Interactions of Membrane Active Peptides with Planar Supported

Mar 14, 2012 - Monitoring and Quantifying the Passive Transport of Molecules Through Patch-Clamp Suspended Real and Model Cell Membranes. Pierluca Mes...
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Interactions of Membrane Active Peptides with Planar Supported Bilayers: An Impedance Spectroscopy Study Janice Lin,† Jennifer Motylinski,† Aram J. Krauson,‡ William C. Wimley,‡,* Peter C. Searson,†,* and Kalina Hristova†,* †

Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States Department of Biochemistry, Tulane University, New Orleans, Louisiana 70112, United States

‡

ABSTRACT: Membrane active peptides exert their biological effects by interacting directly with a cell’s lipid bilayer membrane. These therapeutically promising peptides have demonstrated a variety of activities including antimicrobial, cytolytic, membrane translocating, and cell penetrating activities. Here, we use electrochemical impedance spectroscopy (EIS) on polymer-cushioned supported lipid bilayers constructed on single crystal silicon to study two pairs of closely related membrane active peptides selected from rationally designed, combinatorial libraries to have different activities in lipid bilayers: translocation, permeabilization, or no activity. Using EIS, we observed that binding of a membrane translocating peptide to the lipid bilayer resulted in a small decrease in membrane resistance followed by a recovery back to the original value. The recovery may be directly attributable to peptide translocation. A nontranslocating peptide did not decrease the resistance. The other pair, two membrane permeabilizing peptides, caused an exponential decrease of membrane resistance in a concentration-dependent manner. This permeabilization of the supported bilayer occurs at peptide to lipid ratios as much as 1000-fold lower than that needed to observe effects in vesicle leakage assays and gives new insights into the fundamental peptide−bilayer interactions involved in membrane permeabilization.

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INTRODUCTION Membrane active peptides are small, water-soluble polypeptides that have a variety of biologically important effects arising from their interactions with cell membranes. Classes of membrane active peptides include membrane translocating peptides that can pass through membranes without destabilizing them and membrane permeabilizing peptides with antimicrobial activity, which include some of the best studied membrane active peptides.1−6 These peptides can have numerous applications in biotechnology. For instance, the membrane translocating peptides, which can pass through membranes without causing leakage, could have important utility as drug delivery vehicles. Recently, we described the identification of a family of membrane translocating peptides, selected from a combinatorial peptide library using high-throughput screening methods.7 These peptides pass through synthetic lipid bilayers and cellular membranes without the need for a metabolic energy source. Translocating peptides are distinctly different from the wellknown cell penetrating peptides such as the HIV TAT peptide and polyarginine, which do not translocate spontaneously across bilayers but rather are taken up by cells via endocytosis.7,8 Thus, translocating peptides could be very useful in the delivery of drugs that are not membrane permeable into cells. Yet, the mechanism of spontaneous translocation is not well understood. By virtue of their propensity to bind preferentially to anionic bacterial membranes and to compromise their permeability © 2012 American Chemical Society

barrier, the membrane-destabilizing antimicrobial peptides have broad spectrum sterilizing activity against Gram positive and Gram negative bacteria as well as against fungi.1 At the same time, they have limited toxic effects on mammalian membranes and thus could have utility as broad spectrum antibiotics.4 However, despite numerous studies, there are still unanswered questions pertaining to their mechanism of action.9−11 In part, the lack of understanding of membrane active peptides is due to limited experimental tools to interrogate the molecular mechanisms behind their activity. Bilayer destabilization is traditionally studied by vesicle leakage assays that examine the leakage of polar compounds across lipid vesicle membranes.12 However, these techniques have numerous limitations. For example, the ability to detect membrane disruption is limited by the size of the analyte. Commonly used probes include citrate chelated-Tb3+, dipicolinic acid (DPA), dithionite, fluorescent probes such as carboxyfluorescein, and dextrans. All of these molecular probes have diameters of 10 Å or more, thus requiring an extensive disruption of the bilayer structure for leakage to be observed. At the high peptide concentrations typically needed to observe leakage of these analytes, the fundamental intermolecular interactions can be obscured. Furthermore, most vesicle-based assays report on the Received: January 18, 2012 Revised: March 13, 2012 Published: March 14, 2012 6088

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Table 1. Peptide sequences and their membrane binding characteristicsa peptide Membrane nonpermeabilizing TP2 (Translocating) ONEG (Inactive control) Membrane permeabilizing FSKRGY FSKRGY-L3

ΔG (kcal mol−1)

fraction bound

PLIYLRLLRGQF−CONH2 PLGRPQLRRGQF−CONH2

−3.0 +1.0

2 × 10−4 (POPC+POPG) 2 × 10−6 (POPC+POPG)

RRGFSLKLALAKDGWALMLRLGYGRR−CONH2

−7.4 −6.4 −6.9 −5.9

0.19 0.05 0.14 0.03

sequence

RRGFSLKLALLKDGWLLLRLGYGRR−CONH2

(POPC+POPG) (POPC+Ch) (POPC+POPG) (POPC+Ch)

Partitioning (binding) free energies ΔG were measured by either equilibrium filtration or fluorescence titration spectroscopy as described in the text. Because partitioning of ONEG was unmeasurable, we used the Membrane Protein Explorer31 to estimate its partitioning by comparing its interfacial hydrophobicity to that of TP2. For the membrane permeabilizing peptides, we measured binding to both POPC+POPG membranes and POPC+Ch membranes. For each peptide we show the fraction of bound peptide under the conditions of the impedance spectroscopy experiments (Fraction bound), calculated from ΔG, as described elsewhere.28

a

to increase the hydrophobicity of the peptide. All the studied peptides are expected to partition into the bilayer interface and to not span the membrane.7,27 Membrane active peptides were synthesized and purified (>90%) by Biosynthesis, Inc. (Lewisville, TX). The interactions of these peptides were examined with asymmetrical, supported lipid bilayers representative of bacterial and mammalian cellular membranes, which were prepared via Langmuir−Blodgett (LB) deposition and vesicle fusion onto single crystal silicon. Representative bacterial lipid bilayers (POPC+POPG bilayers) were composed of a 5.9 mol % (1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (PEG-2k) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) lower leaflet and a 5 mol % 1-palmitoyl-2-oleoylsn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 95 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) upper leaflet. Representative mammalian lipid bilayers (POPC+Ch bilayers) were composed of the same 5.9 mol % PEG-2k and DPhPC lower leaflet and a 25 mol % cholesterol (Ch) and 75 mol % POPC upper leaflet. We found previously that utilizing DPhPC lipids in the lower leaflet increases the baseline membrane resistance when a physiologically relevant upper leaflet, containing POPC, POPG, or cholesterol, is constructed on top.25 All lipids were purchased from Avanti (Alabaster, AL). Measurement of Peptide Binding. Mole fraction partition coefficients were calculated as K X = [P] b / ([L])+[P]b/[P]w/([W]+[P]w), where [P]b and [P]w are the molar concentrations of peptides in the bilayer and water phases respectively and [L] and [W] are the molar concentrations of lipid and water. The partition coefficients were measured using two well established methods,28−30 and the free energy of partitioning (binding) was calculated as ΔG = −RT ln Kx . For TP2 and ONEG, we used an equilibrium filtration method, which is optimal when the peptides do not bind very strongly to membranes. Briefly, peptides were incubated with 50−100 mM large unilamellar vesicles (LUVs) for one hour, followed by centrifugation of the solution for 10 min at 5000 g in a 100 000 molecular weight cutoff centricon filtration device (Millipore), which filtered about 25% of the total volume. Peptide concentrations in the filtrate (free peptide) and in the original solution (free + bound peptide) were measured by optical absorbance. We measured the binding of TP2 but found that ONEG binding to membranes was too weak to be measured reliably. Therefore, we predicted the difference (ΔΔG) of interfacial membrane

dissipation of analyte concentration gradients and thus report only on the cumulative effects of membrane active peptides on bilayer structure and integrity. Electrochemical impedance spectroscopy (EIS) is a tool that can be used to study the effects of membrane active peptides in bilayers, yielding direct measurements of bilayer resistance and capacitance. This technique is highly sensitive to the passage of small ions (Na+, K+, Cl−, etc.) and can thus detect subtle changes in bilayer structure. Furthermore, EIS has real-time temporal resolution ranging from about one minute to one hour, such that transient bilayer effects can be monitored. Whereas EIS has been used widely in conjunction with tethered lipid bilayers on gold,13−16 we have recently established a reproducible and robust surface supported polymer-cushioned bilayer platform on silicon that ensures the fluidity of the supported lipid bilayers.17−25 We have further shown that we can vary the lipid composition of this supported bilayer platform without compromising its electrical response.25 Here, we use EIS to compare the electrical response of membranes comprising different lipid compositions to two pairs of peptides with different membrane activities. The first pair includes two nonpermeabilizing peptides: one peptide that translocates across membranes without causing destabilization and a related control peptide that has no membrane activity. The second pair includes two membrane permeabilizing peptides. We show that EIS reveals details of molecular mechanism that are not accessible in lipid vesicle experiments.

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MATERIALS AND METHODS Peptides and Vesicles. Two pairs of membrane active peptides identified in previous vesicle leakage studies were investigated (Table 1). The membrane nonpermeabilizing pair included (1) Translocating Peptide TP2 (PLIYLRLLRGQF− CONH2), which was recently identified from a combinatorial library using an orthogonal high-throughput screen that selected for peptides that pass through lipid bilayers without causing leakage of solutes,7 and (2) Observed NEGative peptide ONEG (PLGRPQLRRGQF−CONH2), identified in the same screen as a peptide that does not translocate or cause leakage of solutes. The membrane permeabilizing pair included (1) FSKRGY (RRGFSLKLALAKDGWALMLRLGYGRR− CONH2), identified from a combinatorial peptide library using a high-throughput screen that is sensitive to Tb3+ leakage26 and (2) a very similar peptide, FSKRGY-L3 (RRGFSLKLLLKDGWLLLRLGYGRR−CONH2), in which three hydrophilic amino acids were substituted with leucines 6089

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grade, Fisher, Waltham, MA). The two leakage-inducing peptides were dissolved in 18 MΩ water (Millipore, Billerica, MA). Solutions of known concentrations of TP2 and ONEG were prepared by dissolving known weights of peptide. Concentrations of FSKRGY and FSKRGY-L3 were determined by UV−vis absorbance using an extinction coefficient of 6800 M−1 cm−1. Peptides were dissolved and used the same day. EIS was performed using a Solartron 1286/1255 Electrochemical Interface/Frequency Response Analyzer. The custom electrochemical cell was configured with a platinum counter electrode and an Ag/AgCl (3 M NaCl) reference electrode. Impedance measurements were made at 0 V over a frequency range from 0.1 to 105 Hz. Each measurement took approximately 2.5 min; scans were repeated at this frequency range at 0 V to obtain quasi-real time data. Prior to the introduction of the peptide, the bilayer impedance was measured to obtain baseline values of membrane resistance and capacitance (Figure 1). For all experiments, the initial

binding for TP2 and ONEG with the Membrane Protein Explorer31 and used this predicted difference to estimate ΔG for ONEG from the measured value for TP2. Control experiments showed that ONEG and TP2 pass through the filter with little or no loss of peptide when lipid is absent and that lipid vesicles do not pass through at all. Because FSKRGY and FSLRGY-L3 binding was much stronger and because they have a tryptophan residue, we measured binding using tryptophan fluorescence titration, a method that is optimal under these conditions.28 In all cases, we used the same buffer (10 mM sodium phosphate and 100 mM potassium chloride) and lipid composition as used in the impedance experiments. Fractional binding of peptides was calculated using the total lipid concentration in the system. Silicon Substrate Preparation. Single side polished, ntype silicon wafers (⟨111⟩, ρ = 0.001 − 0.005 Ω cm, Silicon Quest International, San Jose, CA) were cleaned by sonication for 15 min first in isopropyl alcohol, followed by acetone, and then again in isopropyl alcohol. Each silicon wafer was rinsed thoroughly in deionized water prior to surface treatment in a 30% (v/v) hydrogen peroxide, 70% (v/v) sulfuric acid solution for 1 h. The silicon wafer was then rinsed thoroughly in deionized water and used within 1 h. The silicon wafer was submerged vertically into a Langmuir trough (Nima Technologies, England) with the polished side facing the open trough for LB deposition. Langmuir−Blodgett Deposition. LB deposition was used to form the lower leaflet of the bilayer on the silicon substrate. DPhPC and 5.9 mol % PEG-2k lipids were premixed in chloroform to achieve a total concentration of 1 mg mL−1. 40 μL of the lipid solution was deposited at the air−water interface on the Langmuir trough. After allowing a minimum of 30 min for the chloroform to evaporate, the lipids were compressed to a surface pressure of 32 mN m−1. The silicon wafer was then raised vertically out of the trough at a rate of 15 mm min−1 allowing the deposition of a PEG supported lipid monolayer on the polished side of the silicon wafer.32 A custom Teflon electrochemical cell was assembled on top of the monolayer with the working electrode area (0.814 cm2) defined by an O-ring. An ohmic contact was formed by first etching a small region at the edge of the wafer with 25% (v/v) hydrofluoric acid for 1 min to remove the native oxide and then applying indium gallium eutectic with a Q-tip. Vesicle Fusion. One-hundred nanometer diameter vesicles, made of either POPC+POPG or POPC+Ch, were prepared from a 1 mg mL−1 lipid solution by first evaporating the chloroform under a stream of nitrogen. The lipids were then further dried under vacuum for a minimum of 1 h. A buffer solution of 10 mM sodium phosphate and 100 mM potassium chloride was added to yield a lipid vesicle solution with a final concentration of 1 mg mL−1. The solution was vortexed to ensure that all the lipids were suspended in buffer. The vesicles were extruded through a 100 nm polycarbonate membrane a minimum of ten times. The PEG supported lipid monolayer was then incubated with 450 μL of extruded vesicles for at least 1 h to allow for vesicle rupture and fusion to occur and complete the lipid bilayer. An additional 10 mL of buffer was added to the electrochemical cell prior to impedance measurements. Experiments were performed without removing the excess vesicles to achieve targeted peptide to lipid ratios. Electrochemical Impedance Spectroscopy (EIS). For EIS studies, the translocating peptide, TP2, and the inactive control peptide, ONEG, were dissolved in methanol (ACS

Figure 1. Typical (a) impedance spectrum and (b) phase angle for a supported bilayer before and after (t = 3.5 min) addition of FSKRGYL3 at a molar concentration ratio of 1:5000. The bilayer was formed with a POPC + 5% POPG upper leaflet and a DPhPC + 5.9% PEG-2k lower leaflet. The symbols represent the measured impedance and the solid lines are nonlinear least-squares fits to a model consisting of two RC loops in series with a single resistor. The capacitive response in the frequency range from about 10 Hz to 10 kHz is due to the membrane whereas the response at lower frequencies (0.1 to 1 Hz) is due to the silicon support. The inflection point between the two capacitive responses in the Bode plot indicates the membrane resistance. For the bilayer we obtain: Rs = 38.5 Ω cm2, Cm = 0.86 μF cm−2, and Rm = 43.6 kΩ cm2. After peptide addition, we obtain: Rs = 38.7 Ω cm2, Cm = 0.82 μF cm−2, and Rm = 18.3 kΩ cm2.

resistance was 25 000 ± 19 000 Ω cm2 (SD, N = 24). Whereas the value of the initial resistance varied, it was always sufficiently high to measure the effect of the peptide.21 Furthermore, there was no correlation between the initial resistance and the measured bilayer response to the peptides. The capacitance values were 0.8 to 1.0 μF/cm.2 6090

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95:5 POPC/POPG. This was the only lipid composition examined with this peptide as its behavior does not depend on the lipid composition.7 We tested increasing concentrations of peptide until reaching a total concentration of 5 mol % relative to the fixed lipid concentration (i.e., 1 peptide per 20 lipids total in the EIS cell), which causes small but statistically significant changes in bilayer electrical impedance (p < 0.001 from a Chi squared test34). Because TP2 binds weakly, a 5 mol % total concentration is equivalent to about 1 peptide bound per 105 lipids (Table 1). This peptide has been shown to translocate across POPC and POPC+POPG bilayers without causing measurable leakage of contents from vesicles.7 On introduction of 5 mol % of the translocating peptide to POPC +POPG bilayers in EIS experiments, there was a reproducible 20% decrease in bilayer resistance, followed by a recovery to the initial bilayer resistance within 20 min (Figure 2). This is the same time scale as membrane translocation in vesicles and even across living cell membranes.7

The peptide was injected into the electrochemical cell and, to ensure the peptide was evenly distributed throughout the system, the solution was mixed via pumping ∼10 times with a 1 mL micropipet. Impedance measurements were taken for ∼2 h after introduction of the peptide or until the bilayer’s impedance had stabilized. All experiments were performed at room temperature and in the dark to prevent photoeffects. Using a nonlinear least-squares fit (ZPlot, Scribner Associates, Southern Pines, NC), impedance spectra were fit to a circuit consisting of two RC loops in series with a single resistor to extract values for the membrane resistance, Rm, and capacitance, Cm. Details for the fitting procedure can be found in previous publications.21,22,25 The time-dependent membrane resistance was normalized to the initial resistance, measured prior to the introduction of peptides. Membrane capacitances are reported as absolute values.

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RESULTS AND DISCUSSION In this work, we used electrical impedance spectroscopy (EIS) to study two pairs of closely related peptides, ONEG/TP2 and FSKRGY/FSKRGY-L3, which exhibited distinctly different effects on bilayers in vesicle-based assays (Table 1). The bilayers that were constructed on silicon exposed upper lipid leaflets composed of POPC+POPG, and in some cases, POPC +Ch.7,26 POPC+POPG was used because the high-throughput vesicle leakage assays developed for the selection of these peptides utilized POPC and POPG.7,26 As shown previously, the leakage activity of FSKRGY was affected by lipid composition,27 whereas ONEG/TP2 translocation activity was not.7 Thus, the membrane destabilizing peptides FSKRGY and FSKRGY-L3 were characterized in both POPC+POPG and POPC+Ch bilayers. Membrane Binding. To assess the concentration of peptides bound to the supported bilayers, we measured free energies of peptide partitioning into 95:5 POPC:POPG LUVs and into 75:25 PC:Ch LUVs, as described in Materials and Methods. The results are shown in Table 1. TP2 and ONEG bound weakly to POPC+POPG LUVs. In fact, we could not measure ONEG binding because it was too weak. However, predictions made based on its hydrophobicity using the interfacial hydrophobicity scale31 indicate that ONEG binding should be 4 kcal/mol weaker than that of TP2, whose partitioning free energy is ΔG = −3.0 kcal/mol (Table 1). The binding of FSKRGY and FSLRGY-L3 was much stronger because they are more hydrophobic (Table 1). Although FSLRGY-L3 was synthesized to be more hydrophobic than FSKRGY, its binding was slightly weaker, a finding which is not surprising as hydrophobicity is not the only determinant of binding affinities.33 Nonetheless, they both bound well (ΔG ≈ −7.0 kcal/mol) to POPC+POPG bilayers. For these two peptides, we also measured binding to POPC LUVs containing 25 mol % cholesterol (a composition that mimics that of mammalian cell membranes). Binding was weaker to cholesterol containing membranes but was still significant. The difference in ΔG between FSKRGY and FSKRGY-L3 was the same in the two lipid compositions. On the basis of these binding measurements, in Table 1 we show the calculated fraction of peptide bound under the conditions of the impedance spectroscopy experiments. Effect of the Translocating Peptide TP2 on the Electrical Properties of Bilayers. The first of the four peptides we investigated via EIS was the translocating peptide TP2. The lipid composition of the upper bilayer leaflet was

Figure 2. Effect of the translocating peptide TP2 at 5 mol % on (a) the resistance and (b) capacitance of a representative POPC+POPG lipid bilayer with time. Values shown are experimental means and standard deviations (N = 3). TP2 effected an immediate ∼20% drop in bilayer resistance and then a gradual recovery up to the original bilayer resistance within ∼20 min. The bilayer capacitance was approximately constant at ∼0.85 μF cm−2 indicating that TP2 had no effect on the thickness of the bilayer.

The initial capacitance of the bilayers we used in these studies varies between bilayer preparations over the relatively narrow range of 0.8 to 1.0 μF/cm2. On the basis of the parallel plate capacitor model (C = εε0/d) and assuming ε = 4 for a hydrated, fluid phase bilayer, we calculate a bilayer thickness of 3.6−4.5 nm from these initial capacitances, which is the range expected for an ideal POPC+POPG bilayer.25 This result suggests that here we are probing nearly ideal bilayers. We are most interested in the effect of the peptides on the capacitance (i.e., bilayer thickness) rather than in the absolute capacitance of a particular bilayer. In Figure 3, the capacitance does not change measurably upon the addition of peptide and so we 6091

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translocation occurs in vesicle systems.7 We propose that the recovery of resistance is due to peptide translocation such that the peptide equilibrates between the two lipid monolayers and the two water reservoirs. This suggests that the initial disruption in lipid packing is due to asymmetrically distributed peptides (because of a transbilayer imbalance of charge, mass or surface tension) but that the same peptides do not disrupt the bilayer as much when symmetrically distributed. This idea has been proposed to explain the transient burst of leakage that occurs when some leakage inducing peptides (e.g., antimicrobial peptides) are added to lipid vesicles.11 However, the recovery of bilayer integrity upon dissipation of peptide asymmetry has rarely, if ever, been experimentally validated or observed previously. Effect of the Peptide FSKRGY on the Electrical Properties of Bilayers. Next, we performed EIS experiments with POPC+POPG membranes in the presence of FSKRGY, which was selected from a combinatorial library specifically for its ability to cause large scale permeabilization of POPC + POPG vesicles.26 In vesicles, 1 mol % FSKRGY (1 peptide per 100 lipids) caused measurable but incomplete leakage of polar solutes, including small dextrans, indicating significant structural disruption of the bilayer.26 However at these concentrations, FSKRGY also caused vesicle aggregation and bilayer fusion,27 making a detailed molecular interpretation of the leakage mechanism essentially impossible. In EIS experiments with 1 mol % FSKRGY, the POPC+POPG bilayers were disrupted so severely that the measurements recorded only the response of the silicon support. This highlights the extraordinary sensitivity of EIS. We observed the same effect in EIS experiments when adding the detergent Triton-X100 at 18 mM (0.8 × critical micelle concentration) (results not shown). We therefore decreased the concentration of FSKRGY until we could observe its effect on bilayers by EIS and found that peptide concentrations of 0.01 mol % (1 peptide per 10 000 lipids) or lower were appropriate for EIS studies. Figure 4 shows the results for total peptide concentrations of 0.0025, 0.005, and 0.01 mol % FSKRGY in POPC+POPG membranes. On the basis of the bound fraction calculations shown in Table 1, these concentrations correspond to 1 bound peptide per 5 × 104 to 2 × 105 lipids. This is similar to the bound concentration of the TP2 peptide allowing a direct comparison between TP2 and FSKRGY. Upon addition of FSKRGY, the membrane resistance decreased with a half-time of a few minutes, reaching a steady state value within 5−10 min (Figure 4). This is about the same time scale as leakage from vesicles27 suggesting that the same physical principles could be applicable here. The steady state resistance decreases with increasing peptide concentration, and at 0.01 mol % peptide is only slightly above the series resistance. The decrease in membrane resistance (R) follows an exponential decay as described by:

Figure 3. Effect of ONEG at 5 mol % on (a) the resistance and (b) capacitance of a representative POPC+POPG lipid bilayer with time. Values shown are experimental means and standard deviations (N = 3). Introduction of 5 mol % of this peptide to a POPC+POPG bilayer did not result in a statistically significant change in resistance (p = 0.07 from a Chi squared test34). From the constant capacitance, we infer that the area-averaged thickness of this bilayer did not change with time in the presence of this peptide.

conclude that TP2 does not alter the bilayer thickness, even at 5 mol % peptide. Effect of the Inactive Peptide ONEG on the Electrical Properties of Bilayers. The peptide ONEG is used here as a validated inactive control peptide.7 ONEG was selected from the same library as TP2 for its lack of membrane translocation and lack of membrane permeabilization. Figure 3 shows the effect of ONEG addition on the normalized resistance and capacitance of bilayers having POPC+POPG lipids in the upper leaflet. Upon the introduction of 5 mol % ONEG, we did not see a statistically significant change in resistance (p = 0.07 from a Chi squared test34). No change was observed in the membrane capacitance after ONEG addition, either, which remained at 0.95 ± 0.05 μF cm−2, indicating that there were no changes in bilayer thickness. Yet, the fact that the fractional binding of ONEG is 100 fold lower than TP2 (Table 1) means that we cannot conclude that its bilayer perturbation per peptide is less than TP2 on a per molecule basis. Molecular Mechanism of Peptide Translocation. In vesicle-based assays and in biological assays, neither TP2 nor ONEG disrupt or permeabilize bilayers,7 consistent with our impedance spectroscopy observations. The primary difference between TP2 and ONEG is that TP2 can translocate across bilayers and ONEG cannot.7 Vesicle studies provide no information about how translocation might occur, but impedance spectroscopy results suggest a mechanistic explanation. After the initial interactions of TP2 with the outer monolayer, there is a measurable decrease in membrane resistance suggesting that the peptide disturbs the lipid packing just enough to cause a slight increase in ion flux through the membrane. However, bilayers with TP2 recover their full initial resistance on the same time scale (half time of ∼10 min) as

R = R f + (R 0 − R f )exp( −t /τ)

(1)

where the initial normalized membrane resistance prior to the introduction of peptide is R0, Rf is the membrane resistance at steady state after peptide addition, t is time, and τ is the time constant. The time constants, τ, obtained by fitting the measured membrane resistance to eq 1 are shown in Table 1 and in Figure 8. Although the changes in time constants are small, the changes indicate that the decrease in membrane resistance is slower with lower peptide concentration suggesting a slight cooperativity. The membrane capacitance did not 6092

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Figure 5. Effect of FSKRGY at 0.005 and 0.0067 mol % on (a) resistance at 100 min and (b) capacitance with time of a representative POPC+Ch bilayer. POPC+POPG membrane values are provided as a reference (shown in red). Values shown are experimental means and standard deviations (N = 3). Higher concentrations of the peptide were required to achieve the same effect on the membrane resistance in POPC+Ch bilayers as compared to POPC+POPG bilayers, by ∼1.5 times. The POPC+Ch membrane capacitance did not change over time, and was slightly lower than the capacitance of the POPC+POPG membranes.

Figure 4. Effect of FSKRGY at 0.0025, 0.005, and 0.01 mol % on (a) the resistance and (b) capacitance of a representative POPC+POPG bilayer with time. Values shown are experimental means and standard deviations (N = 3). An immediate decrease in resistance that followed exponential decay kinetics was observed regardless of peptide concentration. The membrane capacitance did not change after introduction of the peptide to the POPC+POPG bilayer suggesting that FSKRGY does not disrupt the overall structure of the bilayer.

change after introduction of FSKRGY to the bilayer regardless of concentration (Figure 4). This suggests that FSKRGY, whereas it is able to create new pathways that result in ion permeation across the bilayer, does not disrupt the overall structure of the bilayer at these concentrations and does not cause a significant change in the thickness of the bilayer. In vesicle experiments, the activity of FSKRGY depends on the lipid composition.27 The peptide is a potent antimicrobial peptide and is much more active in bacteria-like membranes (containing anionic POPG lipids and no sterols) than in mammalian-like membranes (composed of POPC and cholesterol).27 We therefore examined the electrical response of representative mammalian POPC+Ch membranes (POPC with 25 mol % cholesterol in the upper leaflet) in the presence of this peptide (Figure 5). The effect of FSKRGY was similar in POPC+POPG and POPC+Ch membranes, but the decrease in resistance was smaller and the time constant was larger in mammalian membranes for a particular peptide concentration. These observations are consistent with those made in vesicle systems.11 Effect of the FSKRGY-L3 Peptide on the Electrical Properties of Bilayers. FSKRGY-L3, a variant of FSKRGY, is very similar but somewhat more hydrophobic. FSKRGY-L3 was less active than FSKRGY in EIS experiments, and so we investigated its activity at higher concentrations: 0.01, 0.02, and 0.05 mol % in POPC+POPG bilayers (Figure 6). Under these conditions, we had 1 peptide bound per 2 × 103 to 4 × 104 lipids. Upon addition of FSKRGY-L3, the POPC+POPG membrane resistance decreased exponentially with a half-time of a few minutes. This behavior is fundamentally the same as for FSKRGY. The exponential time constants (eq 1), shown in Table 1 and summarized in Figure 8, are also similar to those

for FSKRGY and decrease with increasing peptide concentration. For FSKRGY-L3, the membrane capacitance showed a small but reproducible decrease corresponding to a small increase in bilayer thickness which can be attributed to the binding and insertion of the more hydrophobic FSKRGY-L3 into the bilayer. We also studied the response of a representative mammalian (POPC+Ch) bilayer to FSKRGYL3 (Figure 7). The general features of the response were similar, except that the bilayer effects of FSKRGY-L3 in POPC +Ch membranes are smaller than in POPC+POPG membranes, consistent with our observations with FSKRGY. To estimate the membrane-destabilizing potencies of FSKRGY or FSKRGY-L3, we assumed that the peptides are forming discrete short circuit pathways in the bilayers, and we calculated the normalized conductance per peptide gpep (S) as: g pep =

Y 1 = m R f n pep n pep

(2)

where Ym is the membrane admittance (the inverse of the final membrane resistance, Rf), and npep is the peptide density (cm−2), calculated assuming an area per lipid = 70 Å2. The conductance values calculated per peptide are on the order of 0.1 to 0.2 pS (Figure 9). These values are only 1 to 2 orders of magnitude lower than typical values reported for discrete channels such as gramicidin, which are commonly on the order of 1 to 10 pS.24,35−37 Because FSKRGY and FSKRGY-L3 likely do not form discrete channels across bilayers,9 this result suggests that FSKRGY and FSKRGY-L3 are causing significant disruption of the lipid bilayer structure or lipid packing to allow ion movement. The fact that resistance changes are observed at 6093

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Figure 7. Effect of FSKRGY-L3 at 0.04, 0.02, 0.1, and 0.2 mol % on representative POPC+Ch bilayer (a) resistance at 100 min versus concentration and (b) capacitance with time. Values shown are experimental means and standard deviations (N = 3). POPC+POPG membrane values (shown in red) are provided as a reference. In the POPC+Ch bilayer, the membrane resistance decreased in the presence of the peptide in a similar manner following exponential decay kinetics, however 5 to 10 times the concentration was required to achieve the same decrease in resistance. The membrane capacitance of POPC+Ch bilayers did not vary much with concentration or time.

Figure 6. Effect of FSKRGY-L3 at 0.01, 0.02, and 0.05 mol % on (a) the membrane resistance and (b) capacitance of a representative POPC+POPG lipid bilayer with time. Values shown are experimental means and standard deviations (N = 3). Similar to the results with FSKRGY, the resistance also decreased here following exponential decay kinetics, however at concentrations over an order of magnitude higher. With increasing peptide concentration, we observed a corresponding decrease in time constant, τ, from ∼6.5 min to ∼2.5 min. A slight increase in membrane thickness, inferred from the slight decrease in membrane capacitance during the first 7 min, may be attributed to the initial peptide binding and organization at the bilayer interface.

very low peptide-to-lipid ratios combined with the fact that membrane capacitance is not changed significantly by the peptides indicates that the effects of the peptide on the bilayer are highly localized. In Figure 9, we show that the membrane-destabilizing potencies of FSKRGY and FSKRGY-L3 per bound peptide are higher in POPC+Ch bilayers compared to POPC+POPG bilayers. It is well-known that leakage of vesicle contents due to cationic membrane-permeabilizing peptides is reduced in the presence of cholesterol and in the absence of anionic lipids.10,11 Here, we show this to be true for FSKRGY and FSKRGY-L3 as well (Table 2). It has been speculated that this reduction in activity is either due to reduced peptide binding or due to differences in the material properties of POPC+POPG and POPC+Ch bilayers. Here, we observe that, at the low peptide concentrations assayed by EIS, the difference in overall activity observed in POPC+Ch bilayers, as compared to POPC+POPG bilayers (part A of Figure 5 and part A of 7), is explained entirely by the difference in binding. In fact, we measure higher activity per bound peptide in POPC+Ch bilayers, as compared to POPC+POPG bilayers (Figure 9). We note a decrease in the efficiency of the peptides with increasing concentration (Figure 9). On a log−log plot, this decrease appears linear both in POPC+POPG bilayers and in POPC+Ch bilayers. A similar decrease in efficiency per peptide with concentration has been observed in vesicle leakage assays.11 This effect is likely associated with peptide selfassociation in membranes and indicates that the activity of the

Figure 8. Time constant τ vs concentration from exponential decay fits to FSKRGY (triangles) and FSKRGY-L3 (circles) kinetics for POPC +Ch (filled symbols) and POPC+POPG (open symbols) bilayers. The time constant τ decreases in an exponential fashion with increasing peptide concentration for FSKRGY-L3; the dependence for FSKRGY is not so obvious.

larger multimers is smaller, per peptide, than the activity of monomers or smaller aggregates. Mechanism of Bilayer Destabilization by MembranePermeabilizing Peptides at Low Concentrations. Using FSKRGY and FSKRGY-L3, we showed here that electrical impedance spectroscopy is sensitive to changes in bilayer electrical properties that occur at peptide concentrations that are at least 3 orders of magnitude smaller than the lowest concentrations needed to cause analyte leakage in vesicle studies.11 Thus, by using EIS, we are examining peptide-bilayer interactions in a concentration regime that is essentially infinite dilution compared to vesicle-based assays, providing access to fundamental peptide-bilayer effects that are undetectable in 6094

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disruption increases until leakage of larger analytes can occur as observed in vesicle studies. Note that these peptides are unique as they are beta-sheet-like and not α-helical like many of the known potent membrane-destabilizing peptides.27 Thus, these conclusions may not be generally valid for all membranedestabilizing antimicrobial peptides Comparison of Translocating and Membrane-Permeabilizing Peptides. Because the differences in binding between TP2 and FSKRGY were approximately matched by the differences in peptide concentrations used in the impedance spectroscopy experiments, these two peptides were studied at about the same bound peptide-to-lipid ratios. Their effects on the bilayers, which can be directly compared, are thus fundamentally very different. It appears that translocating peptides such as TP2 create a pathway for themselves through the membrane upon binding to the bilayer. The lack of substantial TP2-induced permeabilization in both vesicles and supported bilayers suggests that the arginine rich TP2 may be chaperoned during its passage across the bilayer by lipids and their phosphate-based headgroups.38 The membrane permeabilizing peptides such as FSKRGY on the other hand, create a path for ions to cross the bilayer and do not allow the bilayer to recover. Even at very low concentrations, they insert into the bilayers and continuously allow ion movement across the bilayers.

Figure 9. Normalized conductance per peptide (gpep) versus peptide mol percent for FSKRGY (triangles) and FSKRGY-L3 (circles) in POPC+POPG (filled symbols) and POPC+Ch (open symbols) lipid bilayers. The conductance was calculated according to eq 2 assuming a discrete conductance pathway for each peptide.

Table 2. Summary of EIS Resultsa concentration

approx. %Rm after 30 min

τ (min)

χ2

TP2 (translocating peptide) 5 mol % 80% (w/ recovery) ONEG (inactive control) 5 mol % ∼100%

membrane POPC +POPG POPC +POPG

FSKRGY (membrane permeabilizing) 0.0025 mol % 55% 3.9 ± 0.17

0.010

0.005 mol %

20%

0.89 ± 0.01

0.00080

0.01 mol %

5%

1.2 ± 0.05

0.0011

0.005 mol % 65% 7.4 ± 0.76 0.0067 mol % 10% 0.71 ± 0.03 FSKRGY-L3 (membrane permeabilizing) 0.01 mol % 25% 6.6 ± 0.66

0.075 0.000066 0.069

0.02 mol %

20%

2.9 ± 0.21

0.021

0.05 mol %

10%

2.5 ± 0.10

0.0055

0.02 mol % 0.04 mol % 0.1 mol % 0.2 mol %

85% 50% 35% 20%

0.14 ± 0.01 2.5 ± 0.23 1.2 ± 0.39 1.5 ± 0.03

0.093 0.029 0.050 0.00055

■

POPC +POPG POPC +POPG POPC +POPG POPC+Ch POPC+Ch

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (W.C.W.), [email protected] (P.C.S.), [email protected] (K.H.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by NSF DMR 1003441 and NSF DMR 1003411.

POPC +POPG POPC +POPG POPC +POPG POPC+Ch POPC+Ch POPC+Ch POPC+Ch

■

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a

Approximate % Rm 30 min after introduction of membrane active peptides to representative bacterial (95% POPC + 5% POPG upper leaflet and DPhPC + 5.9% PEG-2k lower leaflet) or mammalian (75% POPC + 25% cholesterol upper leaflet and DPhPC + 5.9% PEG-2k lower leaflet) lipid bilayers, and the time constant, τ, determined from exponential decay fits (if applicable). The χ2 fit parameter is also reported.

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