Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial

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Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2 Moynul Hasan, Mohammad Abu Sayem Karal, Victor Levadnyy, and Masahito Yamazaki Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04219 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2 Moynul Hasan,a,# Mohammad Abu Sayem Karal,a, #,$ Victor Levadnyy,a,b,# and Masahito Yamazakia,c,d,* a

Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka, 422-

8529, Japan b

Theoretical Problem Center of Physico-Chemical Pharmacology, Russian Academy of Sciences, Коsugina, 4,

117977, Moscow, Russia c

Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University, Shizuoka 422-8529,

Japan d

Department of Physics, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan.

# These authors contributed equally. $

Present address: Department of Physics, Bangladesh University of Engineering and Technology, Dhaka-

1000, Bangladesh

*Correspondence should be addressed to: Dr. Masahito Yamazaki, Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan TEL and FAX: 81-54-238-4741, Email: [email protected]

1 Environment ACS Paragon Plus

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ABSTRACT Antimicrobial peptide magainin 2 forms pores in lipid bilayers, a property that is considered the main cause of its bactericidal activity. Recent data suggest that tension or stretching of the inner monolayer plays an important role in magainin 2-induced pore formation in lipid bilayers. Here, to elucidate the mechanism of magainin 2-induced pore formation, we investigated the effect on pore formation of asymmetric lipid distribution in two monolayers. First,

we

developed

a

method

to

prepare

giant

unilamellar

vesicles

(GUVs)

composed

of

dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), and lyso-PC in the inner monolayer and of DOPG/DOPC in the outer monolayer. We consider that in these GUVs the lipid packing in the inner monolayer was larger than that in the outer monolayer. Next, we investigated the interaction of magainin 2 with these GUVs with asymmetric distribution of lyso-PC using the single GUV method, and found that the rate constant of magainin 2-induced pore formation, kp, decreased with increasing lyso-PC concentration in the inner monolayer. We constructed a quantitative model of magainin 2-induced pore formation, whereby the binding of magainin 2 to the outer monolayer of a GUV induces stretching of the inner monolayer, causing pore formation. A theoretical equation defining kp as a function of magainin 2 surface concentration, X, reasonably explains the experimental relationship between kp and X. This model quantitatively explains the effect on kp of the lyso-PC concentration in the inner monolayer. Based on these results, we discuss the mechanism of the initial stage of magainin 2-induced pore formation. INTRODUCTION Antimicrobial peptides (AMPs), produced by various organisms such as amphibians, insects, plants and mammals, have activities that inhibit or kill bacteria and/or fungi; these AMPs are thought to help defend the producing organisms against their enemies.1,2 AMPs are highly positively charged peptides of 1040 amino acid residues; the high positive charge permits the peptides to bind strongly to the negatively charged outer monolayer of the plasma membrane of bacteria, while avoiding binding to the electrically neutral outer monolayer of the plasma membrane of eukaryotic cells.1 The antimicrobial activity of most AMPs is believed to result from the induction of damage in the bacterial plasma membranes.1,2 Among these AMPs, magainin 2, which was first isolated from the African clawed frog Xenopus laevis,3 has been extensively investigated. Natural magainin 2 is composed of L-amino acids; notably, magainin 2 that is composed of all D-amino acids has the same antibacterial activity as the native peptide.4 Thus, magainin 2’s antibacterial activity does not appear to require specific interactions with chiral


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receptors or proteins, suggesting that this AMP targets the lipid domains of bacterial membranes. Magainin 2 exhibits a high binding constant to negatively charged lipid bilayers,5 and the peptide forms an -helix in the membrane interface, parallel to the membrane surface.6,7 To further elucidate the mechanism of the bactericidal activity of magainin 2, the interactions of this AMP with lipid bilayers have been investigated using suspensions of small liposomes such as large unilamellar vesicles (LUVs)8-10 and using single giant unilamellar vesicles (GUVs) with diameters greater than 10 m.11-13 The membranes of these vesicles are typically composed of lipid mixtures that include negatively charged phosphatidylglycerol (PG) and electrically neutral phosphatidylcholine (PC); this composition mimics bacterial plasma membranes, which are negatively charged1 and (in E. coli) are composed primarily of PG. These studies indicate that magainin 2 induces leakage of internal contents, such as water-soluble fluorescent probes, from the inside of the vesicles, suggesting that magainin 2 induces pore formation in lipid bilayers. Using the single GUV method,14 detailed information on the elementary processes of magainin 2-induced pore formation have been obtained; such as the rate constant of the magainin 2-induced pore formation, kp, and the rate constants of membrane permeation (or leakage) through the magainin 2-induced pores.11-13 Several structural models for the formation of AMP-induced pores in biomembranes have been proposed,2 including the barrel-stave (or helix-bundle) structure15 and the toroidal (or wormhole) structure.16-18 In the barrelstave structure, peptides insert perpendicularly into the lipid membranes and a fixed number of peptides specifically associate with each other to form an -helix bundle that produces a narrow pore with a specific size. In contrast, in the toroidal structure, the outer and inner monolayers bend and merge each other in a toroidal fashion to form a pore in which the inner wall is composed of peptides and lipid head groups. Notably, tension-induced pores (created by various external forces) in lipid bilayers also exhibit toroidal structures.19-22 Generally, the stability of a toroidal structure consisting solely of lipids is not high, given the large line tension (i.e., line free energy per unit length) at the rim of a pore.20-22 However, the toroidal structure of AMP-induced pores may be stabilized by the presence of AMP molecules at the rim of the pore. Several researchers have proposed that magainin 2 forms a toroidal structure,1618

whereas results of molecular dynamic (MD) simulations support a disordered toroidal model.23 Recently, the pores

induced by peptides derived from Bax, a proapoptotic protein, have been convincingly shown to assume toroidal structures.24 However, it is important to keep in mind that these structures represent the final structures of pores at equilibrium, because these structures were obtained by structural analysis of a regular arrangement of pores using Xray diffraction and other physical methods. One can reasonably consider that the initial structures of pores induced


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by AMPs and pore-forming toxin proteins (PFTs) are different from the final structures. Hence, elucidation of the evolution (i.e., process) of pore formation is indispensable for understanding the mechanism of action of AMPs. For magainin 2, the size of pore is large at the initial stage and then decreases to reach a steady, equilibrium size.13 Similar results have been reported for other proteins such as Bax.25,26 To understand the initial structure of pores and the process of pore formation, it is important to know the location of peptides in a lipid bilayer during pore formation. Melittin, a pore-forming toxin-peptide, can translocate across a lipid bilayer to localize in both monolayers, with subsequent pore formation occurring in the lipid bilayer.27 This behavior is similar to that of cell-penetrating peptides (CPPs), which can pass through the plasma membrane of living eukaryotic cells. CPPs can be categorized as arginine-rich or amphipathic molecules.28 Transportan 10 (TP10), one of amphipathic CPPs, has been extensively investigated,29,30 and our recent studies of TP10 indicate that TP10 can translocate across a lipid bilayer and then pore formation occurs in the lipid membrane.31,32 In contrast, magainin 2 localizes only to the outer monolayer until just before pore formation,33 a behavior that is distinct from those of melittin and TP10. Our recent results indicate that the binding of magainin 2 to the outer monolayer of a GUV increases the area of the GUV bilayer, a change that strongly correlates with the rate constant of magainin 2-induced pore formation, kp.33 Moreover, an increase in the tension on a GUV membrane (e.g., by imposition of an external force) also increases the kp.33 On the basis of these results, we have proposed a hypothesis on the mechanism of magainin 2-induced pore formation, such that the stretching of the inner monolayer induced by the binding of magainin 2 to the outer monolayer is a main driving force of the pore formation. The purpose of this report is to test our proposed hypothesis on the mechanism of initial stage of magainin 2induced pore formation and to develop the construction of a quantitative model for this process. For this purpose, we first prepared GUVs with asymmetric distribution of lyso-PC (LPC) in the inner and outer monolayers, which were composed of (respectively) dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC)/LPC (at various ratios) and DOPG/DOPC (at various ratios). In these spherical GUVs, the total number of lipids in the inner monolayer exceeded that of the outer one by the number of LPC molecules; as a result, the optimal total area of the inner monolayer (i.e., the area of the membrane under no stretching and compression) was larger than that of the outer monolayer. It is known that the rate of transbilayer movement (i.e., flip-flop) of lipid molecules with large hydrophilic segments (i.e., polar head groups) is usually very small (~105 s1 depending on kinds of lipids and temperature),34-39 and hence, we can reasonably assume that this asymmetric composition can be maintained for


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enough long time (for several hours) and thus can be used for experiments. As described in S.1 of the Supporting Information (SI), in the case of spherical GUVs the ratio of the membrane thickness to the radius of vesicles is very small, and therefore the ratio of the number of lipids in both monolayers can be approximated as one. To maintain spherical shape of GUVs with asymmetric distribution of LPC, the ratio of the total area of the inner monolayer to that of the outer monolayer should be equal to one. As a result, the lipid packing in the inner monolayer of the lysoPC-containing GUVs is larger than that in the outer one. If our mechanism is correct, we can reasonably infer that in these GUVs the magainin 2-induced stretching of the inner monolayer (or an increase in area of the inner one) would be decreased, and therefore the kp of the magainin 2-induced pore formation would be decreased. We succeeded in preparing these GUVs with asymmetric distribution of LPC, and then investigated the interaction of magainin 2 with these GUVs using the single GUV method. It is worth to underline that recently several laboratories have made significant progress in research on tension-induced rupture of GUVs.20-22,40,41 Using the results of the tension-induced rupture, we developed a quantitative model of magainin 2-induced pore formation and compared this model with our experimental results. Finally, based on the model, we succeeded in quantitatively explaining the effect of asymmetric lipid packing in the inner and outer monolayers on magainin 2-induced pore formation. MATERIALS AND METHODS Chemicals and peptides DOPG, DOPC, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:1 lyso-PC; hereafter LPC) and 1-oleoyl2-hydroxy-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazole-4-yl)

(18:1-NBD-lyso-PE;

hereafter NBD-LPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Calcein was purchased from Dojindo Laboratories (Kumamoto, Japan). Alexa Fluor 647 hydrazide (AF647) was purchased from Invitrogen Inc. (Carlsbad, CA). Bovine serum albumin (BSA) was purchased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan). Magainin 2 was synthesized and purified as described previously.11 Preparation of GUVs and their observation GUVs of DOPG/DOPC/NBD-18:1 lyso-PE (40/(60x)/x; molar ratio) (hereafter, PG/PC/NBD-LPE) and those of DOPG/DOPC/18:1 lyso-PC (40/(60x)/x) (hereafter, PG/PC/LPC) were prepared by the natural swelling method.11 Briefly, 20 L Milli-Q water was added to a dry lipid film (2.0 mol lipid) in a glass vial, and the mixture was incubated at approximately 45 C for about 7 min (prehydration), with subsequent addition of 1 mL of buffer A (10 mM PIPES, pH 7.0, 150 mM NaCl, and 1 mM EGTA) containing 0.10 M sucrose; the resulting mixture was


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incubated for 2 to 3 h at 37 C. To obtain a purified GUV suspension, smaller vesicles and untrapped fluorescent probe were removed using the membrane-filtering method.42 Briefly, the GUVs prepared as above were centrifuged at 13,000  g for 20 min at 20 C and the supernatant was filtered through a nuclepore membrane with 10-mdiameter pores (Whatman, GE Healthcare, UK, Ltd., Buckinghamshire, UK) in buffer A containing 0.10 M glucose for 1 h at a flow rate of 1 mL/min (using a peristaltic pump) at room temperature (20-25 C). The retained suspension (i.e., the volume that did not pass through the filter) then was collected and used as the purified GUV suspension in the following experiments. To prepare GUVs containing calcein or AF647, we used buffer A containing 1 mM calcein or 6.0 M AF647 in the procedure of GUV preparation. After purification, each GUV suspension (~300 L) was transferred into a hand-made microchamber. Each microchamber which had been formed on a glass slide by inserting a U-shaped silicone-rubber spacer (for experiments using a micropipette)43 or two bar-shaped siliconrubber spacers positioned in parallel (for experiments using two micropipettes) between a glass cover slip and a glass slide.33 To prevent strong interactions between the glass surfaces and the GUVs, the inside of the microchamber was coated with 0.10 % (w/v) BSA in the same buffer as used for the experiments.43 The GUVs were observed using an inverted fluorescent differential interference contrast (DIC) microscope (IX-71, Olympus, Tokyo, Japan) with a 20 objective or a confocal laser scanning microscope (CLSM; FV1000-D, Olympus) with a 60 objective at 25  1 C; the temperature was controlled by a stage thermocontrol system (Thermoplate, Tokai Hit, Shizuoka, Japan).31- 33 Measurement of rim intensity of GUVs The fluorescence intensities (FIs) of the GUV membranes (i.e., the rim intensities) before and after the purification of GUVs were measured using the CLSM. We analyzed the resulting data using the poly-line mode in the CLSM software (Fluoview, v. 4.1, Olympus), and calculated mean values of FIs of all channels, which is proportional to the FI per unit length of the membrane. To examine the interaction of the new buffer with individual PG/PC/NBD-LPE (40/59/1)-GUVs, we added buffer A slowly in the vicinity of the GUV through a 20-m-diameter glass micropipette positioned using a micromanipulator. The distance between the GUV and the tip of the micropipette was ~50 m. Details of this method were described in our previous report.33 In the measurement of time course of rim intensity after purification (Fig. 1E), the FI of each GUV was measured for 10 s every 20 min during measurement up to 2 h (the shutter for incident laser was closed without measurement). Measurement of fractional change in the area of a GUV induced by the GUV’s interaction with magainin 2


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A standard micropipette aspiration method40,44 was used to measure the fractional change in the area of a single GUV. A single GUV was held at the tip of micropipette A (a ~10-m-diameter micropipette that had been coated with 0.50 % (w/v) BSA in buffer A containing 0.10 M glucose) for a few minutes by applying aspiration pressure P. This aspiration pressure was defined as P = Pout  Pin, where Pout and Pin were the pressure of the outside and the inside of the micropipette, respectively.44 P was adjusted so that the tension on the GUV membrane, , was 0.50 mN/m.  is defined as a function of P as follows:44

 

Pd P 4(1  d P / DV )

(1)

where dp is the internal diameter of the micropipette and DV is the diameter of the spherical cap segment (on the outside of the micropipette) of the aspirated GUV. Following equilibration of the GUV held at the tip of micropipette A, a magainin 2 solution was continuously added from micropipette B (a separate ~20-m-diameter micropipette) into the vicinity of the GUV. The distance between the GUV and the tip of micropipette B was ~40 m, and the P of the micropipette B was 30 Pa. The P was measured using a differential pressure transducer (DP15, Validyne, CA, USA), a pressure amplifier (PA501, Validyne, CA), and a digital multimeter.43 Glass micropipettes were prepared by pulling 1.0-mm borosilicate glass capillaries (G-1, Narishige, Tokyo, Japan) using a puller (PP-83 or PC-10, Narishige, Tokyo, Japan).43 Magainin 2 concentrations in the vicinity of the GUV were determined by the method described in our previous paper.33 The fractional change in the area of the GUV membrane is defined as  = S/S0, where S0 is the area of the GUV before the interaction with magainin 2 and S is the change in the area of the GUV membrane after the interaction with magainin 2. Several parameters are required to obtain . One parameter is the change in the projection length, L = Leq  L0, where Leq and L0 are the projection lengths of a part of the GUV inside the micropipette at equilibrium after and before (respectively) the interaction with magainin 2. The equation for  under assumption of constant volume is given by:44

 

S d p L 1  d p / Dv   S0 Dv20

(2)

where DV and DV0 are the diameter of the spherical cap segments of the aspirated GUV at equilibrium after and before (respectively) the interaction with magainin 2. The DIC images of the GUV were recorded using a chargecoupled device (CCD; CS230B, Olympus, Tokyo, Japan), and the parameters Leq, L0, DV, and DV0 were measured


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using video converter software (Intervideo WinDVD; Corel, Ottawa, Ontario, Canada). Measurement of the transbilayer movement of NBD-LPE in the GUVs interacting with magainin 2 We prepared PG/PC/NBD-LPE (40/59/1; inner)-PG/PC (40/59; outer)-GUVs containing 6.0 M water-soluble fluorescent probe AF 647 and purified them using the membrane filtering method (see the details in the above section). We investigated interaction of magainin 2 with these single GUVs in PIPES buffer containing 0.10 M glucose at 25 C using CLSM by the same method described in Ref. 33. The rim intensity of each GUV was measured using the method described above. To measure the FI of each GUV lumen due to AF647, we specified a small circle that was ~80% of the diameter of the GUV at the center of the GUV and measured the FI of this area.. Measurement of the rate constant of magainin 2-induced pore formation in single GUVs For this purpose, we used the single GUV method described in our previous papers.11-14 Briefly, an aliquot (300 l) of the purified GUV suspension (using 0.10 M sucrose in buffer A as the internal solution, and 0.10 M glucose in buffer A as the external solution) was transferred into a hand-made microchamber that had been pre-coated with 0.10 % (w/v) BSA in buffer A containing 0.10 M glucose. The GUVs were observed using an inverted fluorescent phase contrast microscope (IX-70, Olympus) with a 20 objective at 25  1 C; the temperature was controlled using a stage thermocontrol system (Thermoplate). Phase contrast and fluorescence images of GUVs were recorded using a high-sensitivity fluorescence EM-CCD camera (C9100-12, Hamamatsu Photonics K.K., Hamamatsu, Japan) with a hard disk. Three ND filters were used to decrease the intensity of the incident light, ensuring that the photobleaching of calcein did not occur. The FI inside each GUV was determined using an AquaCosmos (Hamamatsu Photonics K.K.), and the average intensity per GUV was estimated. Various concentrations of magainin 2 in buffer A containing 0.10 M glucose were added slowly in the vicinity of a GUV through a 20-m-diameter glass micropipette positioned using a micromanipulator. The distance between the GUV and the tip of the micropipette was ~70 m and the P of the micropipette was 30 Pa. The details of this method are described in our previous reports.33,43 To obtain kP for various GUVs, two independent experiments were carried out at each magainin 2 concentration using 20 single GUVs per experiment. Mean values and standard deviations (SDs) of kP values were calculated. Magainin 2 concentrations in the vicinity of the GUV were determined by the method described in our previous paper.33 RESULTS AND DISCUSSIONS Preparation of GUVs with asymmetric distribution of lyso-PC (DOPG/DOPC/lyso-PC (inner monolayer) and


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DOPG/DOPC (outer monolayer)) First, we developed a method to prepare GUVs with asymmetric distribution of lyso-PC (LPC), i.e., PG/PC/LPC (inner monolayer) and PG/PC (outer monolayer). In this method, we first prepared GUVs with symmetric lipid compositions of PG/PC/LPC (40/59/1) in buffer A, and then removed LPC from the outer monolayers of these GUVs by continuously replacing the buffer surrounding the GUVs with new buffer using the same method used for purification of GUVs (i.e., the membrane-filtering method42). Since the critical micelle concentration (CMC) of LPC is 3 M,45 and the insertion of LPC into a membrane from the surrounding aqueous solution and the unbinding of LPC from the membrane into aqueous solution are rapid,46 we reasonably expected that the LPC in the outer monolayer would be rapidly transferred into the aqueous solution. To verify this new method and to confirm the

Figure 1 (A)

(B)

(D)

(C)

4000 Rim intensity of GUV

Rim Intensity of GUV

4000 3000

3000

2000

2000

1000

1000

0

0

0

180 360 540 720 900 1080 Time (s)

(E) Normalised Rim Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

2 5 30 70 Dilution ratio

MP

AF

1.0 0.8 0.6 0.4 0.2 0.0 0

20 40 60 80 100 120 Time (min)

Figure 1. CLSM images of PG/PC/NBD-LPE (40/59/1)-GUVs. (A) One of the GUVs before purification and (B) one of the GUVs after purification. The bar represents 20 m. (C) The effect of interaction of a GUV with new buffer provided through a micropipette on its rim intensity. The GUV which was 5-fold diluted before purification was used. (D) The rim intensities of the GUVs at 30 min after dilution with the buffer in various dilution ratio. MP indicates the GUVs after diluted for 18 min with the buffer provided through micropipette. AF indicates the GUVs after purification using membrane-filtering method. (E) Time course of rim intensity of the GUV after purification.


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composition of the GUVs, we prepared these GUVs using a fluorescent analogue of LPC, NBD-LPE, instead of LPC. Figures 1A and 1B show representative confocal images of a PG/PC/NBD-LPE (40/59/1)-GUV (with fluorescence due to NBD-LPE) before and after (respectively) 1 h of purification. To obtain the images of the GUVs shown in Fig. 1A, the GUV suspension after preparation (but before purification) was diluted 5-fold using buffer A and then it was observed at 30 min after the dilution because the equilibrium was attained at 19 min after the dilution (see Fig. S1 in SI). These images indicated that the FI of the GUV membranes (i.e., the rim intensity) after purification was decreased compared to that before the purification. We measured the rim intensity of multiple GUVs and obtained rim intensity values (mean ± SD) before and after purification of 3570  190 (n = 10 examined GUVs) and 1790  110 (n = 14), respectively. The ratio of the FI after purification to that before purification was 0.49. This result indicated that the concentration of NBD-LPE in the GUV membrane was approximately halved by purification, suggesting that the unbinding of NBD-LPE from the outer leaflet to aqueous solution outside the GUVs occurred and its concentration in the outer leaflet was almost zero. To confirm whether the unbinding of NBD-LPE from the outer leaflet to aqueous solution is rapid, we investigated the interaction of the buffer with a single PG/PC/NBD-LPE (40/59/1)-GUV before purification. First, we selected a PG/PC/NBD-LPE (40/59/1)-GUV under the CLSM, and then the buffer in the neighborhood of the GUV was continuously replaced by flow of new buffer through the micropipette. The rim intensity gradually decreased with time, achieving a plateau of approximately half the original intensity after ~700 s (Fig. 1C). This result indicated that the unbinding of NBD-LPE from the outer leaflet of the GUV into the surrounding aqueous solution was rapid. If the concentration of NBD-LPE in the aqueous solution is held constant, the binding of NBD-LPE to the membrane is in the equilibrium state. Taking this into account, one can presume that the NBD-LPE concentration in the outer monolayer of a GUV would decrease as the volume of the aqueous solution outside the GUVs increased. To confirm this hypothesis, after preparation we diluted the GUV suspension with the buffer at different dilution ratios, and then observed the GUVs using CLSM. As shown in Fig. 1D, rim intensity decreased with increased dilution ratios of the GUV suspension at observation (i.e., increased volumes of the aqueous solution outside the GUVs). Hence, the results shown in Fig. 1D supported the above conclusion regarding equilibrium binding. During the purification of a GUV suspension using the membrane-filtering method, the buffer surrounding the GUVs was rapidly exchanged with a new one that lacks NBD-LPE, which induces unbinding of NBD-LPE from the outer


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monolayer; after 1 h of purification, the NBD-LPE concentration in the outer leaflet approached zero. The rim intensity of the GUVs after purification was a little smaller than that after interaction of the buffer using the micropipette. Using these asymmetric GUVs, we can estimate the rate of transbilayer movement of NBD-LPE from the inner monolayer to the outer one in the GUVs. Figure 1E shows the time course of rim intensity of PG/PC/NBD-LPE (40/59/1; inner monolayer)-PG/PC (40/59; outer monolayer)-GUVs after 1h of purification. The normalized rim intensity was almost constant (≈ 1.0) (i.e., rim intensity did not decrease significantly with time) within 2 h of observation using CLSM, showing that this asymmetric structure of GUVs was almost stable for at least 2 h. On the basis of the result of Fig. 1C, one can reasonably consider that if the transbilayer movement of NBD-LPE from the inner monolayer to the outer one occurs, the NBD-LPE molecules in the outer monolayer rapidly unbind to the aqueous solution outside the GUV, inducing the decrease in rim intensity. The normalized rim intensity at 2 h was 0.981  0.005 (n = 8), indicating that 2% of NBD-LPE transferred from the inner to the outer monolayer for 2h. Therefore, we can conclude that the transbilayer movement of NBD-LPE was negligible under the experimental conditions used in our experiments. Moreno et al.38 measured the rate constant of transbilayer movement of NBDLPE (myristoyl) in palmitoyloleoyl-PC (POPC)-LUVs with 100 nm diameter using the method47 of reduction of the NBD group by dithionite. They obtained that it was 7.5  106 s1 at 25 C (Fig. 2A in ref. 38). This rate constant predicts that the normalized rim intensity after 2 h is 0.95, which is a little smaller than the above result. The rate of transbilayer movement of phosphatidylcholine at 37 C or 30 C was 105106 s1,35,36,39 and that of phosphatidylglycerol was a little larger than that of PC,35 and hence, we can reasonably consider that the transbilayer movement of DOPC and DOPG in PG/PC/NBD-LPE (40/59/1; inner monolayer)-PG/PC (40/59; outer monolayer)GUVs were negligible within 2 h at 25 C. These results support the statement that the asymmetric distribution of NBD-LPE in the two monolayers of these GUVs was almost stable for at least 2 h. Next we estimate the NBD-LPE concentration in the inner monolayer of GUVs after purification. In the inner monolayer, some unbinding of NBD-LPE into the aqueous solution in the GUV lumen may occur, but this NBDLPE is expected to rapidly reach a binding equilibrium. We obtained an apparent binding constant (KB) of 1.1  105 M1 for binding of NBD-LPE to PG/PC (4/6) membranes (see S.3 in SI for details). We used this KB value to calculate the mole fraction of the NBD-LPE in the inner monolayer (i.e., NBD-LPE concentration in the inner monolayer), and it was 9.9  103; this mole fraction was very close to that of the initial value (1.0  102). In contrast, the NBD-


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Page 12 of 35

LPE concentration in the GUV lumen was 8.9  102 M, which was much less than its CMC (12 M; see Fig. S2 in SI for details). Therefore, the purified GUVs possessed asymmetric lipid compositions in the inner (PG/PC/NBDLPE, 40/59/1) and outer (PG/PC, 40/59) monolayers, and the amount of lipid in the inner monolayer exceeded that in the outer one by 1 mol% NBD-LPE. Hence, these GUVs were expected to exhibit compression of the inner monolayer, i.e., the higher lipid packing in the inner one. Using the same method, we prepared GUVs with asymmetric distribution of LPC, i.e., PG/PC/LPC (at various ratios) in the inner monolayer and PG/PC (at various ratios) in the outer monolayer, based on the assumption that the KB of LPC was similar to that of NBD-LPE. Magainin 2-induced area change in individual PG/PC/LPC (inner)-PG/PC (outer)-GUVs In a previous report, we demonstrated that the binding of the magainin 2 to the outer monolayer of PG/PC-GUVs induces an increase in the area of the GUV membranes.33 Here, we investigated the effects of the asymmetric distribution of LPC in GUVs on the magainin 2-induced area change of the GUV membranes. First, a PG/PC/LPC (40/58.5/1.5; inner)-PG/PC (40/58.5; outer)-GUV was held at the tip of micropipette A by aspiration; this aspiration provided a constant tension of 0.5 mN/m to the GUV membrane. After this tension had been maintained for two minutes (which was expected to eliminate problems in hidden areas40,44), magainin 2 solution was continuously delivered into the neighborhood of the GUV via micropipette B.33 As magainin 2 interacted with the GUV, the projection length rapidly increased with time to reach a steady value within 1 min (Fig. S4), indicating that the fractional change in the area of the GUV membrane, , rapidly increased over time to reach a steady value (ST) within 1 min. For the interaction of 5.8 M magainin 2 with asymmetric GUVs, ST was 0.021  0.002 (n = 28) (e.g., Fig. S4A). As a control experiment, we investigated the magainin 2-induced area change of symmetric PG/PC (40/58.5)-GUVs; this control experiment yielded a ST = 0.030  0.002 (n = 31) (e.g., Fig. S4B), a value that was

Table 1: Experimental and estimated values of magainin 2-induced area change, ST, in the presence and the absence of LPC in the inner monolayer. Mean values and SDs of all the examined GUVs are shown (n is the number of the examined GUVs).

lyso

Experimental ST

Magainin 2

LPC conc.

conc. (M)

(mol %)

2.9

0

0

(2.3  0.2)  102 ( n = 35)

2.9

1.5

0.75  102

(1.6  0.1)  102 ( n = 25)

5.8

0

0

(3.0  0.2)  102 ( n = 31)

5.8

1.5

0.75  102

(2.1  0.2)  102 ( n = 28)


Estimated ST

(1.6  0.2)  102 (2.3  0.2)  102

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Langmuir

significantly larger than that obtained with the asymmetric GUVs. Performing the same experiments at 2.9 M magainin 2 yielded similar results (Table 1). The removal of LPC from the GUV outer monolayer decreases the total optimal area of this monolayer. If the initial outer monolayer (i.e., before removal of LPC) is composed of NPG molecules of PG, NPC molecules PC, and NLPC molecules LPC (the total number of lipids is NPG + NPC + NLPC), then its total area S0 is (Nt APC + NLPCAPC/2), where Nt is the total number of PG and PC molecules and APC is the cross-sectional area per molecule of PC and PG, and we approximated the cross-sectional area of an LPC molecule as half that of a DOPC molecule, an estimate that was used in previous reports.48,49 After removal of NLPC molecules of LPC from this outer monolayer (its mole fraction in the monolayer is NLPC / (NLPC + Nt) ≈ NLPC / Nt = 0.015), the total area of the outer monolayer S1 becomes Nt APC. The fractional change in the area of the outer monolayer in the absence of LPC (due to the removal of LPC) compared with that in the presence of LPC, lyso, can be calculated as lyso = (S1  S0)/ S0 = NLPC/2 Nt = 0.0075. In the presence of LPC, the binding of magainin 2 to the outer monolayer increases the area of the GUV bilayer, and its fractional change of the area of the GUV bilayer can be expressed by mag. The absence of LPC in the outer monolayer decreased the magainin 2-induced effective fractional change in the area of the outer monolayer, thereby that of the inner monolayer, intotal, by lyso (i.e., intotal = mag + lyso), because in the spherical GUVs the area of the outer monolayer is the same as that of the inner monolayer. Thus, ST (1.5 % LPC) = intotal = ST (0 % LPC) +

lyso, which provides an estimated value for ST (Table 1). We found that the experimental value of ST (1.5 % LPC) agreed with the estimated value, within experimental error. Magainin 2-induced pore formation in individual PG/PC/LPC (inner)-PG/PC (outer)-GUVs Next, we examined the effects of the asymmetric distribution of LPC in GUVs on magainin 2-induced pore formation. For this purpose, we first investigated magainin 2-induced membrane permeation of the fluorescent probe calcein from individual PG/PC/LPC (40/59.5/0.5; inner)-PG/PC (40/59.5; outer)-GUVs. The interaction of magainin 2 with individual GUVs containing 1.0 mM calcein was performed in PIPES buffer containing 0.10 M glucose at 25 C, and the resulting vesicles were analyzed using the single GUV method.12,14 A representative experimental result for the effect of 31 M magainin 2 on the calcein concentration within a single GUV is shown in Fig. 2A. Prior to magainin 2 addition, the GUV displayed high contrast in a phase-contrast microscopic image (Fig. 2A (I)) due to the difference in refractive index produced by the difference in the concentrations of sucrose and glucose between the inside (0.10 M sucrose) and the outside (0.10 M glucose) of the GUV. A fluorescence microscopic image of the same


Langmuir

Figure 2 (A)

(C)

1.0 0.8 0.6 0.4 0.2 0

Pintact (t)

Fluorescence intensity

(B)

0

(D)

100 200 Time (s)

300

1.0 0.8 0.6 0.4 0.2 0

0

100 200 300 400 Time (s)

kp (s-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.01

0.001

0.00 0.25 0.50 0.75 1.00 Lyso PC (mol%)

Figure 2. Magainin 2-induced leakage of calcein from single asymmetric GUVs in the presence of LPC in the inner monolayer. (A) Interaction of 31 M magainin 2 with PG/PC/LPC (40/59.5/0.5; inner)-PG/PC (40/59.5; outer)GUVs. Fluorescence images (II) show that the calcein concentration inside the GUV progressively decreased after the addition of magainin 2. The numbers above each image show the time in seconds after the magainin 2 addition was started. Also shown are phase contrast images of the GUV at time 0 (I) and at 370 s (III). The bar corresponds to 30 m. (B) Time course of the change of the fluorescence intensity of the GUV shown in (A). (C) Time course of Pintact of single GUVs of PG/PC/LPC (40/(60x)/x; inner)-PG/PC (40/(60x); outer) in the interaction of 31 M magainin 2, where x is the LPC concentration in the inner monolayer: (○) x = 0.50, () 0.25, and (□) 0 mol% LPC. The red solid lines represent the best fit curves of eq. 3. The obtained values of kP were 0.0035, 0.0058, and 0.0098 s-1 for 0.50, 0.25, and 0 mol% LPC, respectively. (D) Dependence of kP on LPC concentration in the inner monolayer (mol%), x. (○) 62 M, and (●) 31 M magainin 2. The mean values and SDs of kp are shown.

GUV (Fig. 2A (II)) showed a high concentration of calcein inside the GUV at this time. The FI inside the GUV remained almost constant over the first 210 s following the addition of the magainin 2 solution; the FI decreased gradually thereafter (Fig. 2A (II)). Figure 2B shows a time course of normalized FI (=I(t)/I(0)), where I(t) and I(0) are the FI of the inside of the GUV at time t and that of the intact GUV before initiation of membrane permeation by calcein, respectively. After 360 s, the FI inside the GUV (I(t)) had decreased to less than 10 % of I(0), although a phase-contrast image of the same GUV (Fig. 2A(III)) showed that the GUV structure did not change; any detectable breaks, associations, membrane fusion of GUVs, and large shape changes were not observed during this interval. As discussed in our previous reports on the interaction of magainin 2 with single GUVs,11-13 the observed decrease in


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Langmuir

FI occurred as a result of the membrane permeation of calcein (i.e., calcein leakage) from the inside to the outside of the GUV through magainin 2-induced pores in the membrane. Thus, the time at which the FI began to decrease corresponds to the time when pore formation started in the membrane. Furthermore, a comparison of the phasecontrast images in Figs. 2A(I) and 2A(III) showed that there was a substantial decrease in the phase contrast of the GUV, indicating that, during the membrane permeation of calcein, sucrose and glucose also passed through the same pores. When the same experiments were carried out in 20 individual GUVs, we observed that, although the membrane permeation of calcein from a GUV initiated stochastically, the time courses of the changes in the FIs of individual GUVs were highly similar. This observation indicated that pores were formed stochastically. We next analyzed the magainin 2-induced pore formation more quantitatively. During the initial time when the FIs inside the GUVs were almost constant, magainin 2 molecules were bound to the outer monolayer of the GUV membrane, but the GUV integrity retained (i.e., the GUV was intact) before the sudden pore formation. As demonstrated in our previous reports,11-14 when estimating the rate constant of pore formation at its initial stage, it is important to know the time course of the fraction of intact GUV from which calcein did not start to leak, i.e., no pores in the GUV membrane, among the population of GUVs examined, Pintact(t). In the case of magainin 2, we considered a two-state transition model from the intact state to the initial pore state. We found that the Pintact of GUVs in the presence of various concentrations of magainin 2 were well fit by a single exponential decay function, defined as follows:11,12

Pintact (t )  exp k P (t  t eq )

(3)

where kP is the rate constant of the two-state transition, which can be considered the rate constant of magainin 2induced pore formation at its initial stage, and teq is a fitting parameter. Figure 2C shows that the decrease in Pintact over time was fit by a single exponential decay function defined by Eq. 3, and this fitting yielded a kp value of 3.5  10−3 s−1. Two independent experiments (N = 2) similar to the experiment shown in Fig. 2C were carried out to obtain mean values and SDs for kP. The mean ± SD value of kP for 0.50 mol% LPC was (3.2 ± 0.5)  10−3 s−1, a value that was smaller than the kP in the absence of LPC ((8.6 ± 1.6)  10−3 s−1). The kP decreased with an increase in the concentration of LPC in the inner monolayer of GUVs (Fig. 2D). When we investigated the interaction of 62 M magainin 2 with the asymmetric GUVs, we obtained a similar result (Fig. 2D). We performed the same experiments using NBD-LPE (instead of LPC) in the inner monolayer, i.e., we examined the magainin 2-induced pore formation in asymmetric GUVs composed of PG/PC/NBD-LPE (inner)-PG/PC (outer). We found that the kP decreased with


Langmuir

increasing NBD-LPE concentration (Fig. S5), which is similar to the result of the effect of LPC described above. Estimation of the transbilayer movement of NBD-LPE in the GUVs interacting with magainin 2. To examine the transbilayer movement of NBD-LPE from the inner monolayer to the outer one in the GUVs interacting with magainin 2, we investigated magainin 2-induced membrane permeation of the fluorescent probe, AF647, from individual PG/PC/NBD-LPE (40/59/1; inner)-PG/PC (40/59; outer)-GUVs. The interaction of 31 M magainin 2 with individual GUVs containing 6.0 M AF647 was performed in PIPES buffer containing 0.10 M glucose at 25 C using CLSM. Among 30 examined GUVs, the leakage of AF647 occurred (i.e., pore formation occurred) in 11 GUVs and in other 19 GUVs no leakage of AF647 occurred (i.e., no pore formation occurred) within 10 min. Figure 3A shows a representative experimental result that no pore formation occurred in the GUV. The FI inside the GUV due to AF647 remained almost constant over 10 min of interaction of magainin 2, indicating no leakage of AF647. The rim intensity due to NBD-LPE also remained almost constant during 10 min, indicating that the transbilayer movement of NBD-LPE did not occur significantly. Therefore, this result clearly indicates that only the binding of magainin 2 to the GUV membrane did not increase the rate of transbilayer movement of NBD-LPE.

Figure 3 (A) 1.2

Normalised lumen intensity


3000

1.0

2500

0.8

2000

0.6

1500

0.4

1000

0.2

500 0

0.0 0

100

(B)

200

300

400

Time (s)

500

600

3000

1.2

Normalised lumen intensity


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0

2500

0.8

2000

0.6

1500

0.4

1000

0.2

500 0

0.0 0

100

200

300

400

500

600

Time (s)

Figure 3: Relationship between the time course of the rim intensity of the GUVs due to NBD-LPE and that of the fluorescence intensity of the GUV lumen due to AF647 during the interaction of 31 M magainin 2 with PG/PC/NBD-LPE (40/59/1; inner)-PG/PC (40/59; outer)-GUVs. (A) The case of no pore formation, (B) the case of pore formation.


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Langmuir

On the other hand, Figure 3B shows a representative experimental result that pore formation occurred in the GUV. The FI inside the GUV due to AF647 remained almost constant over the first 197 s following the addition of the magainin 2 solution; the FI decreased gradually thereafter. This result demonstrates that magainin 2-induced pore formation started to occur at 197 s. The rim intensity due to NBD-LPE also remained almost constant over the first 208 s and then decreased gradually, indicating that transbilayer movement of NBD-LPE started to occur at 208 s. The difference between the time when the rim intensity started to decrease (tf) and the time when pore formation started to occur (tp), i.e., t = tf  tp, was 11 s. We analyzed the values of t in all the examined GUVs with pore formation, and obtained the mean value of t was 12  3 s (n = 11). This result indicates that the rate of the transbilayer movement of NBD-LPE started to increase when the pore formation started. One can conclude from these results that the state of asymmetric distribution of NBD-LPE in the GUVs during the interaction of magainin 2 remained stable until pore formation occurs. The above results clearly testify that only the binding of magainin 2 to the membrane did not increase the rate of transbilayer movement of NBD-LPE but appearance of the pore in membrane increased it (Fig. 3). It has been reported in the studies using the LUV suspension method that the interactions of AMPs and venom peptides with lipid vesicles increased the rate of transbilayer movement of lipids.8,16,50,51 Matsuzaki et al. suggested using this method a strong correlation between the magainin 2-induced leakage of internal contents and the flip-flop of lipids,6 supporting our results obtained by the single GUV method. Note that it is very difficult (and maybe impossible) to determine the elementary processes of such transbilayer movement of lipids in the framework of the LUV suspension method. In contrast, our method using single GUVs clearly revealed these processes. The rate of transbilayer movement of NBD-LPE after the pore formation (Fig. 3B) was much larger than that in the normal lipid bilayer shown in Fig. 1E, indicating that the mechanism of the transbilayer movement of NBD-LPE after the pore formation is different from that of normal transbilayer movement. Now we discuss the possible mechanism for this acceleration. It is reported that the structures of pores induced by peptides such as magainin 2 and Bax-derived peptide at equilibrium are toroidal structures.17,18,24 One can assume that the structure of the magainin 2-induced pore at initial stage is also a toroidal structure. It means that after pore formation a continuous integrated lipid monolayer (composed of inner monolayer, outer monolayer, and monolayer forming the pore wall) arises. It is reasonable to consider that lipid molecules (including NBD-LPE) can rapidly diffuse along this integrated lipid monolayer from the inner leaflet to the outer one through the wall of the pore (i.e., inducing rapid transbilayer


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movement). Although there are no direct experimental evidences on the structure of the pores at their initial stage, this scenario seems to be most probable currently. Model of magainin 2-induced pore formation In our previous paper,33 we demonstrated that the binding of magainin 2 to the interface of the GUV outer monolayer increases the fractional change in the area of the GUV bilayer, as defined by the equation  = S/S0, (where S0 and S are the initial area and the area change of the bilayer, respectively). We also showed that  is proportional to the magainin 2 surface concentration in the outer monolayer, such that  = m X, where X (mol/mol) is the molar ratio of magainin 2 bound to the membrane interface to total lipids of the outer monolayer, and m is a proportionality constant that has a value of m = 0.58  0.01. We further demonstrated that magainin 2 molecules were localized only in the outer monolayer, just before pore formation.33 These results indicated that the binding of magainin 2 to the outer monolayer increases the area of GUV bilayer, inducing a stretching of the inner monolayer, resulting in the lateral tension (in) in the inner monolayer. Generally, in is proportional to the fractional change in the area of the inner monolayer in (i.e.,  in = 1/2KAin, where KA is the elastic compressibility modulus of the bilayer). For spherical GUVs, we can reasonably presume that the total area of the inner monolayer is the same as that of the outer monolayer, such that in = . Using the value of KA (141  5 mN/m) obtained for PG/PC (4/6)-GUVs, we calculated the lateral tension in the inner monolayer of the GUV, in (= 1/2KA). X can be converted to in using

 = 0.58X: thus in = 70.5 = 41X (mN/m) = b X (where b is a constant, b = 41).33 Recent studies indicate that stretching of lipid bilayers or lateral tension on lipid bilayers due to external forces induces pore formation in the lipid bilayers or rupture of GUVs.20-22,40,41 As described in the above paragraph, the binding of magainin 2 induces stretching of the inner monolayer. Based on these results, we proposed a model of the magainin 2-induced pore formation: the binding of magainin 2 in the outer monolayer induces the stretching of the inner monolayer, which induces pore formation in the bilayer.33 Here, we consider this model quantitatively. It is well known that thermal fluctuations of lateral density of lipid molecules (local condensation and local rarefaction) exists in lipid membranes. The probability of formation of a local rarefaction is larger in a stretched membrane or when tension is applied. When the size of such rarefaction reaches a critical value, this area converts into a hydrophilic prepore with an effective radius, which pierces the lipid bilayer (i.e., a transmembrane prepore).41 If the radius of a prepore is less than the critical radius r*, the prepore closes quickly. However, if the radius expands and reaches r*, the prepore transforms into a pore. This is the mechanism of tension-induced rupture of GUVs due to


Page 19 of 35

Figure 4 (A)

(B)

20

B

A

C

15

D

U(r)/kT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

10

E

5 0

0

1

2

3 4 5 r (nm)

6

7

8

Figure 4. (A) A scheme of a half-prepore in the inner monolayer stretched by the bound magainin 2 in the outer monolayer. The radius of a half-prepore, r, is indicated. Magainin 2 is indicated by a rectangle. The direction of the stretching of the inner monolayer is indicated by arrows. See the text for a detailed explanation. (B). Dependence of the free energy of a half-prepore in PG/PC (4/6) membrane, U (r), on the half-prepore radius for various surface concentration of magainin 2, X: (A) 0, (B) 50, (C) 70, (D) 80, and (E) 95 mmol/mol. U (r) is calculated according to eq. 7 using U0 = 1.1 kT, B = 1.8 mN/m, b = 41,  = 12.4 pN,  = 2.8 mN/m.

pore formation in their membranes. 20-22,40,41 In contrast, in the interaction of magainin 2 with GUVs, only the inner monolayer is stretched. Therefore, a prepore is expected to form only in the inner monolayer, as shown in Fig. 4A, forming what we refer to as a “half-prepore”. This half-prepore has a certain effective size, which can be approximated as a circle with radius r. The behavior of such half-prepores can be described in a framework of the theory of tension-induced pore formation.19,21,22,41,52 Generally, the free energy of a prepore, U (r), consists of two terms: one (r2) that is associated with lateral tension

(), favoring expansion of the prepore; and another (2r)

that is associated with the line tension (Γ) (i.e., the line free energy per unit length) of the prepore edge, favoring prepore closure.19,20 To consider the free energy of this half-prepore in the inner monolayer, it is necessary to take into account that the length of a half-prepore is half of that of a transmembrane prepore, i.e., half of the bilayer thickness. Hence the contribution of line tension in the free energy of a half-prepore would be r. Additionally it is necessary to take into account that the terminal hydrocarbon chains of lipid molecules in the outer monolayer located opposite to the half-prepore partly contact with water in the inside of the GUV. Thus, this site produces an extra free energy due to exposure of the hydrocarbon chains to water. We describe the interfacial free energy per unit area of this site as . The extra free energy of this site would therefore be  r 2 . Hence the free energy of a halfprepore can be described as follows.


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U r, X   U 0   r   in    r 2  U el  U 0   r  bX    r 2  U el

Page 20 of 35

(4)

where U0 is a term that does not depend on tension,41 and Uel is the change of the electrostatic energy of the charged GUV as result of half-prepore formation.53 For charged membranes such as PG/PC, the effects of electrostatic interactions on tension-induced pore formation must be considered. For a transmembrane prepore, the Uel was considered in detail in Ref. 53. Using the same approach, one can represent Uel for a half-prepore as follows:

U el  r 2 B , ,

(5)

2Ω

ln

(6)

where B is a term due to electrostatic interactions arising from surface charges in a monolayer, the surface charge density of the GUV membrane  equals eY/A0; e is the elementary charge; Y is the mole fraction of charged lipid in the membrane; A0 is the cross-sectional area of a lipid molecule in the bilayer before magainin 2 binding (here we assume that A0 of a DOPG molecule is the same as A0 of a DOPC molecule (72.5 Å2));54 p  2BY A0 1    ,

q  1  p 2 ; B is the Bjerrum length in water ( B  e 2 4kT 0 w  0.716 nm at 25 C); D is the Debye length ( D  1    0 w kT 2Сe ); C is salt concentrations in the buffer, w is the relative dielectric constant of 2

water; and 0 is the permittivity of free space. To generalize this calculation, we assumed that the surface charge density of the half-prepore wall, p, is less than that of the GUV surface, and that p = a (where a is an adjustable parameter). Therefore, the total free energy of a half-prepore in a charged membrane with a fixed Y is:

U r   U 0   r  bX  B    r 2

(7)

U (r) has a maximum of Ua at r  r*   2bX    B  as follows:

U a  U r *  U0   2 4bX    B 

(8)

where Ua corresponds to the activation energy of the tension-induced pore formation. If the radius of a half-prepore is less than the critical radius r*, the half-prepore closes quickly. However, if the radius expands and reaches r*, the half-prepore transforms into a half-pore, and finally into a transmembrane pore. Note that the radius of a half-prepore is much smaller than that of a transmembrane pore. Figure 4B shows examples of the free energy profiles U(r) for different values of X, showing that as magainin 2 surface concentration X increases, the free energy barrier of the activation energy (Ua) decreases. This analysis indicates that the rate constant of magainin 2-induced pore formation


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(kP) should increase with X. In our previous paper,41 we demonstrated that the Arrhenius equations for the rate constant kr of constant-tensioninduced rupture of GUVs (calculated using a theoretical equation for Ua and incorporating parameter values determined by the analysis of the results of temperature dependence of kr) fit well to the experimental data for the tension dependence of kr for PG/PC-GUVs. Here, we applied this equation to the rate constant of magainin 2-induced pore formation, kp, using Eq. 8 as follows.

k P  AF exp(U a / kT )  AF exp(

   U0   2 ) exp  kT  4kT bX    B 

(9)

where AF is the pre-exponential factor, which has a meaning of the frequency factor. For the constant-tension-induced pore formation in PG/PC (4/6) bilayers in buffer A,41 we obtained the best fit values for the parameters by the analysis of the activation energy of constant-tension-induced pore formation as follows:41 B = 1.8 mN/m (i.e., a = 0.49),  = 12.4 pN, U0 = 9.0 pN nm, and AF = 5.0 104 s−1. We can use the same values of B, , and AF for magainin 2-induced pore formation where the stretching of only inner monolayer is considered, because we used in the magainin 2 experiments the same PG/PC (4/6) membranes under the same conditions (such as buffer, salt concentration, and temperature) as used in the constant-tension-induced rupture of GUVs41 and also we already established the fact that the inner monolayer does not contain magainin 2 before pore formation and thus it is the pure PG/PC monolayer.33 As described in S.4 of the SI, the rate constant kr of tension-induced rupture of GUVs of PG/PC/LPC (40/(60-x)/x)– GUVs containing different mol fraction of LPC (0  x  1.0) were almost the same within experimental error. This result indicates that the effect of LPC on constant-tension-induced pore formation is negligible at these low mole fractions of LPC and thus we can apply Eq. 9 to the magainin 2-induced pore formation in these GUVs. It is noted that the value of U0 for a half-prepore in the inner monolayer may be half that for a prepore in the bilayer (4.5 pN nm), because U0 is the nucleation free energy required to form a hydrophilic half-prepore. If we use these values, Eq. 9 has only one fitting parameter, i.e., . The experimental data of kp vs. X (Fig. 1D in Ref. 33) were well fit by Eq. 9 with these parameter values, providing a best-fit value of  = 2.8 mN/m (Fig. 5A). This fitting suggested that the proposed model of magainin 2-induced pore formation is valid. Using all the parameter values and the relation

in = b X, Eq. 9 is converted to following equation;


Langmuir

Figure 5 0.1

kp (s-1)

(A)

0.01

0.001 50

(B)

60

70 80 90 X (mmol/mol)

100

0.1

kp and kr (s-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.01

0.001

0

1

2 3 in (mN/m)

4

Figure 5. (A) The rate constant, kp, of magainin 2-induced two-state transition from the intact state to the pore state in PG/PC (4/6)-GUVs as function of X. The circle indicates the experimental values of kp. The solid line shows the best-fit curve using the theoretical eq. 9 for AF = 5.0 104 s−1, U0 = 1.1 kT, B = 1.8 mN/m,  = 12.4 pN,  = 2.8 mN/m. (B) The comparison between the rate constant of the magainin 2-induced initial pore formation, kp, and that of constant-tension-induced pore formation, kr, as a function of in. Experimental results of the magainin 2-induced pore formation (black open circle) and the constant-tension-induced rupture of PG/PC (4/6)-GUVs (blue solid circle) are reprinted from refs. 7 and 8 with permission, respectively.

  1 29.4 kP  16600 exp  (s )   1 . 04 [ mN/m ]   in  

(10)

Under these conditions, the values of the critical radius r* can be estimated to 2.7 nm for X = 0.080 and 3.0 nm for X = 0.075. Figure 4A shows that the radius of prepore r is the summation of the length of lipids in the rim of the half-prepore and the radius of a water region. Therefore, the radius of water region in prepores is much smaller than r*. Figure 5B (black open circles) shows the experimental data of kp vs. in. To compare the values of kp with those of the rate constant of constant-tension-induced rupture, kr, we plotted the kr values (blue solid circles) in Fig. 5B. It is evident that the dependence of these rate constants (kp and kr) on tension in the inner monolayer, in, are almost the same, which supports the validity of our model of magainin 2-induced pore formation. Although the parameter ε has the same physical origin as γ, the value of ε is much less than that of γ (39 mN/m)55.


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While there exist several possible explanations for this difference, we focus here on an explanation that addresses the nanosize of the half-prepore. Specifically, Glaser et al.56 showed that the interfacial tension between hydrophobic lipid tails and water inside narrow hydrophobic pores is proportional to pore radius r and becomes very small in pores with radius ~1 nm. Molecular dynamic simulations also showed pronounced decreasing effects of interfacial tension in small pores.57 Furthermore, we can presume that the terminal hydrocarbon chains in a half-prepore are partly covered by distorted lipids of the inner monolayer, such that the hydrophilic segments of these lipids are exposed to water (i.e., exposing the more hydrophilic surface) in the half-prepore. Thus, lipid molecules are expected to cover the hydrophobic surface of the rim of a prepore to make the prepore more hydrophilic, thereby decreasing the free energy (e.g., as would be seen with a quasi-toroidal structure). GENERAL DISCUSSION In the present work, we have developed a new method of preparation of GUVs with asymmetric distribution of LPC and NBD-LPE in the two monolayers. We can reasonably consider that the density of lipids in the inner leaflet of the GUVs is higher than that in the outer one, and therefore, these GUVs have the asymmetric lipid packing in the monolayers. The asymmetric distribution of NBD-LPE was stable at least two hours because the transbilayer movement of NBD-LPE from the inner monolayer to the outer one and also the unbinding of NBD-LPE from the inner leaflet to aqueous solution in the GUV lumen were not observed within 2 h (Fig. 1E). Using MD simulation, Esteban-Martin et al. demonstrated the stability of asymmetric lipid bilayers composed of mainly dipalmitoyl-PC (DPPC).58 They have shown that the bilayers were stable if the differences in number of lipids in both monolayer were enough small. The reason of such stability is that the area per lipid in the leaflet with smaller number of lipids increases (i.e., stretching occurs) and that in the leaflet with larger number of lipids decrease (i.e., compression occurs) so that the area of both monolayer equals. They also demonstrated that the asymmetric distribution of lipids with intrinsic positive curvature (i.e., positive spontaneous curvature of its monolayer) such as diC8PC and LPC in DPPC bilayers was stable if the mole fraction of these lipids in the bilayer was small.58 Their results support our conclusion on the stability of GUVs in our experiments where the differences in number of lipids in both leaflets were small and the mole fraction of lipids with intrinsic positive curvature (LPC and NBD-LPE) was small. As demonstrated in Figs. 1E and 3, the GUVs with asymmetric distribution of NBD-LPE can be used in the estimation of the rate of transbilayer movement of lipids. So far all experiments to estimate the rate of transbilayer movement of lipids have been performed using the suspensions of LUVs and small unilamellar vesicles (SUVs) (i.e.,


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the LUV suspension method43) by various physicochemical methods such as electron spin resonance, fluorescence spectroscopy, and neutron scattering.34-39 Using the LUV suspension method, it has also been demonstrated that the interactions of AMPs and venom peptides with lipid vesicles increased the rate of transbilayer movement of lipids.8,16,50,51 However, one can obtain only ensemble averages of physical quantities such as fluorescence intensity of all vesicles using the LUV suspension method,43 and therefore, it is difficult to obtain detailed information on elementary processes such as the relationship between the transbilayer movement and peptide-induced pore formation. In contrast, the results shown in Fig. 3 clearly indicate that only the binding of magainin 2 to GUV membranes before pore formation did not induce the transbilayer movement of NBD-LPE while after pore formation the movement started to occur. This is the first direct experimental evidence that the transbilayer movement of lipids is increased through only the pores induced by AMPs as far as we know. However, the present method using single GUVs has low time-resolution, because the transbilayer movement of lipids can be detected after the lipids in the outer monolayer unbinds to aqueous solution, which rate constant is estimated as 3.8  10−3 s−1 (Fig. 1C). However, this new method would provide indispensable information when (or at which elementary process) transbilayer movement of lipids occurs. In the present work, we investigated magainin 2-induced initial pore formation in GUVs harboring LPC in the inner lipid monolayer but not in the outer one. Figure 2D indicates that kp decreased with an increase in LPC concentration in the inner monolayer. It is generally considered that because LPC forms micelles, its monolayer has a positive spontaneous curvature, which favors pore formation in the membranes. Therefore, kp would increase with an increase in LPC concentration. However, the result of Fig. 2D was the opposite one. Here we propose that the asymmetric packing of lipids in the two leaflets (i.e., the higher-density packing of lipids in the inner monolayer than that in the outer one) yielded a decreased kp. As described in the Result section, this asymmetric packing of lipids decreased the area change of the membrane (Table 1), inducing the decrease in the stretching of the inner monolayer (i.e., the area increase from the optimum area) when magainin 2 bound to the outer leaflet. As described in our previous paper,33 the stretching of the inner monolayer plays an important role in the magainin 2-induced pore formation: kp increases as the stretching of the inner monolayer increases. Therefore, we can explain qualitatively the effect of the asymmetric packing on kp: the higher packing of the inner monolayer decreases its stretching on the binding of magainin 2 to the outer leaflet, leading in turn to decreased kp. In the present report, we also constructed a theoretical equation for kp (Eq. 9) and demonstrated that this equation could quantitatively explain our experimental


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Table 2. Theoretical and experimental values of rate constant of magainin 2-induced pore formation kP in the presence of LPC in the inner monolayer. Mean values and SDs of two independent experiments are shown. Here, we approximated the area of LPC as the half of that of DOPC.48,49 (A) 31 M magainin 2 LPC conc.

lyso

total

(mol%)

intotal

Theoretical

Experimental kP

[mN/m]

kP [s1]

[s1]

0

0

4.39  102

3.10

(8.6  1.6)  103

0.25

1.25  103

4.27  102

3.01

5.5  103

(5.2  0.9)  103

0.50

2.5  103

4.14  102

2.92

2.7  103

(3.2  0.5)  103

lyso

total

intotal

Theoretical

Experimental kP

[mN/m]

kP [s1]

[s1]

(B) 62 M magainin 2 LPC conc. (mol%) 0

0

4.77  102

3.37

(4.5  0.7)  102

0.50

2.5  103

4.52  102

3.19

1.9  102

(1.1  0.2)  102

1.0

5.0  103

4.27  102

3.01

5.5  103

(5.3  0.7)  103

results. Here, using this model, we explain the magainin 2-induced pore formation in the presence of LPC in the inner monolayer. If we do not consider the mechanical coupling of both monolayers and focus instead on the total optimum area, the presence of LPC in the inner leaflet (and not in the outer one) means that the optimum area of the inner leaflet of such a GUV will be larger than that of the outer one; the difference in fractional optimum area of the outer leaflet from the inner one can be expressed as lyso (< 0). When magainin 2 binds to the outer monolayer, the fractional change of the area of the GUV bilayer, mag, will be increased in proportion to the magainin 2 surface concentration, X, such that mag = mX. The presence of LPC in the inner monolayer decreases the magainin 2-induced effective, total fractional change in the area of the inner one, intotal, by lyso, (i.e., intotal = mag + lyso). Therefore, the effective total tension in the inner monolayer, intotal, can be calculated using intotal, such that intotal = 70.5intotal. Table 2 summarizes the values of lyso, intotal, and intotal under all of the experimental conditions. Using Eq. 10, we calculated the theoretical values of kp (Table 2). The experimental values of kp were similar to the theoretical values of kp. This result supports the validity of our model of magainin 2-induced initial stage of pore formation. Our model of the mechanism of initial stage of magainin 2-induced pore formation can reasonably explain the result of dependence of kp on magainin 2 surface concentration (X) and the effect of asymmetric packing of lipids on kp. Based on this model, magainin 2-induced pore formation occurs via the mechanism different from the pore


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formation induced by other membrane-active peptides such as toxin-peptide, melittin, as well as a CPP, TP10. One of the distinct characteristics of magainin 2-induced pore formation is that the binding of magainin 2 in the outer monolayer induces a rapid increase in the area of the GUV bilayer until the area achieves a steady value, which remains constant for an extended interval (e.g., 50  400 s, depending on conditions); this “silent” stage persists until pore formation occurs, which is evidenced (in our system) by the leakage of water-soluble fluorescent probe.11-13,33 For melittin and TP10, pore formation occurs before the area change reaches a steady value with no “silent” stage.27,33 In the case of magainin 2, during this “silent” stage, constant stretching of the inner monolayer continues (or constant tension is applied) in the inner monolayer, and then stochastically pore formation occurs. This situation is similar to the constant-tension-induced rupture of GUVs.40,41,53 In that phenomenon, constant tension is applied to a GUV bilayer via aspiration of the GUV; the GUV persists in this state, with no apparent change for an extended interval (e.g., 50  300 s, depending on conditions), until the GUV ruptures due to pore formation in the GUV membrane. Our model of magainin 2-induced pore formation can explain this “silent” stage. We hypothesize that during this stage, thermal fluctuation of lipid density occurs in the inner monolayer, and defects with lower lipid densities or rarefaction, i.e., half-prepores, form transiently and disappear. If the radius of a half-prepore reaches the critical radius, r*, required to overcome the activation energy, the half-prepore transforms into a transmembrane pore and leakage of the fluorescent probe starts. Therefore, our model suffices to explain the initial “silent” stage and the rate constant of magainin 2-induced pore formation. However, our model is still one of the hypotheses, and therefore, more experiments and theoretical analysis are necessary to reveal the mechanism. As described in the introduction to this paper, the evolution of the magainin 2-induceed pore rapidly occurs after initiation of pore formation; the size of pore is large at the initial stage and then decreases to a steady equilibrium size.13 At present, this evolution of pore, which is the second stage of magainin 2-induced pore formation, cannot be explained by our model. It is worth noting that our model is based on experimental data that proved that there are no magainin 2 molecules in the inner monolayer before pore formation.33 As described above, this property is not seen with all peptides. The mechanisms of pore formation by melittin and TP10, which localize in both monolayers before pore formation, cannot be explained by our model. A threshold concentration of AMPs is considered a prerequisite for the antimicrobial activity of these peptides.2 From an equilibrium point of view, Huang and colleagues proposed a model for peptide-induced pore formation.59,60 When peptides bind to membrane interface, which creates distortion of lipid hydrocarbon chains under the bound


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peptides and decreases the thickness of membranes. This increases the free energy of the membrane. With an increase in the peptide to lipid ratio, P/L, the membrane thickness decreases linearly and reaches a steady value at its threshold ratio (P/L)*. Pore formation occurs only if P/L > (P/L)*. In their model, a state of no pores (S phase) and a state of multiple pores (I phase) are in equilibrium, and they obtained the threshold concentration of peptide required for pore formation using the principle that the chemical potentials of lipid in both phases are equal.59,60 On the other hand, our model is a kinetic one; the initial stage of magainin 2-induced pore formation is described as an irreversible two-state transition from an intact GUV state to the pore state, which explains reasonably the stochastic pore formation in single GUVs. The pore state in our model is not an equilibrium state, as supported by the experimental results showing that the pore size changes over time to reach a steady size, which is almost the same as the equilibrium size.13 We consider that the distortion energy due to the binding of magainin 2 in the outer leaflet does not play an important role in the initial stage of pore formation because the experimental data of the dependence of kp for the magainin 2-induced initial pore formation on in is almost the same as that of the rate constant of constanttension-induced rupture of GUVs on in (Fig. 5B) and the application of external tension due to the aspiration of a GUV increased the kp for the magainin 2-induced pore formation.33 In our kinetic model for the expression of kp, the threshold concentration of AMPs depends on the time scale. If we consider the interaction of magainin 2 with a GUV for an infinite time interval, the magainin 2 surface concentration that induces an infinite activation energy, Ua, corresponds to a threshold concentration Xth. Based on Eq. 8, Xth = (  B)/b = 0.024. Thus, the relationship between Ua and X indicates that Ua increases rapidly as X approaches Xth (Fig. S7). The physical meaning of the result shown in Fig. S7 is that even if we observe magainin 2-induced pore formation for an infinite time, magainin 2 surface concentrations at or below Xth will never be sufficient to induce pore formation. However, practically, it is important to consider the threshold concentration under a specific time scale. If Ua  20 kT, which corresponds to a kp  1.0  104 s1, we can consider that no significant pore formation will occur within 3 h. The value of X corresponding to Ua = 20 kT is 0.065, which is a threshold concentration for 3 h reaction. However, if we consider the limitation of measurement time (i.e., ~1000 s) of the current method, a value of X of 0.068 (corresponding to kp of 0.001 s1) can be considered a realistic threshold concentration of magainin 2 for 1000 s reaction. Recently, MD simulations on the interactions of some AMPs such as maculatin and PGLa with lipid bilayers have been reported,61,62 indicating that maculatin and PGLa can translocate across lipid bilayers without pore formation. Apparently these simulation results are contradict with our experimental results on magainin 2. However,


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it is generally accepted that the interactions of peptides with lipid bilayers greatly depend on the kinds of peptides (such as amino acid composition and hydrophobicity) and the kinds of lipid bilayers (such as membrane thickness and mechanical strength). For example, we found using the single GUV method that TP10, a CPP composed of 21 amino acid including 4 positively charged Lys which composition is similar to magainin 2 (23 amino acid including 4 Lys), can translocate across lipid bilayers with most lipid compositions (including pure PC membranes) to enter the GUV lumens without pore formation and then after some lag times pore formation occurs in the GUV membranes.31 The comparison of the results of TP10 with magainin 2 indicates that the mode of interaction of peptides with lipid bilayers is completely different due to some small factors such as hydrophobicity and amino acid sequence, which mechanisms are not clearly revealed yet. In contrast, similar concentrations of TP10 cannot translocate across lipid bilayers containing high concentrations of cholesterol, whereas higher concentrations of TP10 can induce pore formation in these bilayers containing cholesterol and subsequently TP10 enters the GUV lumen by passing through the pores, which is almost the same mode as observed in magainin 2-induced pore formation in PG/PC-GUVs.32 These results clearly indicate that the mode of interaction of peptides with lipid bilayers also greatly depends on the kind of lipid bilayers. On the other hand, it is well known that the time range and the area of membrane used in all-atom MD simulations are very limited in their current stage. Therefore, due to these limitations it is difficult to simulate all phenomena in the fluctuations of lipid bilayer structures such as prepore formation and the interactions of peptides with lipid bilayers.33 It is well known that plasma membranes have asymmetric lipid compositions in both monolayers, which plays various physiological roles. In this report, we demonstrated that the asymmetric packing of lipids in the outer and inner monolayers of GUVs greatly affects magainin 2-induced pore formation. To the best of our knowledge, this work represents the first experimental evidence of the effect of asymmetric packing of lipids on the function of proteins/peptides in biomembranes/lipid membranes. Here the asymmetric packing of lipids were produced by the asymmetric distribution of LPC, but in principle we can prepare GUVs with asymmetric packing using the difference in the number of typical phospholipids such as PC in both monolayers. We can reasonably expect that these GUVs with asymmetric packing also would affect greatly the magainin 2-induced pore formation. Moreover, the asymmetric lipid packing of plasma membrane of bacteria could affect the magainin 2-induced pore formation and hence its bactericidal activity. In plasma membranes and organelle membranes, asymmetric lipid packing in both monolayers would appear transiently and locally, which can affect functions of membrane proteins and membrane


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dynamics. Further studies on asymmetric packing of lipids will reveal new aspects of its roles in biomembrane functions. CONCLUSION In this report, we succeeded in preparing GUVs with asymmetric lipid composition of LPC and NBD-LPE in the two monolayers. We can reasonably consider that the density of lipids in the inner leaflet of the GUVs is higher than that in the outer one, and therefore, these GUVs have the asymmetric lipid packing in the monolayers. We demonstrated that the rate constant of magainin 2-induced pore formation, kp, decreased with increasing LPC concentration in the inner monolayer. On the basis of these results, we proposed that the asymmetric packing of lipids in the two leaflets (i.e., the higher-density packing of lipids in the inner monolayer than that in the outer one) yielded a decreased kp. On the other hand, based on the theory of tension-induced pore formation and the calculated values of relevant parameters, we constructed a quantitative model expressing kp as a function of X (the magainin 2 surface concentration). The theoretical results obtained using this model were consistent with the experimental relationship between kp and X. This model quantitatively explains the effect of asymmetric lipid packing on kp: the higher density packing of the inner monolayer decreases the magainin 2-induced stretching of the inner monolayer, which in turn decreases kp. Moreover, using single GUVs with asymmetric lipid composition of NBD-LPE, we succeeded in estimating the rate of transbilayer movement of lipids in the absence and in the presence of magainin 2. Only the binding of magainin 2 to the GUV membrane did not increase the rate of transbilayer movement of NBDLPE but appearance of the pore in membrane increased it. These results also demonstrate the advantage of the method using single GUVs with the asymmetric lipid composition.

Supporting Information: Measurement of CMC of NBD-LPE. Estimation of NBD-LPE concentration in the inner monolayer. Effect of NBD-LPE on kp. Effect of LPC in the membrane on constant-tension-induced rupture of GUVs. This material is available free of charge in the Internet at http://pubs.acs.org.. Acknowledgements: This work was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 15H04361) from the Japan Society for the Promotion of Science (JSPS) to M.Y. This work was also supported in part by the Cooperative Research Project of Research Center for Biomedical Engineering.


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TOC Graphic Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial Peptide Magainin 2 Moynul Hasan, Mohammad Abu Sayem Karal, Victor Levadnyy, and Masahito Yamazaki


Mechanism of Initial Stage of Pore Formation Induced by Antimicrobial

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