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Research Center Borstel, Leibniz Center for Medicine and Biosciences, Department of Immunochemistry and Biochemical Microbiology, Division of Biophysics, D-23845 Borstel, Germany
Correspondence: Address reprint requests to Dr. Thomas Gutsmann, Tel.: +49-4537-188291; Fax: +49-4537-188632; E-mail: tguts{at}fz-borstel.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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on various asymmetric planar lipid bilayers using the inner field compensation method. The results are compared to the respective ones of inner membrane potential differences 
determined from ion carrier transport measurements. Finally, the time courses of membrane capacitances and of have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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A powerful tool to investigate interactions between membranes and peptides are symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method (1972). These membranes can be used to perform electrical measurements for the investigation of pore formation induced by antimicrobial peptides or membrane proteins. In the case of pore-forming peptides, from the time-resolved determination of membrane current, the size and the lifetime of membrane lesions and their current voltage (I/U) characteristics can be deduced. In those cases, in which the accumulation of the peptide at the membrane surface or the intercalation into the lipid matrix does not lead to membrane permeabilization—either in the initial phase of peptide-membrane interaction or for nonpore-forming peptides—other membrane properties such as membrane capacitance and inner membrane potential difference can be used to characterize the interaction process.
The membrane capacitance yields information about area, thickness, and composition of the lipid bilayer (Gutsmann et al., 2001
). The accumulation of peptides on the bilayer surface or their intercalation into the lipid bilayer causes changes of these parameters and with that of membrane capacitance. The interaction with charged molecules such as polycationic antimicrobial peptides may also lead to changes in the electrostatic potential profile across the bilayer (Schoch et al., 1979). The three main contributions to the intrinsic membrane potential (Fig. 1) arise from i), the charged groups of lipid molecules (as phosphate, carboxylate, amino groups) expressed at the membrane surface and generating a surface potential, which—according to Gouy and Chapman (for review see (Cevc, 1990))—decreases exponentially within a distance of a few nanometers from the membrane surface (Gouy-Chapman potential VG); ii) the dipole potential (VD) inside the lipid bilayer caused by oriented dipoles of bound water molecules (Zheng and Vanderkooi, 1992); and iii), the Born self-energy (Born potential, VB), which is the energy necessary to transfer a charge from a medium with a high dielectric constant 
(water) to one with a low (membrane) (Schoch et al., 1979). VB is smoothed near the interface by interaction with induced dipoles (Neumcke and Läuger, 1969; Parsegian, 1975). Differences of the potentials between the two bilayer leaflets composed of different lipids lead to an inner membrane potential difference.
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) is based on the ion carrier-mediated membrane permeability. Under the assumption of a trapezoidal potential profile (Fig. 1 A), the potential difference between the two corners is given by
 | (1) |
; Seydel et al., 1992). This method has, however, two major drawbacks: i), the bilayer system has to be doped with a carrier molecule, which might influence the measurement and ii), a time-resolved measurement of is not possible because the recording of a I/U curve takes several minutes.
These disadvantages can be overcome by applying the inner field compensation (IFC) method (Sokolov and Kuz'min, 1980). It is known that membrane capacitance is a function of the effective potential (Babakov et al., 1966; White, 1970). This potential, is composed of two components, the externally applied voltage U and the intrinsic potential , which is due to differences in the surface potentials VG and VD on the two sides of the bilayer (Fig. 1 B)
 | (2) |
 | (3) |
) and 
is a constant for each individual membrane. For planar bilayers according to Montal and Mueller, was found to be 
0.02 V-2 (Alvarez and Latorre, 1978). Therefore, CM adopts its minimum, when the intrinsic potential is compensated by the externally applied potential U. Thus, is also called capacitance minimization potential. Experimentally, the bilayer is excited with a voltage composed of a dc and a sinusoidal contribution yielding a capacitive current response at the second harmonic of the stimulus, which vanishes at U = - (Fig. 3, B and C) (Carius, 1976; Pohl et al., 2000, 1997, 1998).
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The cell envelope of Gram-negative bacteria consists of the cytoplasmic membrane, the peptidoglycan layer, and an additional barrier, the outer membrane (OM), which is strictly asymmetric with respect to its lipid composition (Lugtenberg and Van Alphen, 1983). Whereas the inner membrane is composed on both sides of phospholipids, the OM consists of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. LPS consists of an oligo- or polysaccharide portion covalently linked to a lipid component, termed lipid A, which anchors the molecule in the membrane (Rietschel et al., 1994). In wild-type strains, the polysaccharide portion consists of an O-specific chain and the core oligosaccharide. Rough mutant strains do not express the O-specific chain, but retain core oligosaccharides of varying length. Deep-rough mutant LPS (Re LPS) represents the minimal structure of LPS consisting of only lipid A and two 3-deoxy-
-manno-oct-2-ulosonic acid (Kdo) monosaccharides (Holst, 1999) (Fig. 2).
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20% of the total phospholipids, carries one negative charge, whereas each LPS molecule (outer leaflet) carries at least 3–4 negative charges. The resulting surface charge densities are -3.25 e0/nm2 for Re LPS from Escherichia coli strain F515 (LPS F515) and -0.31 e0/nm2 for the phospholipid mixture (PL)—resembling the inner leaflet of the OM, respectively, since the molecular cross section of an LPS F515 molecule is 1.23 nm2 and that of a diacyl phospholipid is 0.55 nm2 (determined from monolayer isotherms with a film balance).
These negative charges at the LPS molecules (Fig. 2) induce a negative surface potential at the outer leaflet of the OM, which leads to an up-concentration of polycationic antimicrobial peptides, such as polymyxin B (Schindler and Teuber, 1975; Wiese et al., 1998; Kubesch et al., 1987; Boggs and Rangaraj, 1985; Peterson et al., 1985) or cathelicidins (Gutsmann et al., 1999, 2000, 2001). Cathelicidins are endogenous peptides, which are in the front line of host defense against invading microorganisms, i.e., Gram-positive and Gram-negative bacteria, mycobacteria, and fungi, by direct physicochemical attack on the surface membranes of these pathogens. Being highly positively charged and amphiphilic, these peptides are perfectly suited to interact with negatively charged membranes, in particular with the LPS leaflet of the OM of Gram-negative bacteria, to cause in a final step disruptive changes in membrane permeability (Gutsmann et al., 2001).
The bioactive part of human cathelicidin hCAP18, an 18 kDa cationic antimicrobial peptide, is a fragment of 37 amino acids (LL37, hCAP18106–142) (Larrick et al., 1994). Respective peptides have also been found in various other mammals, such as sheep (SMAP29) (Mahoney et al., 1995) and rabbit (rCAP18) (Larrick et al., 1991). Bioactivity has been found against Gram-negative bacteria such as E. coli, Salmonella enterica serovar Minnesota and Typhimurium, and Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. There is, however, no activity of CAP18 against the Gram-negative strain of Proteus mirabilis, fungi, and multidrug resistant strains of Mycobacterium avium and Mycobacterium tuberculosis (Larrick et al., 1993). For the action of various CAP18 fragments, we have established a model of action based on the determination of membrane capacitance, membrane current, and inner membrane potential difference (Gutsmann et al., 2001).
In this article, we establish a setup for the time-resolved determination of the capacitance minimization potential on the basis of the IFC method. We determine for different asymmetric membranes and compare the results with the respective values of determined in ion carrier experiments. Further on, we use this technique to characterize the interaction between different asymmetric bilayers mimicking outer membranes of Gram-negative bacteria and CAP18-derived antibacterial peptides.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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; Rietschel et al., 1992, 1994; Vinogradov et al., 1994; Wiese et al., 1998; Holst, 1999) were used. The chemical structure of the respective LPS is given in Fig. 2. LPS was extracted by the phenol/chloroform/petroleum ether method (Galanos et al., 1969), purified, lyophilized, and transformed into the triethylamine salt form. The amounts of nonstoichiometric substitutions by fatty acids, 4-amino-4-deoxyarabinose (Ara4N), and phosphoethanolamine were analyzed by MALDI-TOF mass spectrometry. Thus, in the LPS R595, the 16:0 fatty acid linked to the amide-linked 3-OH-14:0 in position 2 of the reducing glucosamine was present only at a level of 30% and Ara4N linked to the 4'-phosphate at a level of 65%. In LPS R45, 50% of the phosphates linked to the 4'-phosphate of lipid A and 50% of the first Kdo were substituted with Ara4N. Phosphatidylethanolamine (PE) from E. coli, phosphatidylglycerol (PG) from egg yolk lecithin (sodium salt), synthetic diphosphatidylglycerol (DPG), phosphatidylserine (PS) from porcine brain (sodium salt), and synthetic diphytanoylphosphatidylcholine (DPhyPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification.
Synthetic CAP18-derived peptides were prepared by Merrifield synthesis as previously described (Larrick et al., 1994) and stored in 0.01% acetic acid. Three fragments of hCAP18 with modified amino acid sequence as compared to the original sequence (m1hCAP1898–117, m2hCAP1898–117, mhCAP18104–123), and three fragments of rCAP18 (rCAP18106–137, rCAP18106–125, and rCAP1898–117) were used (Table 1).
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Planar membranes
Planar bilayers according to the Montal-Mueller technique (1972) were prepared as described before (Wiese and Seydel, 1999; Gutsmann et al., 1999). Briefly, asymmetric bilayers were formed by opposing two lipid monolayers each prepared on an aqueous bathing solution from chloroformic solution of the lipids at a small aperture in a thin Teflon septum.
For the formation of planar membranes, phospholipids were dissolved in chloroform (2.5 mg/ml) and LPS in chloroform/methanol (v/v = 10:1) (2.5 mg/ml) by heating to 95°C for 2 min. The PL leaflet of asymmetric LPS/PL membranes consisted of a mixture of PE, PG, and DPG in a molar ratio of 81:17:2 resembling the phospholipid composition of the inner leaflet of the outer membrane of S. enterica serovar Typhimurium (Osborn et al., 1972) being composed of the same constituents as that of E. coli (Shaw, 1974).
For electrical measurements, membranes were voltage-clamped via a pair of Ag/AgCl electrodes (type IVM E255, Advanced Laboratory Research, Franklin, MA). All signals were digitized with a PCI-200428W-1 analog input board (Intelligent Instrumentation, Leinfelden-Echterdingen, Germany) and further analyzed by a microcomputer system.
All measurements were performed with bathing solutions consisting of 100 mM KCl and 5 mM MgCl2 at a temperature of 37°C, except for the measurements showing the influence of nonactin on , where KCl was replaced by NaCl. The bathing solutions were buffered with 5 mM HEPES. Mg2+ was used to improve the stability of the membranes. Peptides (100 µg/ml) were added in aliquots of 30 µl to the cis side (named first) of the bilayer, e.g., LPS F515/PL.
Capacitance measurements
Membrane capacitance was determined from the first harmonic of the current response to an ac excitation U1 with a dc bias U0 according to Carius (1976). To this end, a sine-wave voltage with a frequency 
of 500 Hz and an amplitude of 500 mVrms was applied to the membrane. The capacitive membrane current I1 was converted into a voltage, low-pass filtered with a four-pole Bessel filter (model 4302, Ithaco, Ithaca, NY) having a cutoff-frequency of 750 Hz, and sent to a microcomputer system. Membrane capacitance C0 was calculated from the equation
 | (4) |
C = ± 5 pF) to calibrate our system.
Inner field compensation
was determined by the IFC method according to Sokolov and Kuz'min (1980). The membrane capacitance CM as a function of the clamp voltage is given by Eq. 3 (Babakov et al., 1966; White, 1970). The current response of a symmetric membrane ( = 0 mV) to an ac excitation with a dc bias U = U0 + U1 sin(t) is
 | (5) |
; Sokolov and Kuz'min, 1980). In the case that U0 = 0 mV, the second harmonic 
vanishes. For an asymmetric membrane ( 
0 mV), I2 vanishes in case that the inner membrane potential difference is compensated by an external voltage (Fig. 3, B and C). In our setup, the current response to an ac excitation with a dc bias was analyzed by a lock-in amplifier (7265 DSP lock-in amplifier, EG&G, Berks, UK). The ac excitation with a frequency of 500 Hz and an amplitude of 50 mVrms was generated by the built-in function generator. The dc bias was applied by an analog output board (PCI-200428W-1, Intelligent Instrumentation).
For time-dependent IFC measurements, the dc bias was controlled by a feed-back loop using the phase of the second harmonic as the control parameter. The phase has an inflection point in the case that the inner membrane potential difference is compensated by the bias voltage.
Data is stored on the microcomputer system with a frequency of 10 Hz.
Carrier transport measurements
was derived from I/U curves obtained from carrier-doped bilayers. For these investigations, the electrodes were connected to the head stage of an L/M-PCA patch-clamp amplifier (List Medical, Darmstadt, Germany). In all cases, the compartment opposite (trans compartment) to which the peptide (cis compartment) was added, was grounded. Current was defined positive, when cation flux was directed toward the grounded compartment. Membrane current and clamp voltage were low-pass filtered with a four-pole Bessel filter (Ithaco model 4302).
K+-carrier nonactin was added to both compartments before membrane preparation in a final concentration of 5 x 10-7 M, and membranes were prepared as described above. The evaluation of the I/U curves was carried out according to procedures described previously (Seydel et al., 1992). Briefly, the current I as a function of the clamp voltage U is obtained from the Schoch equation
 | (6) |
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RESULTS |
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and of different asymmetric membranes and for various asymmetric membranes composed of LPS F515, LPS R595, or LPS R45 as well as PS, PG, or DPhyPC on the one and PL on the other side are depicted. (dark gray bars) was determined by IFC measurements, (light gray bars) by carrier transport measurements using nonactin-doped membranes. In case of phospholipid/PL membranes, the absolute values for and differ, but have the same polarity, and with that the inner membrane potential has the same slope, whereas in the case of LPS/PL membranes, and differ as well in absolute value as in polarity.
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were determined properly, we induced a defined change of the transmembrane potential by applying a Mg2+ concentration gradient across PS/DPhyPC membranes. For a symmetric distribution of Mg2+ ions (2 mM on both sides), we observed a value of = 66 mV. Changing the Mg2+ concentration to 10 mM on the PS side led to a reduction of by (24 ± 3) mV (data not shown), which is consistent with the expected value of 22 mV according to the Goldman equation.
The influence of nonactin on of LPS R45/PL membranes was investigated by IFC. It is only possible to determine in the absence of a net current, therefore NaCl was used to avoid the transport function of nonactin. The transport rate for Na+ is reduced by a factor of 1000 as compared to that of K+. Measurements were performed in electrolyte solution containing 100 mM NaCl and 5 mM MgCl2 buffered with 5 mM HEPES and adjusted to pH 7. After addition of 5 µl, 10 µl, and 30 µl nonactin, no effect on could be seen (data not shown).
Change of the inner membrane potential difference of various asymmetric membranes after addition of rCAP18106–137
The influence of rCAP18106–137 on was observed by IFC measurements. rCAP18106–137 was added to the cis side of various asymmetric membranes composed on one side of PG, DPhyPC, or LPS F515, LPS R595, or LPS R45 and on the other of PL. In Fig. 5, typical potential traces (black) are depicted exemplarily for LPS F515/PL (A), LPS R45/PL (B), and DPhyPC/PL (C) membranes. Peptide was added 5 min after membrane preparation (indicated by arrows). The gray traces represent relative changes in membrane capacitance C/Ci recorded simultaneously to . Ci is the initial membrane capacitance determined before peptide addition.
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decreases drastically from 40 mV to -100 mV. A sharp but reduced decrease in could also be observed for LPS R45/PL (from 40 mV to -60 mV) and DPhyPC/PL (from -30 mV to -70 mV) membranes (Fig. 5, B and C). Only in the case of LPS F515/PL membranes, decreases to a minimal value and increases then to a final value of -40 mV. Approximately 5 min after peptide addition, a steady state of could be observed for all membranes. In Table 2, the total changes of for LPS F515/PL, LPS R595/PL, LPS R45/PL, PG/PL, and DPhyPC/PL membranes, calculated as the sum of the decrease and the increase in , are given.
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135% of the initial value and a subsequent drop to 80% were observed. For LPS R45/PL membranes (Fig. 5 B), a slight increase to 110% of the initial value followed by a decrease to the initial value occurred. No significant changes were detectable for DPhyPC/PL membranes (Fig. 5 C).
It should be mentioned that both—the changes in as well as those in C/Ci—were concentration-dependent. Absolute changes in and C/Ci increased with increasing CAP18 concentration (data not shown). In most cases, CAP18 concentrations above 20 µg/ml led to membrane rupture.
Change of the capacitance and the inner membrane potential difference of F515 LPS/PL membranes after addition of various CAP18 fragments
The influence of various CAP18-derived peptides on of LPS F515/PL membranes was observed by IFC measurements. Different fragments of CAP18 were added to the cis side of asymmetric LPS F515/PL membranes. In Fig. 6, typical potential traces (black) are depicted exemplarily for three fragments of rabbit CAP18; the gray traces represent the relative change in membrane capacitance C/Ci. Peptide was added 5 min after membrane preparation (indicated by arrows).
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from -40 mV to -30 mV. Addition of the same amount of rCAP18106–125 (Fig. 6 B) led to a decrease to -80 mV. In comparison to rCAP18106–137, the rCAP18106–125-induced decrease in is not followed by a subsequent increase. In Table 3, the total changes in calculated as the sum of the decrease and the increase in after addition of 2 µg/ml m1hCAP1898–117, m2hCAP1898–117, mhCAP18104–123, rCAP1898–117, rCAP18106–125, or rCAP18106–137 to the LPS side of an LPS F515/PL membrane are given.
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120% of the initial value, which is 15% less then the change caused by rCAP18106–137 (Fig. 5 A). In contrast to the trace recorded after addition of rCAP18106–125, rCAP18106–137 led to a decrease in capacitance before a steady state was reached.
For all fragments of hCAP18 and rCAP18, a concentration-dependent increase of the absolute changes in and C/Ci could be observed (data not shown). At CAP18 concentrations above 20 µg/ml, membrane rupture occurred only for rCAP18106–125 and rCAP18106–137.
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DISCUSSION |
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on the basis of the IFC method. In our setup, the phase of the second harmonic has been used to control the feedback loop. The phase of the second harmonic has a sharp transition by 180°, and at the same voltage its amplitude drops to 0 pA. Moreover, in contrast to the amplitude, the phase contains information for a directed feedback and with that opens up the possibility to achieve a good time resolution of the apparatus.
In contrast to the determination of from carrier-doped membranes, the IFC method yields several advantages, e.g., it does not require any probe molecules (like nonactin or valinomycin) and is not ion-specific. The major advantage of the IFC method is the possibility to perform time-resolved measurements of , which opens up the possibility to achieve additional information on the timescale of interaction. Furthermore, the IFC allows investigating the influence on and C simultaneously.
It should, however, be pointed out that both methods do not allow observing an electrically undisturbed system. In both cases, relatively high voltages as compared to the transmembrane voltage in vivo are applied to the membrane, which might induce reorientation of peptides on the membrane surface and intercalation into the lipid matrix and subsequently the formation of lesions. Furthermore, it should be mentioned that an influence of the ion carrier nonactin on in a K+ free system even at high nonactin concentrations could not be observed.
and were determined for various asymmetric lipid bilayers. In the focus of our interest was the asymmetric LPS/PL bilayer. LPS (Fig. 2) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria and is, therefore, a primary target for several antimicrobial peptides such as polymixin B (Wiese et al., 1998), defensins (Sahly et al., 2003), and cathelicidins (Gutsmann et al., 1999). First, we determined the values of and for several ReLPS/PL and phospholipid/PL membranes and compared the respective values (Fig. 4). Interestingly, a positive linear correlation between and was only found for phospholipid/PL membranes. Furthermore, and are linearly correlated with the charge density of the phospholipid leaflet. In contrast to this, we found no correlation between and in case of LPS/PL membranes.
Interestingly, the values for and are different for PS/PL and PG/PL membranes. differs by a value of 14 mV and by 13 mV (Fig. 4). Both lipids, PG and PS, carry one negative net charge, but have a different size (determined from monolayers isotherms). Based on the Gouy-Chapman theory, values of -150 mV and -141 mV were calculated for the PG and the PS layer, respectively, leading to a difference of 9 mV. Additional contributions to the differences might arise from the Born and the dipole potentials of the two monolayers.
We can only speculate on the overall potential profile of LPS/PL membranes. On the basis of the model from Schoch et al. (1979) (Fig. 7 A) for phospholipid membranes we propose a more complex model for LPS/PL bilayers (Fig. 7 B) leaving the trapezoidal shape of the overall potential profile and the PL side of the potential profile unchanged. Due to the more complex structure of the LPS headgroup (Fig. 2) in comparison to that of phospholipids, in particular its size and the distribution of positive and negative charges in the lipid A portion and the Kdo disaccharide, we expanded the region of the LPS headgroup in the potential profile. The Gouy-Chapman model for the surface potential—based on the assumption that the membrane surface is plane on a molecular scale—could be affected. In the model in Fig. 7 B, we only assumed that the Gouy-Chapman potential is negative and the absolute value is smaller than that of the dipole potential; therefore, the potential increases in the LPS headgroup region from negative to positive values. Due to the lack of knowledge on the exact potential profile in the headgroup region, we used a linear increase as a first approximation.
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(Fig. 4). In contrast to the carrier transport, the voltage dependence of the capacitance bases on the electromechanical properties (Alvarez and Latorre, 1978) of the entire membrane. Therefore, the potential difference might be detected between the positions 
and 
within the membrane. In the case of the phospholipid/PL membranes, the positions n1 and and also n2 and differ only slightly, and subsequently the difference between and is small (Fig. 7 A). In contrast to this, in the case of LPS/PL membranes, the positions n1 and have to differ significantly with located in the LPS headgroup region (Fig. 7 B). This assumption is backed by the results depicted in Fig. 4 showing that and vary significantly, e.g., they differ in polarity. The two methods, IFC and carrier transport measurements, are complementary and the combination of the results lead to more detailed descriptions of the potential profile.
Characterization of peptide-membrane interaction by IFC
It has been shown earlier that the composition of the lipid matrix of the OM of Gram-negative bacteria plays an important role for the interaction mechanisms with a huge variety of peptides/proteins, membrane proteins (Hagge et al., 2002), as well as antimicrobial peptides (Gutsmann et al., 1999; Wiese et al., 1998). Therefore, we focused on the investigation of interactions of antimicrobial peptides with asymmetric LPS/PL membranes to point out the role of the IFC method for the characterization of peptide-membrane interactions.
First, we performed measurements on the interaction of rCAP18106–137 with several lipid bilayers composed on one side of ReLPS from various sensitive (E. coli, S. Minnesota) and resistant (P. mirabilis) Gram-negative species, as well as several phospholipids and of PL on the other side to study the influence of the lipid matrix. In Fig. 5, typical traces of rCAP18106–137-induced changes of and C/Ci are depicted.
Applying time-resolved measurements of via the IFC technique, we could—for a first time—show that the addition of rCAP18106–137 to the LPS side of an LPS F515/PL (Fig. 5 A) membrane mimicking the outer membrane of a sensitive species led to rapid changes in as well as in C/Ci indicating a very fast peptide membrane interaction. The interaction process might even be faster because of the still limited sampling rate (0.1 Hz resulting from the velocity of feedback loop). Furthermore, after starting the IFC measurement, the feedback loop changed the clamp voltage with a maximum speed of 50 mV/min to finally compensate , which is about the same rate as the observed peptide-induced changes in (Fig. 5). The observed effects were reduced when rCAP18106–137 was added to the LPS side of membranes mimicking the outer membrane of a resistant species such as P. mirabilis (Fig. 5 B). This observation is in agreement with earlier results showing a reduced capability of the peptide to form lesions in the respective membranes (Gutsmann et al., 1999).
Interestingly, in the case of DPhyPC/PL (Fig. 5 C), rCAP18106–137 has only slight influence on the relative membrane capacitance C/Ci whereas changed drastically. The latter observation is in contrast to the conclusion published earlier that rCAP18106–137 does not interact with PC membranes (Gutsmann et al., 1999, 2000, 2001). These earlier observation were based on the fact that neither the membrane capacitance obtained from planar lipid bilayers nor the film area in monolayer experiments changed significantly. Furthermore, the minimal clamp voltage needed to induce pore formation in DPhyPC/PL membranes is significantly higher as compared to the respective values of LPS/PL membranes. This underlines the role of the IFC method as a very sensitive tool for the investigation of peptide-membrane interactions.
We found that the CAP18-induced changes in are linearly correlated with the charge density of the LPS side of the bilayer (Fig. 8), indicating that electrostatic interactions between peptides and membranes play an important role for the accumulation of peptide on the membrane surface and/or the intercalation into the lipid matrix. Similar results for magainin-derived peptides obtained from in vivo studies and liposome assays have been published earlier (Dathe et al., 2001). Interestingly, the results obtained from carrier-doped membranes (Gutsmann et al., 1999) show different final values for as compared to . This is in agreement with the model discussed above that the inner membrane potential differences determined by the two methods are different because of the different positions n1 and 
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; Travis et al., 2000; Pathak et al., 1995). Therefore we studied the influence of several fragments of rabbit and human CAP18—varying in net charge, hydrophobic moment, hydrophobicity, and -helical content (Gutsmann et al., 2001)—on C/Ci and of LPS F515/PL membranes (Figs. 5 A and (6). The results are summarized in Table 3. In Fig. 9, the correlation between net charge and change in are given. We found that the change in increases with increasing net charge. This also underlines the role of electrostatic interactions in peptide-membrane interactions. This result is in accordance to those found earlier in biological experiments as well as biophysical investigations on different model systems (liposomes, monolayers, planar bilayers) (Gutsmann et al., 2001). Dathe et al. (2001) could show that a modification of charge led to an optimization of the antimicrobial activity of magainin peptides against E. coli and Bacillus subtilis. These authors found that the antimicrobial activity increases with increasing net charge; however, increase upon a certain threshold led to an increase in hemolytic activity and subsequently a loss of selectivity (Dathe et al., 2001).
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, which does not allow studying the kinetics of the interaction. In this article, we could show that the determination of the inner membrane potential by IFC measurements is a helpful and in some cases very sensitive tool for the characterization of properties of lipid bilayers as well as interactions between peptides and membranes and their kinetics. Even though the IFC yields several advantages as compared to the carrier transport measurements, the latter can provide additional information on the system. Therefore, the combination of both methods—IFC and carrier transport measurements—leads to a more detailed description of the interaction mechanism. We are indebted to Mr. James W. Larrick from the Palo Alto Institute of Molecular Medicine (Mountain View, CA) for providing the CAP18-derived peptides.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Submitted on July 25, 2003; accepted for publication October 7, 2003.
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REFERENCES |
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of the side of peptide addition and change of the inner membrane potential difference 