries within the membrane might serve as points of micelle detachment, whereas in the bulk of the membrane the lateral pressure prohibits such processes to happen. This preference can be explained in terms of the variations in surface tension at such sites. As composition does also have a strong effect on surface tension, this may underlie the observed lipid specificity.
Specific and Selective Peptide-Membrane Interactions Revealed Using Quartz Crystal Microbalance
In contrast, in the lipid mixture, aurein interacts via partial insertion Fig. This implies that in the lipid mixtures, maculatin may form pores. Representation for the interactions of aurin a and b , maculatin c and d , and caerin e and f peptides with supported phospholipids membranes: We found that caerin, the longest of the three peptides, incorporated into phospholipid bilayers in a transmembrane manner, a mechanism that was qualitatively independent of concentration and lipid composition. Maculatin, however, switched from transmembrane incorporation to membrane lysis in a concentration-dependent manner while also exhibiting specificity toward phospholipids.
Aurein associated with the upper leaflet of the membrane and acted in a carpet-like manner, also showing threshold behavior. Notably, the threshold concentration changed for different phospholipid compositions. In light of these observations, we suggest that specificity-enhanced mutants of maculatin and aurein could serve as future drug candidates and offer enormous potential for new antibiotics. To view all of the supplemental files associated with this article, visit www.
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