Kinetic Pathway of Antimicrobial Peptide Magainin 2-Induced Pore Formation in LipidMembranes

Yukihiro Tamba,†,|,⊥ Hirotaka Ariyama,†,| Victor Levadny,†,‡ and Masahito Yamazaki*,†,§

Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka UniVersity, 836 Oya,Suruga-ku, Shizuoka 422-8529, Japan; Theoretical Problems Center of Physico-Chemical Pharmacology,Russian Academy of Sciences, Kosugina, 4, 117977, Moscow, Russia; and Department of Physics, Faculty ofScience, Shizuoka UniVersity, Shizuoka 422-8529, Japan

ReceiVed: May 18, 2010; ReVised Manuscript ReceiVed: August 15, 2010

The pore formation in lipid membranes induced by the antimicrobial peptide magainin 2 is considered to bethe main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important toelucidate the kinetic pathway of magainin 2-induced pore formation in lipid membranes. In this report, toexamine the change in pore size over time during pore formation which can monitor its kinetic pathway, weinvestigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced poresin single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphos-phatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10 000, Texas-Reddextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initialstage followed by a stage of slow leakage. In contrast, magainin 2 induced a transient, but very small (10-20%),leakage of fluorescent probes of a larger size such as Texas-Red dextran 40 000 and FITC-BSA. These resultsindicate that magainin 2 molecules initially induce a large, transient pore in lipid membranes following whichthe radius of the pore decreases to a stable smaller size. We estimated the radius of these pores, which increaseswith an increase in magainin 2 concentration. On the basis of these data, we propose a hypothesis on themechanism of magainin 2-induced pore formation.

1. Introduction

Antimicrobial peptides with bactericidal and fungicidal activ-ity have been discovered in, and isolated from, a wide varietyof organisms including amphibians, invertebrates, plants, andmammals.1,2 Among these antimicrobial peptides, magainin 2,which was first isolated from the African clawed frog XenopuslaeVis,3,4 has been extensively investigated. All-D amino acidmagainin 2 had the same antibacterial activity as that of thenatural, all-L amino acid magainin 2.5 Since specific interactionof magainin 2 with chiral receptors or proteins is not requiredfor its antibacterial activity, this observation indicates that thetarget of magainin 2 is the lipid membrane regions of bacterialand fungal biomembranes. The interactions of magainin 2 withbiological lipid membranes have been investigated using variousmethods such as the large unilamellar vesicle (LUV) suspensionmethod6-9 and X-ray diffraction.10,11 On the basis of theseresults, magainin 2-induced pore formation in lipid membranesis considered to be the main mechanism underlying its bacte-ricidal activity. Several structural models for the antimicrobialpeptide-induced pore in biomembranes have been proposed:12

the barrel-stave (or helix bundle) model and the toroidal (orwormhole) model. In the former model, peptides insert perpen-dicularly into the lipid membranes and a fixed number of

peptides specifically associate with each other to form anR-helical bundle that produces a narrow pore with a specificsize. In contrast, in the toroidal model, the external and internalmonolayer membranes bend and merge in a toroidal fashion tocreate a pore of which the inner wall is composed of R-helicalpeptides and lipid head groups. Several researchers considerthat magainin 2 forms a toroidal structure.10,11 Results ofmolecular dynamics simulations suggest the disordered toroidalmodel.13 Recently, the pore induced by Bax-derived peptideshas been convincingly shown to be a toroidal structure.14 In atoroidal pore, there may be no specific interactions betweenR-helical peptides, which makes it difficult to understand thesize and the stability of the pore. Moreover, there is noinformation regarding how toroidal pores are formed in lipidmembranes nor regarding which factor determines the size ofthese pores. Therefore, both the kinetic pathway and themechanism of pore formation remain unknown.

So far, almost all studies of interactions of antimicrobialpeptides with lipid membranes have been done using a suspen-sion of many small-size vesicles such as LUVs (the LUVsuspension method). In these studies, the average values of thephysical parameters of vesicles have been obtained from a largenumber of vesicles, and thereby much information has been lost.Recently, we proposed a novel method, the single giantunilamellar vesicle (GUV) method. In this method, we observeand measure the changes of structure and physical propertiesof single GUVs with a diameter of g10 µm during theinteraction of substances such as antimicrobial peptides, andanalyze these results over many “single GUVs” statistically.15,16

Using this method, we previously succeeded in observing themagainin 2-induced pore formation in lipid membranes of eachsingle GUVs and estimating the rate constant of the pore

* To whom correspondence should be addressed. Tel./Fax: +81-54-238-4741. E-mail: spmyama@ipc.shizuoka.ac.jp.

† Integrated Bioscience Section, Graduate School of Science andTechnology, Shizuoka University.

‡ Russian Academy of Sciences.§ Department of Physics, Faculty of Science, Shizuoka University.| These authors contributed equally.⊥ Present address: General Education, Suzuka National College of

Technology, Shiroko-cho, Suzuka, Mie 510-0294, Japan.

J. Phys. Chem. B 2010, 114, 12018–1202612018

10.1021/jp104527y 2010 American Chemical SocietyPublished on Web 08/30/2010

formation.17,18 Moreover, the use of this method allows us toseparate the step of pore formation in membranes from the stepof fluorescent probe leakage through the pores, which enablesan accurate estimation of the leakage rate from single GUVs.We also previously found that the surface concentration ofmagainin 2 in the external monolayer of a GUV is the maindeterminant of the rate of pore formation in GUVs of variousmembranes.18 Hence the single GUV method can provide a greatdeal of new information that cannot be obtained by theconventional LUV suspension method.

To address the above questions on the magainin 2-inducedpore formation, it is important to elucidate the kinetic pathwayof pore formation in lipid membranes. In this report, in orderto examine the change in pore size during pore formation whichcan monitor its kinetic pathway, we investigated the rate of theleakage of various sized fluorescent probes through the magainin2-induced pores in single GUVs of 50% dioleoylphosphati-dylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC)membrane (i.e., 50% DOPG/DOPC-GUVs). The fluorescentprobes we used were Texas-Red dextran (TRD) of variousmolecular weights, fluorescein isothiocyanate bovine serumalbumin (FITC-BSA), and trypsin inhibitor from soybean AlexaFluor 488 conjugate (AF-SBTI). We show that the size of thepores changes over time and also that the size and the numberof the pores depends on the concentration of magainin 2. Apart of the preliminary results of this research was published inthe proceedings of an international conference.19

2. Materials and Methods

2.1. Materials and Peptide Synthesis. DOPC and DOPGwere purchased from Avanti Polar Lipids Inc. (Alabaster, AL).Texas-Red Dextran 3000 (TRD-3k), Texas-Red Dextran 10 000(TRD-10k), Texas-Red Dextran 40 000 (TRD-40k), Texas-RedDextran 70 000 (TRD-70k), FITC-BSA, and AF-SBTI werepurchased from Invitrogen Inc. (Carlsbad, CA). These TRDmolecules were used without further purification for mostexperiments. However, some experiments were then repeatedusing TRD molecules that were purified by gel chromatographyusing a Sephadex G-10 column. Bovine serum albumin (BSA)was purchased from Wako Pure Chemical Industry Ltd. (Osaka,Japan). Magainin 2 was synthesized by the FastMoc methodusing a 433A peptide synthesizer (PE Applied Biosystems,Foster City, CA). The sequence of magainin 2 (23-mer) isGIGKFLHSAKKFGKAFVGEIMNS with an amide-blocked Cterminus. The methods for purification and identification of thepeptides were described in our previous paper.17

2.2. Experiments Using the Single GUV Method. DOPG/DOPC-GUVs were prepared in buffer A (10 mM PIPES, pH7.0, 150 mM NaCl, and 1 mM EGTA) containing 0.1 M sucroseand various fluorescent probes by the natural swelling of a drylipid film at 37 °C.17 The concentrations of the fluorescent probeswere as follows: 10 µM for TRD-10k, TRD-40k, and TRD-70k, 30 µM for TRD-3k and FITC-BSA, and 8 µM for AF-SBTI. To obtain a pure GUV solution, untrapped fluorescentprobes were removed as described in our previous report.17

The interaction of magainin 2 with single GUVs was carriedout in buffer A containing 0.1 M glucose at 25 °C and wasanalyzed by fluorescence phase contrast microscopy.15-18 300µL of the purified GUV solution (0.1 M sucrose in buffer A asthe internal solution; 0.1 M glucose in buffer A as the externalsolution) was transferred into a handmade microchamber.15-18

A slide glass and a cover glass in a microchamber wereprecoated with 0.1% (w/v) BSA in buffer A containing 0.1 Mglucose to prevent the direct contact of the GUVs with the glass

surface.15-18 It is well demonstrated in the micropipet aspirationmethod to measure the tension and the elastic modulus of themembrane of a GUV accurately that the BSA coating on theglass surface of the slide glass and the micropipet preventsthe contact between the glass and the GUV from inducing theexternal tension in the GUV membrane.20 Various concentrationsof magainin 2 solution in buffer A containing 0.1 M glucosewere continuously added in the vicinity of a GUV through a20 µm diameter glass micropipet positioned by a microman-ipulator. The distance between the GUV and the tip of themicropipet was ∼70 µm. Thereby, the equilibrium magainin 2concentration near the GUV is considered almost the same asthat in the micropipet.15,17

Phase contrast and fluorescence images of GUVs wererecorded using a high-sensitivity EM-CCD camera (C9100-12,Hamamatsu Photonics K.K., Hamamatsu, Japan) with a harddisk. Neutral density filters were used to decrease the intensityof the incident light, resulting in conditions where almost nophotobleaching of fluorescent probes in a GUV occurred duringthe interaction of the magainin 2 solution with single GUVs.Therefore, the decrease in fluorescence intensity inside a GUVcan be considered as the result of leakage of the fluorescentprobes from the inside to the outside of the GUV. Thefluorescence intensity inside the GUVs was determined usingthe AquaCosmos software (Hamamatsu Photonics K.K.,Hamamatsu, Japan), and the average intensity per GUV wasestimated. The details of this method were described in ourprevious reports.15-18

To obtain the rate constant of the magainin 2-induced poreformation in lipid membranes, kP, for various GUVs, threeindependent experiments were carried out for each magainin 2concentration. For each experiment, 10-20 single GUVs wereanalyzed to evaluate the rate constant. Average values andstandard deviations of the rate constant among the threeexperiments were calculated.

3. Results

3.1. Induction of Leakage of TRD-3k from 50% DOPG/DOPC-GUVs by Magainin 2. In order to examine the changein the size of the magainin 2-induced pores in lipid membranes,we first investigated the leakage of the small fluorescent probeTRD-3k (average molecular weight, Mw, is 1500 according toInvitrogen Inc., and Stokes-Einstein radius, RSE, is 1.4 nm21,22)through magainin 2-induced pores in single 50% DOPG/DOPC-GUVs. Figure 1A shows a typical experimental result of theeffect of the interaction of 7 µM magainin 2 with single GUVson the TRD-3k concentration within a GUV. Prior to magainin2 addition, a phase contrast microscopic image of the GUVindicated a high contrast in the GUV (Figure 1A-1) due to thedifference in the concentration of sucrose and glucose betweenthe inside (0.1 M sucrose) and the outside (0.1 M glucose) ofthe GUV. A fluorescence microscopic image of the same GUV(Figure 1A-2) showed a high concentration of TRD-3k insidethe GUV at this time. During the addition of the 7 µM solutionof magainin 2, the fluorescence intensity inside the GUV wasalmost constant over the first 170 s, following which thefluorescence intensity decreased rapidly (Figure 1A-2,B). After220 s, a low fluorescence intensity, which was less than 10%of the original intensity, was detected inside the GUV, althougha phase contrast image of the same GUV (Figure 1A-3) showedthat the GUV structure was still intact with no detectable breaks.As discussed in our previous report,17 the rapid decrease influorescence intensity occurred as a result of the leakage of thefluorescent probe through the magainin 2-induced pore in

Antimicrobial-Peptide-Induced Pore Formation J. Phys. Chem. B, Vol. 114, No. 37, 2010 12019

the lipid membranes, i.e., due to the diffusion of TRD-3kfrom the inside to the outside of the GUV through the porein the membrane. Thus, the time at which the fluorescenceintensity began to rapidly decrease corresponds to the timeat which the pore was formed in the membrane. Furthermore,a comparison of the phase contrast images in Figure 1A-1,A-3 also showed that there was a substantial loss in thephase contrast of the GUV, indicating that, during the leakageof TRD-3k, sucrose and glucose also passed through the samepore. When the same experiments were carried out usingmany single GUVs, we observed that a similar rapid leakageof TRD-3k from a GUV started stochastically. These resultsindicate that the pores were formed stochastically and that∼90% of the TRD-3k had leaked from single GUVs over aperiod of ∼100 s after pore formation (Figure 1C).

As we demonstrated in our previous papers,17,18 the rateconstant of the magainin 2-induced pore formation in lipidmembranes can be obtained by analyzing the time course ofthe fraction of intact GUVs, Pintact(t), among the populationof GUVs examined, from which the fluorescent probe didnot leak over time t. Figure 1D shows that the value of Pintact

of 50% DOPG/DOPC-GUVs decreased with time during theinteraction with 7 µM magainin 2. As shown in Figure 1D,the curve of the time course of Pintact was well fitted bya single-exponential decay function defined by eq 1 asfollows:

where kP is the rate constant of the magainin 2-induced poreformation and teq is a fitting parameter. In this fitting, weneglected a few unstable GUVs in which leakage easilyoccurred. Three independent experiments similar to theexperiment shown in Figure 1D were carried out to obtainthe value for kP. The average value of kP was then calculatedusing the results of all of the independent experiments. Theaverage value of kP for 7 µM magainin 2 was 0.014 ( 0.001s-1, which is almost the same value as that obtained inprevious experiments of magainin 2-induced calcein leak-age.17 The fact that similar values of kP were obtained forTRD-3k and calcein is in agreement with the fact thatmagainin 2-induced pore formation does not depend on thetype of the fluorescent probe within GUVs.

3.2. Induction of Leakage of TRD-10k from 50% DOPG/DOPC-GUVs by Magainin 2. We next investigated theinteraction of magainin 2 with single 50% DOPG/DOPC-GUVscontaining TRD-10k (Mw distribution is 9000-11 000 accordingto Invitrogen Inc., RSE ) 2.7 nm21). Figure 2A shows time courseof the change in the normalized fluorescence intensity of severalsingle GUVs containing TRD-10k induced by 7 µM magainin2. A rapid leakage of TRD-10k was observed from each singleGUV that started in a stochastic manner, and then the rate ofthe leakage decreased and the resulting slower leakage contin-ued. The phase contrast microscope images of the single GUVsshowed complete leakage of sucrose, indicating that magainin2 induced a pore (or pores) in the GUV membrane. These resultsindicate that pores were formed stochastically and that ∼60%of the TRD-10k leaked from single GUVs over a period of ∼100s after pore formation. When the same experiments were carriedout using many single GUVs (the number of the examinedGUVs, n ) 26), a similar two-stage leakage (i.e., an initial rapidand transient leakage followed by a slow leakage) was observedfrom a GUV.

The effect of magainin 2 concentration on the leakage ofTRD-10k was also investigated. When 4 µM magainin 2 wasused (Figure 2B), the two stages of leakage were more clearlyobserved. Thus, initially, a rapid leakage occurred over ∼5 s,resulting in a ∼20% leakage of TRD-10k. This initial leakagewas then followed by a very slow leakage. Only ∼40% of theTRD-10k leaked from single GUVs over a period of ∼100 safter pore formation. In contrast, when 15 µM magainin 2 was

Figure 1. Leakage of TRD-3k from single 50% DOPG/DOPC-GUVsinduced by 7 µM magainin 2 in buffer A at 25 °C. (A) Fluorescenceimages (2) show that the TRD-3k concentration inside the GUVprogressively decreased after the addition of magainin 2. The numbersabove each image show the time in seconds after the magainin 2addition was started. Also shown are phase contrast images of the GUVat time 11 (1) and 245 s (3). The bar corresponds to 10 µm. (B) Timecourse of the change in the normalized fluorescence intensity of theGUV shown in (A). We defined the normalized fluorescence intensityof the intact GUV before the initiation of the leakage as 1. (C) Otherexamples of the time course of the change in the normalizedfluorescence intensity of several single GUVs under the same conditionsas in (A). (D) Time course of Pintact of 50% DOPG/DOPC-GUV. Asolid line represents the best-fitted curve of eq 1.

Pintact(t) ) exp{-kP(t - teq)} (1)

Figure 2. Leakage of TRD-10k from single 50% DOPG/DOPC-GUVsinduced by magainin 2 in buffer A at 25 °C. Time course of the changein the normalized fluorescence intensity of several single GUVs inducedby (A) 7 µM, (B) 4 µM, and (C) 15 µM magainin 2.

12020 J. Phys. Chem. B, Vol. 114, No. 37, 2010 Tamba et al.

used (Figure 2C), the initial rapid leakage that occurred within∼5 s resulted in a leakage of ∼70% of the TRD-10k, whichwas then followed by a slow leakage. During the next ∼50 sfollowing pore formation, ∼90% of the TRD-10k leaked fromsingle GUVs.

3.3. Induction of Leakage of FITC-BSA, TRD-40k, andTRD-70k from 50% DOPG/DOPC-GUVs by Magainin 2.We further investigated the interaction of magainin 2 with single50% DOPG/DOPC-GUVs containing fluorescent probes of alarger molecular size such as FITC-BSA (RSE ) 3.6 nm23), TRD-40k (Mw distribution is 35 000-50 000 according to InvitrogenInc., RSE ) 5.0 nm21), and TRD-70k (Mw distribution is60 000-90 000 according to Invitrogen Inc., RSE ) 6.4 nm21).Figure 3A shows time course of the change in the fluorescenceintensity of several single GUVs containing FITC-BSA inducedby 15 µM magainin 2. A small decrease in the fluorescenceintensity inside the GUV was observed for a short time, butsubsequently the fluorescence intensity remained almost con-stant. The phase contrast microscope images of the single GUVsshowed complete leakage of sucrose, indicating that magainin2 did induce a pore in the GUV membrane. When the sameexperiments were carried out using many single GUVs (n )15), we observed only a similar transient, but very small (∼10%)leakage of FITC-BSA.

We then investigated the interaction of magainin 2 with single50% DOPG/DOPC-GUVs containing TRD-40k or TRD-70kand obtained almost the same results as those using FITC-BSA.Thus, using 15 µM magainin 2, an initial, transient, smalldecrease in the fluorescence intensity inside the GUV wasobserved over several seconds, but subsequently the fluorescenceintensity remained almost constant (data not shown). When thesame experiments were carried out using many single GUVs(n ) 21), a similar transient but very small (∼10%) leakage ofTRD-40k was observed.

We further carried out similar experiments using purifiedTRD-40k and TRD-70k. The results were similar to thoseobtained using the unpurified molecules, indicating that con-tamination with smaller sized TRDs was very small andtherefore did not affect the results of the leakage experimentsdescribed above.

3.4. Induction of Leakage of AF-SBTI from 50% DOPG/DOPC-GUVs by Magainin 2. We also investigated theinteraction of magainin 2 with single 50% DOPG/DOPC-GUVscontaining AF-SBTI (RSE ) 2.8 nm24). Figure 3B shows timecourse of the change in the fluorescence intensity of severalsingle GUVs containing AF-SBTI induced by 15 µM magainin2. We observed two stages of leakage similar to that of TRD-10k. The initial rapid leakage was followed by a very slowleakage. Approximately 70% of the AF-SBTI leaked from singleGUVs over a period of ∼100 s after pore formation (n ) 23).

In contrast, in the case of 7 µM magainin 2, we observed asimilar leakage of AF-SBTI as that of FITC-BSA; an initial,transient, small decrease in the fluorescence intensity inside theGUV was observed over several seconds, but subsequently thefluorescence intensity remained almost constant (n ) 16). Inthe case of 4 µM magainin 2, we observed a similar leakage ofAF-SBTI as that of 7 µM magainin 2 (n ) 7).

4. Discussion

The results presented in this paper show that magainin2-induced leakage of fluorescent probes from single GUVsdepends on the size of the probe molecules. Moreover, thereare two different patterns of leakage. For probes with a relativelysmall molecular size (such as TRD-10k, TRD-3k, and AF-SBTI), a transient, rapid leakage was observed at the initial stageof pore formation that was followed by a slow leakage (patternA). In contrast, for probes with a larger molecular size (such asTRD-40k, TRD-70k, and FITC-BSA), leakage occurred over avery short period of time (less than 10 s) during which only asmall amount of the GUV contents (∼10-20%) leaked fromthe GUV (pattern B). It is widely accepted that magainin 2induces pore formation in lipid membranes.9 The probe leakagefrom single GUVs that was observed in our current study hasbeen shown to be due to magainin 2-induced pore formation.17,18

Our results in this paper indicate for the first time that the sizeof the magainin 2-induced pore (or a few pores) changes overtime; at the beginning of the pore formation the pore size isvery large, but this pore then rapidly decreases to a smaller size.Hence, the large probes could only leak from the GUV interiorduring the initial stage of pore formation (pattern B). In contrast,the smaller probes could leak from the GUV over the entireperiod of pore formation (pattern A). The observed leakagesoccur at two different rates: the transient, initial, rapid leakageand the slow leakage at the final stage indicate that magainin 2molecules initially induces a transient, large pore in the lipidmembrane following which the radius of the pore decreases toa stable, smaller size.

The rate constant of the leakage of the fluorescent probe froma GUV, kleak, is determined by the following experimentalformula,

where Cin(t) (mol/m3) and C0in are the concentration of the

fluorescent probe inside of a GUV at time t after, and before,initiation of the leakage, respectively. We can determine thenormalized concentration of the fluorescent probe inside a GUV,Cin(t)/C0

in, experimentally because the probe concentration insidethe GUV is roughly proportional to the fluorescence intensityof the GUV, I(t), i.e., Cin(t)/C0

in ) I(t)/I(0), where I(0) is thefluorescence intensity of the intact GUV before the initiationof the leakage. If we plot the log of normalized fluorescenceintensity, FI ()I(t)/I(0)), vs time (s), we can obtain the rate con-stant of the leakage kleak quantitatively. Figure 4 shows the typicalbehavior of Cin(t)/C0

in observed in our experiments. There aretwo types of the rate constant kleak: kleak

initial of the rapid leakage atthe initial stage and kleak

steady of the slow leakage at the final steadystage (see also Table 1). In the case of 4 µM magainin 2-inducedleakage, for TRD-3k, kleak

initial ) 1.2 × 10-1 s-1 and kleaksteady ) 4.5

× 10-3 s-1, and for TRD-10k, kleakinitial ) 5.5× 10-2 s-1 and kleak

steady

) 2.2 × 10-3 s-1. For each probe, the value of kleakinitial is 20-40

times greater than that of kleaksteady under the same experimental

conditions. For the same probe, both kleak values increased as

Figure 3. Leakage of (A) FITC-BSA and (B) AF-SBTI from single50% DOPG/DOPC-GUVs induced by 15 µM magainin 2 in buffer Aat 25 °C. Time course of the change in the normalized fluorescenceintensity of several single GUVs.

Cin(t) ) C0in exp(-kleakt) (2)

Antimicrobial-Peptide-Induced Pore Formation J. Phys. Chem. B, Vol. 114, No. 37, 2010 12021

the magainin 2 concentration increased. For the same magainin2 concentration, kleak increased as the RSE of the TRD decreased,i.e., with an increase in the D value of the probe. In the case ofthe leakage of a small fluorescent probe, calcein (Mw is 623,RSE) 0.74 nm),18 we found that only in the 4 µM magainin2-induced pore the leakage of calcein occurred in two stages: atransient rapid leakage in the initial stage followed by a stageof slow leakage (Table 1). At higher concentrations of magainin2, the calcein leaked almost completely in one stage.

We can obtain the relationship between the rate constant ofthe leakage of the fluorescent probes and the cross-sectionalarea of the magainin 2-induced pores using a theoreticalequation. We assume that the fluorescent probes can diffuseonly through the magainin 2-induced pores from the inside tothe outside of the GUV. Generally, the membrane permeabilitycan be expressed by a flux of a substance per unit area of pores,J (mol/(m2 · s)), which follows Fick’s law:25

where P (m/s) is permeability coefficient of the substance inthe pore and Cout(t) (mol/m3) is concentration of the substanceoutside of the GUV at time t. P is equal to D/h, where D (m2/s)is diffusion coefficient of the substance in the pores and h (m)is the effective length of the pore, which is almost the same asthe membrane thickness (h ) 3.5 nm). Thereby, the rate ofleakage of the fluorescent probe from a GUV can be expressedas follows (here we assume Cout ) 0 for any time, because thevolume of the outside the GUV is very large),

where Sp (m2) is the effective cross-sectional area of pores (oneor several) in each GUV and V (m3) is the volume of each GUV.Using the kleak values obtained experimentally described above,we estimated the values of Sp using the eq 5, i.e., Sp ) kleakhV/D(Table 1). For this calculation, we used the D values of theprobesinwater.TherelationshipbetweenDandtheStokes-Einsteinradius of the fluorescent probe, RSE (nm), is determined byEinstein-Stokes equation at 25 °C as follows:

We determined the D values of various TRD molecules usingthe experimentally determined RSE values.21 On the other hand,we determined the RSE values of FITC-BSA, AF-SBTI, andcalcein using the experimentally determined D values.23,24,26

As shown in Table 1A, the Sp values at the initial leakagestage for the same magainin 2 concentration were almost thesame irrespective of the kinds of fluorescent probes tested, andfor all the probes tested the Sp values at the initial leakage stageincreased with magainin 2 concentration. At the final stage ofthe leakage, for only probes of a smaller size (TRD-3k and TRD-10k) the Sp values were determined, since no leakage of thelarger probe was observed (Table 1B). The Sp values for bothof these probes at this stage were almost the same for the samemagainin 2 concentration. Taking into account that AF-SBTIcould not leak at the final stage for 7 (or 4) µM magainin 2, weconclude that the radius of the pore induced by 7 (or 4) µMmagainin 2 at the final stage is smaller than 2.8 nm (RSE ofSBTI), but is larger than 1.4 nm (RSE of TRD-3k). This valueagrees with that of the magainin 2-induced pores in multilayermembranes determined using neutron in-plane scattering (1.9nm).10 In contrast, TRD-10k leaked slowly at the final stagefor 7 (or 4) µM magainin 2 although its radius is similar to thatof AF-SBTI. This result can be explained by the presence ofTRD-10k molecules with smaller radius than the average one,since there is some distribution of molecular weight of dextran(i.e., 9000-11 000). On the other hand, the data of the AF-SBTI leakage show that the radius of the pores at the final stageinduced by 15 µM magainin 2 is larger than 2.8 nm (RSE ofSBTI), but is smaller than 3.6 nm (RSE of BSA), which is largerthan that induced by 7 (or 4) µM magainin 2. These resultsclearly indicate that the radius of the magainin 2-induced poreat the final stage increases with an increase in magainin 2concentration. This conclusion can also explain the data of theleakage of TRD-3k and TRD-10k; i.e., the Sp values at the finalsteady stage increase with an increase in magainin 2 concentra-tion (Table 1B). However, we cannot discard the possibility ofthe increase in the number of the pores as magainin 2concentration.

Generally, the D values of the probes in the pore can differfrom those in bulk water due to some frictions and someinteractions between the probes and the pore. Especially, in thepermeation of the fluorescent probes through the small porewhose diameter is similar to the probes at the final stage, the Dvalues of the probes in the pore may have different values fromthose in water because there are some frictions between theprobes and the pore wall and thereby the quantitative values ofSp may have some errors. However, we did not use these Sp

values to evaluate the radius of the pore at the final stagedescribed above. On the other hand, the pores at the initial statehave much larger radius than the fluorescent probes and therebywe can assume that the D values of the probes in the pore arealmost the same as those in bulk water (i.e., we can use thesimple diffusion equation (eq 3) to estimate the Sp values ofthe pore). Especially in the case of several TRD molecules withdifferent molecular weights which have the same chemicalproperties and no net charges, their interactions with the poreare similar and thereby we can compare the Sp values obtainedby the leakage data of the various TRD molecules. As shownin Table 1A, the Sp values at the initial leakage stage of theTRD in the same magainin 2 concentration were almost thesame irrespective of the molecular weight.

Figure 4. Time course of the logarithm of the normalized fluorescenceintensity, FI, of single 50% DOPG/DOPC-GUVs containing TRD-3kor TRD-10k during the interaction of 4 µM magainin 2 with the GUVin buffer A at 25 °C.

J ) -P(Cin(t) - Cout(t)) ) -Dh

(Cin(t) - Cout(t))

(3)

VdCin

dt) -D

hSpC

in

∴Cin(t)

C0in

) exp(-kleakt)(4)

where kleak )DSp

hV(5)

D ) kT6πηRSE

) 2.45 × 10-19

RSEm2 s-1 (6)

12022 J. Phys. Chem. B, Vol. 114, No. 37, 2010 Tamba et al.

So far, there have been no experimental methods to monitorthe change in the effective cross-sectional area, Sp, or the radius,rp, of the nanometer-size pores in the vesicles such as GUVswith time, although there are some methods to determine the

radius of the pore at the equilibrium state.10,11 In this report, weproposed for the first time the new physicochemical method tomonitor the change in Sp or rp of the nanometer-size pores inthe vesicles with time, and succeeded in revealing the great

TABLE 1: Rate Constants of the Magainin 2-Induced Leakage of Various Fluorescent Probes and the Effective Cross-SectionalArea of the Magainin 2-Induced Pores in Single 50% DOPG/DOPC-GUVs Whose Radius Was 5 ( 1 µm at 25 °C: (A) theInitial Stage, and (B) the Final Steady Stage

(A) The Initial Stagea

fluorescent probe/RSE (nm) 4 µM magainin 2 7 µM magainin 2 15 µM magainin 2

calcein/0.74kleak

initial (s-1) (3.6 ( 1.0) × 10-1 N.D.c N.D.Sp (nm2) (1.9 ( 0.4) × 103

n 7rlp (nm) 24 ( 2TRD-3k/1.4kleak

initial (s-1) (1.2 ( 0.1) × 10-1 (1.9 ( 0.1) × 10-1 (7.2 ( 0.5) × 10-1

Sp (nm2) (1.0 ( 0.1) × 103 (2.1 ( 0.1) × 103 (6.6 ( 0.5) × 103

n 9 29 12rlp (nm) 18 ( 1 26 ( 1 46 ( 2TRD-10k/2.7kleak

initial (s-1) (5.5 ( 0.5) × 10-2 (8.2 ( 0.8) × 10-2 (2.4 ( 0.3) × 10-1

Sp (nm2) (1.2 ( 0.1) × 103 (2.0 ( 0.1) × 103 (4.9 ( 0.7) × 103

n 11 12 12rlp (nm) 20 ( 1 25 ( 1 40 ( 3AF-SBTI/2.8kleak

initial (s-1) (3.6 ( 0.7) × 10-2 (1.2 ( 0.1) × 10-1 (2.5 ( 0.2) × 10-1

Sp (nm2) (8 ( 2) × 102 (2.2 ( 0.3) × 103 (4.7 ( 0.4) × 103

n 6 14 17rlp (nm) 16 ( 3 26 ( 2 39 ( 2TRD-40k/5.0kleak

initial (s-1) N.D. (4.0 ( 0.3) × 10-2 (1.2 ( 0.1) × 10-1

Sp (nm2) (1.8 ( 0.3) × 103 (4.6 ( 0.4) × 103

n 10 17rlp (nm) 24 ( 2 38 ( 2FITC-BSA/3.6kleak

initial (s-1) N.D. 4.8 ( 0.3) × 10-2 (1.9 ( 0.2) × 10-1

Sp (nm2) (1.3 ( 0.1) × 103 (6 ( 1) × 103

n 9 7rlp (nm) 20 ( 1 44 ( 4

(B) The Final Steady Stageb

fluorescent probe/D (m2 s-1) 4 µM magainin 2 7 µM magainin 2 15 µM magainin 2

calcein/3.3 × 10-10

kleaksteady (s-1) (5.6 ( 2.1) × 10-2 N.D.c N.D.

Sp (nm2) (3.3 ( 1.2) × 102

n 7TRD-3k/1.7 × 10-10

kleaksteady (s-1) (4.5 ( 0.4) × 10-3 (1.0 ( 0.1) × 10-2 (2.0 ( 0.6) × 10-2

Sp (nm2) (3.8 ( 0.5) × 101 (1.1 ( 0.1) × 102 (1.9 ( 0.5) × 102

n 9 28 12TRD-10k/9.1 × 10-11

kleaksteady (s-1) (2.2 ( 0.5) × 10-3 (3.3 ( 0.4) × 10-3 (7.6 ( 0.9) × 10-3

Sp (nm2) (5 ( 1) × 101 (7.7 ( 0.7) × 101 (1.7 ( 0.3) × 102

n 11 12 12AF-SBTI/8.8 × 10-11

kleaksteady (s-1) no leakage no leakage (2.6 ( 0.4) × 10-3

Sp (nm2) (6 ( 1) × 101

n 6 12 16TRD-40k/4.9 × 10-11

kleaksteady (s-1) N.D. no leakage no leakage

Sp (nm2)n 10 17FITC-BSA/6.8 × 10-11

kleaksteady (s-1) N.D. no leakage no leakage

Sp (nm2)n 9 7

a kleakinitial (s-1): the rate constants of the leakage. Sp (nm2): the effective cross-sectional area of the pores. n: the number of GUVs examined. r1p

(nm): the radius of the pore. b kleaksteady (s-1): the rate constants of the leakage. Sp (nm2): the effective cross-sectional area of the pores. n: the

number of GUVs examined. c N.D.: not determined.

Antimicrobial-Peptide-Induced Pore Formation J. Phys. Chem. B, Vol. 114, No. 37, 2010 12023

change in Sp or rp of the magainin 2-induced pore with time.This method can provide only qualitative information of thetime course of Sp or rp of the pore, which is, however, the firstinformation of the kinetic pathway of the antimicrobial peptide-induced pore formation in the lipid membranes.

On the basis of a combination of the above data with previouspublished data, we propose a hypothesis on the followingmechanism for magainin 2-induced pore formation in lipidmembranes (Figure 5). First, magainin 2 in aqueous solutionbinds to the membrane interface of the external monolayer ofa GUV. This binding rapidly achieves equilibrium, and we callthis state the Bex state. In the Bex state, magainin 2 forms anR-helix that lies parallel to the membrane interface12 and insertsdeeply into the membrane interface due to high interfacialhydrophobicity.17 This binding compresses the external mono-layer, inducing some negative internal tension in the externalmonolayer σex (<0), which increases the area of the externalmonolayer. Here we define the internal tension in a compressedmembrane as negative and tension in a stretched membrane aspositive. Since the shape of the GUV kept the spherical duringits interaction of magainin 2, the increase of the externalmonolayer area stretches the internal monolayer to increase itsarea due to the strong coupling between the external and internalmonolayers in the GUV (step (i) in Figure 5); i.e., the increaseof the external monolayer area acts on the internal monolayeras an external mechanical tension to stretch it. This externaltension increases with an increase in the surface concentrationof magainin 2 in the external monolayer Xex. Then, the stretchingof the internal monolayer induces a positive internal tension inthis monolayer σin (>0), which tends to decrease the area ofthis monolayer and counterbalances with the external tension.Our previously published experimental results described thisincrease in area and in tension following the magainin 2 binding.For example, the binding of magainin 2 molecules induced ashape change in a DOPG/DOPC-GUV from a prolate to a shapecomposed of two spheres connected by a narrow neck, indicatingthat the area of the GUV membrane had been increasedfollowing the binding of magainin 2 to the membrane.17 We

also previously observed that, in the interaction of magainin 2with a single spherical GUV, the binding of magainin 2suppressed the undulating motion of the GUV membrane,indicating an increase in the tension of the membrane. It is well-known that the tension due to an external force induces theformation of a pore in the lipid membranes of GUVs as a resultof thermal fluctuation of the lipid membrane lateral density.27-29

The rate of the pore formation increases as the tensionincreases.29 As we discussed above, in the interaction ofmagainin 2 with lipid membranes, the binding of magainin 2induces an increase in external mechanical tension in the internalmonolayer, which stretches the internal monolayer, raising theprobability of pore formation.18 As a result, a pore forms in themembrane stochastically17,18 (step (ii)) and the rate constant ofthe pore formation increases with Xex. We can consider thechange of the pore size over time as follows. The formation ofa transmembrane pore decreases the stretch of the internalmonolayer (resulting in a decrease in σin) and also induces acompression of the external monolayer (thereby increasing |σex|).Initially, σin > |σex| and thereby the radius of the pore increaseswith time, which decreases σin and at the same time increases|σex|, and finally at σin ) |σex| the pore growth stops. Theunbalance of the tension in both the monolayers may inducethe transfer of lipid molecules from the external to the internalmonolayers through the rim of the pore, which decreases thedifference in the absolute value of the tension of thesemonolayers to zero. Next, magainin 2 molecules in the externalmonolayer also transfer into the internal monolayer through therim of the pore (steps (iii) and (iv)). It increases the surfaceconcentration of magainin 2 in the internal monolayer, Xin, whichchanges the balance of tensions of both the monolayers. Itincreases the area of the internal monolayer, inducing thedecrease in the pore radius. Hence, as the difference in surfaceconcentration of magainin 2 in both the monolayers, Xex - Xin,decreases, the pore radius decreases; i.e., Xex - Xin is a keyfactor in determination of the pore radius. During this step, thelarge pore may transform to several smaller stable pores by therearrangement of magainin 2 molecules in the rim of the largepore (step (v)). The stability of these final pores may bedetermined by the interaction free energy between magainin 2molecules and the total free energy of the lipid membranescontaining the pores. However, at present, we do not know itsmechanism in detail. It is reported that the transfer (i.e., flip-flop) of lipid molecules and the translocation of magainin 2molecule from the outside to the inside of vesicles occurred inthe magainin 2-induced pore formation,6 which supports ourhypothesis.

In the above scenario, the magainin 2-induced pore in thelipid membrane was produced because of the thermal fluctuationof the lipid membrane in the presence of tension, and therebythe pore was produced stochastically, which agrees well withour experimental results.17,18 On the basis of this scenario, onlyone large pore is formed in the initial stage of the leakage,because once a pore is formed the tension of the lipid membraneimmediately decreases and this means that the possibility ofthe occurrence of other pores becomes very low. Assuming thatthis is true, we determined the radius of the large pore at theinitial leakage stage, rlp, under various conditions using the Sp

values for GUVs with a radius of 5 ( 1 µm (Table 1A).Irrespective of the fluorescent probe used, the values of rlp aresimilar for the same magainin 2 concentration. Moreover, asthe magainin 2 concentration is increased, the value of rlp alsoincreases (Table 1A). With an increase in magainin 2 concentra-tion in solution, its concentration in the external monolayer Xex

Figure 5. Hypothesis on the mechanism of magainin 2-induced poreformation in lipid membranes. Step (i): The binding of magainin 2 tothe external monolayer increases its area, which stretches the internalmonolayer, inducing an external tension in this monolayer indicatedby arrows. Step (ii): The increase in tension in the internal monolayerinduces pore formation in the membrane stochastically. Step (iii): Theformation of a transmembrane pore decreases the stretch of the internalmonolayer (resulting in a decrease in σin) and also induces a compres-sion of the external monolayer (thereby increasing |σex|). This imbalanceof the tension may induce the transfer of lipid molecules from theexternal to the internal monolayers through the rim of the pore, whichdecreases the difference in the tension of these monolayers to zero.Step (iv): The transfer of magainin 2 into the rim of the pore and theninto the internal monolayer increases its area, which decreases the radiusof the pore. Step (v): During the decrease in the pore size, the poremay rearrange to form several smaller stable pores.

12024 J. Phys. Chem. B, Vol. 114, No. 37, 2010 Tamba et al.

increases. As described above, the higher the Xex value and thegreater the tension, the larger the radius of the induced pore.This prediction agrees well with the above experimental results.On the other hand, the value of rlp increases with the radius ofthe GUV, R, in the same magainin 2 concentration (Figure 6).The values of the radius of the pore including rlp are determinedby the rate constants of the transfer of lipid molecule andmagainin 2 from the external to the internal monolayers, andthereby we need more experimental data to analyze the resultsof Figure 6 quantitatively. Here, it is noted that the radius ofbacterial cells is much smaller than that of a GUV (e.g., theradius of most bacteria is ∼0.5 µm), and therefore, the radiusof the transient pore in bacterial membrane produced bymagainin 2 is much smaller than that observed in the 50%DOPG/DOPC-GUVs. The hypothesis on the mechanism of themagainin 2-induced pore formation described above can providea rational, qualitative explanation of the obtained experimentalresults, although we need more detailed experimental evidenceto prove this hypothesis. We are now developing a quantitativetheory to describe this model to analyze the experimental resultsin more detail.

On the basis of the hypothesis on the mechanism of poreformation described in this paper, magainin 2 induces poreformation in lipid membranes by using the intrinsic propertyof thermal fluctuation of lipid membranes. Formation of a poreby such a mechanism can provide a rational explanation as tohow toroidal pores are formed in lipid membranes. Moreover,the following consideration of the surface concentration ofantimicrobial peptides required for their pore formation in lipidmembranes would support the hypothesis. For the magainin2-induced pore formation, high surface concentrations ofmagainin 2 molecules at and above Xex ) 60 mmol/mol (molarratio of the magainin 2 to lipid in the membrane surface) wererequired.18 It is also reported that in other antimicrobial peptidesvery high surface concentrations of peptides in lipid membranesor biomembranes were required for their pore formation andtheir activity to kill bacteria (represented by the minimuminhibitory concentration, MIC).12 If the antimicrobial peptidesinduce the pore in the lipid membranes by a specific mannersuch as ionic channel and pore-forming toxin proteins, highsurface concentrations of peptides are not necessary for the poreformation. In contrast, the mechanism we proposed above is aphysical model for the pore formation, which can reasonablyexplain why high surface concentrations are required for thepore formation. It is likely that, apart from magainin 2, otherpeptides and proteins can use a similar strategy to induce theformation of a toroidal pore.14

5. Conclusion

To our knowledge, this is for the first time that the change inSp or rp of the antimicrobial peptide-induced pores in the lipid

membranes with time has been monitored. We demonstratedthe new physicochemical method for this purpose. This methodcan provide only qualitative information of the time course ofSp or rp of the pore, which will be valuable and helpful toelucidate the mechanism of the magainin 2-induced poreformation. On the basis of the results obtained using this method,we conclude that magainin 2 molecules initially induce a large,transient pore, which then rearranges to form smaller, stablepores at the final stage. Theoretical analysis of the leakage rateat the initial stage provided values for the radius of the pore.This radius increases with magainin 2 concentration and alsoincreases with the radius of the GUV. These results provide arational explanation for the mechanism of magainin 2-inducedpore formation that we have proposed. We estimated the radiusof the pores at the final steady stage, which increases withmagainin 2 concentration. These data provide the first informa-tion concerning the kinetic pathway of magainin 2-induced poreformation in lipid membranes.

Acknowledgment. This work was supported in part by aGrant-in-Aid for Scientific Research (B) (No. 21310080) fromthe Japan Society for the Promotion of Science (JSPS), by aGrant-in-Aid for Scientific Research on Priority Areas (SystemCell Engineering by Multiscale Manipulation) (No.20034023),and also by a Grant-in-Aid for Scientific Research on PriorityAreas (Soft Matter Physics) (No. 21015009) from the Ministryof Education, Culture, Sports, Science and Technology (MEXT)of Japan to M.Y. and in part by a JSPS invitation fellowship(S-08108) to V.L. This work was partially carried out usinginstruments at the center for Instrumental Analysis of ShizuokaUniversity.

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Figure 6. Relationship between the radius of the large pore at theinitial stage and the radius of the GUV. These data were obtained byanalysis of the leakage of FITC-BSA induced by 7 µM (0) or 15 µM(O) magainin 2.

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