Kinetic Pathway of Antimicrobial Peptide Magainin 2-Induced Pore

Aug 30, 2010 - Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, J...
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J. Phys. Chem. B 2010, 114, 12018–12026

Kinetic Pathway of Antimicrobial Peptide Magainin 2-Induced Pore Formation in Lipid Membranes 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 of Science, 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 be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10 000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage 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 results indicate that magainin 2 molecules initially induce a large, transient pore in lipid membranes following which the radius of the pore decreases to a stable smaller size. We estimated the radius of these pores, which increases with an increase in magainin 2 concentration. On the basis of these data, we propose a hypothesis on the mechanism of magainin 2-induced pore formation. 1. Introduction Antimicrobial peptides with bactericidal and fungicidal activity have been discovered in, and isolated from, a wide variety of organisms including amphibians, invertebrates, plants, and mammals.1,2 Among these antimicrobial peptides, magainin 2, which was first isolated from the African clawed frog Xenopus laeVis,3,4 has been extensively investigated. All-D amino acid magainin 2 had the same antibacterial activity as that of the natural, all-L amino acid magainin 2.5 Since specific interaction of magainin 2 with chiral receptors or proteins is not required for its antibacterial activity, this observation indicates that the target of magainin 2 is the lipid membrane regions of bacterial and fungal biomembranes. The interactions of magainin 2 with biological lipid membranes have been investigated using various methods such as the large unilamellar vesicle (LUV) suspension method6-9 and X-ray diffraction.10,11 On the basis of these results, magainin 2-induced pore formation in lipid membranes is considered to be the main mechanism underlying its bactericidal activity. Several structural models for the antimicrobial peptide-induced pore in biomembranes have been proposed:12 the barrel-stave (or helix bundle) model and the toroidal (or wormhole) model. In the former model, peptides insert perpendicularly into the lipid membranes and a fixed number of * To whom correspondence should be addressed. Tel./Fax: +81-54-2384741. E-mail: [email protected]. † Integrated Bioscience Section, Graduate School of Science and Technology, 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.

peptides specifically associate with each other to form an R-helical bundle that produces a narrow pore with a specific size. In contrast, in the toroidal model, the external and internal monolayer membranes bend and merge in a toroidal fashion to create a pore of which the inner wall is composed of R-helical peptides and lipid head groups. Several researchers consider that magainin 2 forms a toroidal structure.10,11 Results of molecular dynamics simulations suggest the disordered toroidal model.13 Recently, the pore induced by Bax-derived peptides has been convincingly shown to be a toroidal structure.14 In a toroidal pore, there may be no specific interactions between R-helical peptides, which makes it difficult to understand the size and the stability of the pore. Moreover, there is no information regarding how toroidal pores are formed in lipid membranes nor regarding which factor determines the size of these pores. Therefore, both the kinetic pathway and the mechanism of pore formation remain unknown. So far, almost all studies of interactions of antimicrobial peptides with lipid membranes have been done using a suspension of many small-size vesicles such as LUVs (the LUV suspension method). In these studies, the average values of the physical parameters of vesicles have been obtained from a large number of vesicles, and thereby much information has been lost. Recently, we proposed a novel method, the single giant unilamellar vesicle (GUV) method. In this method, we observe and measure the changes of structure and physical properties of single GUVs with a diameter of g10 µm during the interaction of substances such as antimicrobial peptides, and analyze these results over many “single GUVs” statistically.15,16 Using this method, we previously succeeded in observing the magainin 2-induced pore formation in lipid membranes of each single GUVs and estimating the rate constant of the pore

10.1021/jp104527y  2010 American Chemical Society Published on Web 08/30/2010

Antimicrobial-Peptide-Induced Pore Formation formation.17,18 Moreover, the use of this method allows us to separate the step of pore formation in membranes from the step of fluorescent probe leakage through the pores, which enables an accurate estimation of the leakage rate from single GUVs. We also previously found that the surface concentration of magainin 2 in the external monolayer of a GUV is the main determinant of the rate of pore formation in GUVs of various membranes.18 Hence the single GUV method can provide a great deal of new information that cannot be obtained by the conventional LUV suspension method. To address the above questions on the magainin 2-induced pore formation, it is important to elucidate the kinetic pathway of pore formation in lipid membranes. In this report, in order to examine the change in pore size during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced pores in single GUVs of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane (i.e., 50% DOPG/DOPC-GUVs). The fluorescent probes we used were Texas-Red dextran (TRD) of various molecular weights, fluorescein isothiocyanate bovine serum albumin (FITC-BSA), and trypsin inhibitor from soybean Alexa Fluor 488 conjugate (AF-SBTI). We show that the size of the pores changes over time and also that the size and the number of the pores depends on the concentration of magainin 2. A part of the preliminary results of this research was published in the proceedings of an international conference.19 2. Materials and Methods 2.1. Materials and Peptide Synthesis. DOPC and DOPG were 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-Red Dextran 70 000 (TRD-70k), FITC-BSA, and AF-SBTI were purchased from Invitrogen Inc. (Carlsbad, CA). These TRD molecules were used without further purification for most experiments. However, some experiments were then repeated using TRD molecules that were purified by gel chromatography using 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 method using a 433A peptide synthesizer (PE Applied Biosystems, Foster City, CA). The sequence of magainin 2 (23-mer) is GIGKFLHSAKKFGKAFVGEIMNS with an amide-blocked C terminus. The methods for purification and identification of the peptides 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, pH 7.0, 150 mM NaCl, and 1 mM EGTA) containing 0.1 M sucrose and various fluorescent probes by the natural swelling of a dry lipid film at 37 °C.17 The concentrations of the fluorescent probes were as follows: 10 µM for TRD-10k, TRD-40k, and TRD70k, 30 µM for TRD-3k and FITC-BSA, and 8 µM for AFSBTI. To obtain a pure GUV solution, untrapped fluorescent probes were removed as described in our previous report.17 The interaction of magainin 2 with single GUVs was carried out in buffer A containing 0.1 M glucose at 25 °C and was analyzed by fluorescence phase contrast microscopy.15-18 300 µL of the purified GUV solution (0.1 M sucrose in buffer A as the internal solution; 0.1 M glucose in buffer A as the external solution) was transferred into a handmade microchamber.15-18 A slide glass and a cover glass in a microchamber were precoated with 0.1% (w/v) BSA in buffer A containing 0.1 M glucose to prevent the direct contact of the GUVs with the glass

J. Phys. Chem. B, Vol. 114, No. 37, 2010 12019 surface.15-18 It is well demonstrated in the micropipet aspiration method to measure the tension and the elastic modulus of the membrane of a GUV accurately that the BSA coating on the glass surface of the slide glass and the micropipet prevents the contact between the glass and the GUV from inducing the external tension in the GUV membrane.20 Various concentrations of magainin 2 solution in buffer A containing 0.1 M glucose were continuously added in the vicinity of a GUV through a 20 µm diameter glass micropipet positioned by a micromanipulator. The distance between the GUV and the tip of the micropipet was ∼70 µm. Thereby, the equilibrium magainin 2 concentration near the GUV is considered almost the same as that in the micropipet.15,17 Phase contrast and fluorescence images of GUVs were recorded using a high-sensitivity EM-CCD camera (C9100-12, Hamamatsu Photonics K.K., Hamamatsu, Japan) with a hard disk. Neutral density filters were used to decrease the intensity of the incident light, resulting in conditions where almost no photobleaching of fluorescent probes in a GUV occurred during the interaction of the magainin 2 solution with single GUVs. Therefore, the decrease in fluorescence intensity inside a GUV can be considered as the result of leakage of the fluorescent probes from the inside to the outside of the GUV. The fluorescence intensity inside the GUVs was determined using the AquaCosmos software (Hamamatsu Photonics K.K., Hamamatsu, Japan), and the average intensity per GUV was estimated. The details of this method were described in our previous reports.15-18 To obtain the rate constant of the magainin 2-induced pore formation in lipid membranes, kP, for various GUVs, three independent experiments were carried out for each magainin 2 concentration. For each experiment, 10-20 single GUVs were analyzed to evaluate the rate constant. Average values and standard deviations of the rate constant among the three experiments 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 change in the size of the magainin 2-induced pores in lipid membranes, we first investigated the leakage of the small fluorescent probe TRD-3k (average molecular weight, Mw, is 1500 according to Invitrogen Inc., and Stokes-Einstein radius, RSE, is 1.4 nm21,22) through magainin 2-induced pores in single 50% DOPG/DOPCGUVs. Figure 1A shows a typical experimental result of the effect of the interaction of 7 µM magainin 2 with single GUVs on the TRD-3k concentration within a GUV. Prior to magainin 2 addition, a phase contrast microscopic image of the GUV indicated a high contrast in the GUV (Figure 1A-1) due to the difference in the concentration of sucrose and glucose between the inside (0.1 M sucrose) and the outside (0.1 M glucose) of the GUV. A fluorescence microscopic image of the same GUV (Figure 1A-2) showed a high concentration of TRD-3k inside the GUV at this time. During the addition of the 7 µM solution of magainin 2, the fluorescence intensity inside the GUV was almost constant over the first 170 s, following which the fluorescence intensity decreased rapidly (Figure 1A-2,B). After 220 s, a low fluorescence intensity, which was less than 10% of the original intensity, was detected inside the GUV, although a phase contrast image of the same GUV (Figure 1A-3) showed that the GUV structure was still intact with no detectable breaks. As discussed in our previous report,17 the rapid decrease in fluorescence intensity occurred as a result of the leakage of the fluorescent probe through the magainin 2-induced pore in

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Figure 2. Leakage of TRD-10k from single 50% DOPG/DOPC-GUVs induced by magainin 2 in buffer A at 25 °C. Time course of the change in the normalized fluorescence intensity of several single GUVs induced by (A) 7 µM, (B) 4 µM, and (C) 15 µM magainin 2. Figure 1. Leakage of TRD-3k from single 50% DOPG/DOPC-GUVs induced by 7 µM magainin 2 in buffer A at 25 °C. (A) Fluorescence images (2) show that the TRD-3k 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 11 (1) and 245 s (3). The bar corresponds to 10 µm. (B) Time course of the change in the normalized fluorescence intensity of the GUV shown in (A). We defined the normalized fluorescence intensity of the intact GUV before the initiation of the leakage as 1. (C) Other examples of the time course of the change in the normalized fluorescence intensity of several single GUVs under the same conditions as in (A). (D) Time course of Pintact of 50% DOPG/DOPC-GUV. A solid line represents the best-fitted curve of eq 1.

the lipid membranes, i.e., due to the diffusion of TRD-3k from the inside to the outside of the GUV through the pore in the membrane. Thus, the time at which the fluorescence intensity began to rapidly decrease corresponds to the time at which the pore was formed in the membrane. Furthermore, a comparison of the phase contrast images in Figure 1A1,A-3 also showed that there was a substantial loss in the phase contrast of the GUV, indicating that, during the leakage of TRD-3k, sucrose and glucose also passed through the same pore. When the same experiments were carried out using many single GUVs, we observed that a similar rapid leakage of TRD-3k from a GUV started stochastically. These results indicate that the pores were formed stochastically and that ∼90% of the TRD-3k had leaked from single GUVs over a period of ∼100 s after pore formation (Figure 1C). As we demonstrated in our previous papers,17,18 the rate constant of the magainin 2-induced pore formation in lipid membranes can be obtained by analyzing the time course of the fraction of intact GUVs, Pintact(t), among the population of GUVs examined, from which the fluorescent probe did not leak over time t. Figure 1D shows that the value of Pintact of 50% DOPG/DOPC-GUVs decreased with time during the interaction with 7 µM magainin 2. As shown in Figure 1D, the curve of the time course of Pintact was well fitted by a single-exponential decay function defined by eq 1 as follows:

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

(1)

where kP is the rate constant of the magainin 2-induced pore formation and teq is a fitting parameter. In this fitting, we neglected a few unstable GUVs in which leakage easily occurred. Three independent experiments similar to the experiment shown in Figure 1D were carried out to obtain the value for kP. The average value of kP was then calculated using the results of all of the independent experiments. The average value of kP for 7 µM magainin 2 was 0.014 ( 0.001 s-1, which is almost the same value as that obtained in previous experiments of magainin 2-induced calcein leakage.17 The fact that similar values of kP were obtained for TRD-3k and calcein is in agreement with the fact that magainin 2-induced pore formation does not depend on the type 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 the interaction of magainin 2 with single 50% DOPG/DOPC-GUVs containing TRD-10k (Mw distribution is 9000-11 000 according to Invitrogen Inc., RSE ) 2.7 nm21). Figure 2A shows time course of the change in the normalized fluorescence intensity of several single GUVs containing TRD-10k induced by 7 µM magainin 2. A rapid leakage of TRD-10k was observed from each single GUV that started in a stochastic manner, and then the rate of the leakage decreased and the resulting slower leakage continued. The phase contrast microscope images of the single GUVs showed complete leakage of sucrose, indicating that magainin 2 induced a pore (or pores) in the GUV membrane. These results indicate that pores were formed stochastically and that ∼60% of the TRD-10k leaked from single GUVs over a period of ∼100 s after pore formation. When the same experiments were carried out using many single GUVs (the number of the examined GUVs, n ) 26), a similar two-stage leakage (i.e., an initial rapid and transient leakage followed by a slow leakage) was observed from a GUV. The effect of magainin 2 concentration on the leakage of TRD-10k was also investigated. When 4 µM magainin 2 was used (Figure 2B), the two stages of leakage were more clearly observed. Thus, initially, a rapid leakage occurred over ∼5 s, resulting in a ∼20% leakage of TRD-10k. This initial leakage was then followed by a very slow leakage. Only ∼40% of the TRD-10k leaked from single GUVs over a period of ∼100 s after pore formation. In contrast, when 15 µM magainin 2 was

Antimicrobial-Peptide-Induced Pore Formation

J. Phys. Chem. B, Vol. 114, No. 37, 2010 12021 In contrast, in the case of 7 µM magainin 2, we observed a similar leakage of AF-SBTI as that of FITC-BSA; an initial, transient, small decrease in the fluorescence intensity inside the GUV was observed over several seconds, but subsequently the fluorescence intensity remained almost constant (n ) 16). In the case of 4 µM magainin 2, we observed a similar leakage of AF-SBTI as that of 7 µM magainin 2 (n ) 7). 4. Discussion

Figure 3. Leakage of (A) FITC-BSA and (B) AF-SBTI from single 50% DOPG/DOPC-GUVs induced by 15 µM magainin 2 in buffer A at 25 °C. Time course of the change in the normalized fluorescence intensity of several single GUVs.

used (Figure 2C), the initial rapid leakage that occurred within ∼5 s resulted in a leakage of ∼70% of the TRD-10k, which was then followed by a slow leakage. During the next ∼50 s following pore formation, ∼90% of the TRD-10k leaked from single GUVs. 3.3. Induction of Leakage of FITC-BSA, TRD-40k, and TRD-70k from 50% DOPG/DOPC-GUVs by Magainin 2. We further investigated the interaction of magainin 2 with single 50% DOPG/DOPC-GUVs containing fluorescent probes of a larger molecular size such as FITC-BSA (RSE ) 3.6 nm23), TRD40k (Mw distribution is 35 000-50 000 according to Invitrogen Inc., RSE ) 5.0 nm21), and TRD-70k (Mw distribution is 60 000-90 000 according to Invitrogen Inc., RSE ) 6.4 nm21). Figure 3A shows time course of the change in the fluorescence intensity of several single GUVs containing FITC-BSA induced by 15 µM magainin 2. A small decrease in the fluorescence intensity inside the GUV was observed for a short time, but subsequently the fluorescence intensity remained almost constant. The phase contrast microscope images of the single GUVs showed complete leakage of sucrose, indicating that magainin 2 did induce a pore in the GUV membrane. When the same experiments 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 single 50% DOPG/DOPC-GUVs containing TRD-40k or TRD-70k and obtained almost the same results as those using FITC-BSA. Thus, using 15 µM magainin 2, an initial, transient, small decrease in the fluorescence intensity inside the GUV was observed over several seconds, but subsequently the fluorescence intensity remained almost constant (data not shown). When the same experiments were carried out using many single GUVs (n ) 21), a similar transient but very small (∼10%) leakage of TRD-40k was observed. We further carried out similar experiments using purified TRD-40k and TRD-70k. The results were similar to those obtained using the unpurified molecules, indicating that contamination with smaller sized TRDs was very small and therefore did not affect the results of the leakage experiments described above. 3.4. Induction of Leakage of AF-SBTI from 50% DOPG/ DOPC-GUVs by Magainin 2. We also investigated the interaction of magainin 2 with single 50% DOPG/DOPC-GUVs containing AF-SBTI (RSE ) 2.8 nm24). Figure 3B shows time course of the change in the fluorescence intensity of several single GUVs containing AF-SBTI induced by 15 µM magainin 2. We observed two stages of leakage similar to that of TRD10k. The initial rapid leakage was followed by a very slow leakage. Approximately 70% of the AF-SBTI leaked from single GUVs over a period of ∼100 s after pore formation (n ) 23).

The results presented in this paper show that magainin 2-induced leakage of fluorescent probes from single GUVs depends on the size of the probe molecules. Moreover, there are two different patterns of leakage. For probes with a relatively small molecular size (such as TRD-10k, TRD-3k, and AFSBTI), a transient, rapid leakage was observed at the initial stage of pore formation that was followed by a slow leakage (pattern A). In contrast, for probes with a larger molecular size (such as TRD-40k, TRD-70k, and FITC-BSA), leakage occurred over a very short period of time (less than 10 s) during which only a small amount of the GUV contents (∼10-20%) leaked from the GUV (pattern B). It is widely accepted that magainin 2 induces pore formation in lipid membranes.9 The probe leakage from single GUVs that was observed in our current study has been shown to be due to magainin 2-induced pore formation.17,18 Our results in this paper indicate for the first time that the size of the magainin 2-induced pore (or a few pores) changes over time; at the beginning of the pore formation the pore size is very large, but this pore then rapidly decreases to a smaller size. Hence, the large probes could only leak from the GUV interior during the initial stage of pore formation (pattern B). In contrast, the smaller probes could leak from the GUV over the entire period of pore formation (pattern A). The observed leakages occur at two different rates: the transient, initial, rapid leakage and the slow leakage at the final stage indicate that magainin 2 molecules initially induces a transient, large pore in the lipid membrane following which the radius of the pore decreases to a stable, smaller size. The rate constant of the leakage of the fluorescent probe from a GUV, kleak, is determined by the following experimental formula,

Cin(t) ) Cin 0 exp(-kleakt)

(2)

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 the normalized concentration of the fluorescent probe inside a GUV, Cin(t)/Cin0 , experimentally because the probe concentration inside the GUV is roughly proportional to the fluorescence intensity of the GUV, I(t), i.e., Cin(t)/C0in ) I(t)/I(0), where I(0) is the fluorescence intensity of the intact GUV before the initiation of the leakage. If we plot the log of normalized fluorescence intensity, FI ()I(t)/I(0)), vs time (s), we can obtain the rate constant of the leakage kleak quantitatively. Figure 4 shows the typical behavior of Cin(t)/C0in observed in our experiments. There are initial of the rapid leakage at two types of the rate constant kleak: kleak steady the initial stage and kleak of the slow leakage at the final steady stage (see also Table 1). In the case of 4 µM magainin 2-induced initial steady ) 1.2 × 10-1 s-1 and kleak leakage, for TRD-3k, kleak ) 4.5 initial steady ) 5.5× 10-2 s-1 and kleak × 10-3 s-1, and for TRD-10k, kleak initial -3 -1 ) 2.2 × 10 s . For each probe, the value of kleak is 20-40 steady under the same experimental times greater than that of kleak conditions. For the same probe, both kleak values increased as

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D)

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

the magainin 2 concentration increased. For the same magainin 2 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 of the leakage of a small fluorescent probe, calcein (Mw is 623, RSE) 0.74 nm),18 we found that only in the 4 µM magainin 2-induced pore the leakage of calcein occurred in two stages: a transient rapid leakage in the initial stage followed by a stage of slow leakage (Table 1). At higher concentrations of magainin 2, the calcein leaked almost completely in one stage. We can obtain the relationship between the rate constant of the leakage of the fluorescent probes and the cross-sectional area of the magainin 2-induced pores using a theoretical equation. We assume that the fluorescent probes can diffuse only through the magainin 2-induced pores from the inside to the outside of the GUV. Generally, the membrane permeability can be expressed by a flux of a substance per unit area of pores, J (mol/(m2 · s)), which follows Fick’s law:25

D J ) -P(Cin(t) - Cout(t)) ) - (Cin(t) - Cout(t)) h

(3) where P (m/s) is permeability coefficient of the substance in the pore and Cout(t) (mol/m3) is concentration of the substance outside 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 as the membrane thickness (h ) 3.5 nm). Thereby, the rate of leakage of the fluorescent probe from a GUV can be expressed as follows (here we assume Cout ) 0 for any time, because the volume of the outside the GUV is very large),

dCin D ) - SpCin dt h Cin(t) ∴ in ) exp(-kleakt) C0

V

where

kleak )

DSp hV

(4)

(5)

where Sp (m2) is the effective cross-sectional area of pores (one or 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 the probesinwater.TherelationshipbetweenDandtheStokes-Einstein radius of the fluorescent probe, RSE (nm), is determined by Einstein-Stokes equation at 25 °C as follows:

kT 2.45 × 10-19 2 -1 ) m s 6πηRSE RSE

(6)

We determined the D values of various TRD molecules using the experimentally determined RSE values.21 On the other hand, we determined the RSE values of FITC-BSA, AF-SBTI, and calcein using the experimentally determined D values.23,24,26 As shown in Table 1A, the Sp values at the initial leakage stage for the same magainin 2 concentration were almost the same irrespective of the kinds of fluorescent probes tested, and for all the probes tested the Sp values at the initial leakage stage increased with magainin 2 concentration. At the final stage of the leakage, for only probes of a smaller size (TRD-3k and TRD10k) the Sp values were determined, since no leakage of the larger probe was observed (Table 1B). The Sp values for both of these probes at this stage were almost the same for the same magainin 2 concentration. Taking into account that AF-SBTI could not leak at the final stage for 7 (or 4) µM magainin 2, we conclude that the radius of the pore induced by 7 (or 4) µM magainin 2 at the final stage is smaller than 2.8 nm (RSE of SBTI), but is larger than 1.4 nm (RSE of TRD-3k). This value agrees with that of the magainin 2-induced pores in multilayer membranes determined using neutron in-plane scattering (1.9 nm).10 In contrast, TRD-10k leaked slowly at the final stage for 7 (or 4) µM magainin 2 although its radius is similar to that of AF-SBTI. This result can be explained by the presence of TRD-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 AFSBTI leakage show that the radius of the pores at the final stage induced by 15 µM magainin 2 is larger than 2.8 nm (RSE of SBTI), but is smaller than 3.6 nm (RSE of BSA), which is larger than that induced by 7 (or 4) µM magainin 2. These results clearly indicate that the radius of the magainin 2-induced pore at the final stage increases with an increase in magainin 2 concentration. This conclusion can also explain the data of the leakage of TRD-3k and TRD-10k; i.e., the Sp values at the final steady stage increase with an increase in magainin 2 concentration (Table 1B). However, we cannot discard the possibility of the increase in the number of the pores as magainin 2 concentration. Generally, the D values of the probes in the pore can differ from those in bulk water due to some frictions and some interactions between the probes and the pore. Especially, in the permeation of the fluorescent probes through the small pore whose diameter is similar to the probes at the final stage, the D values of the probes in the pore may have different values from those in water because there are some frictions between the probes and the pore wall and thereby the quantitative values of Sp may have some errors. However, we did not use these Sp values to evaluate the radius of the pore at the final stage described above. On the other hand, the pores at the initial state have much larger radius than the fluorescent probes and thereby we can assume that the D values of the probes in the pore are almost the same as those in bulk water (i.e., we can use the simple diffusion equation (eq 3) to estimate the Sp values of the pore). Especially in the case of several TRD molecules with different molecular weights which have the same chemical properties and no net charges, their interactions with the pore are similar and thereby we can compare the Sp values obtained by the leakage data of the various TRD molecules. As shown in Table 1A, the Sp values at the initial leakage stage of the TRD in the same magainin 2 concentration were almost the same irrespective of the molecular weight.

Antimicrobial-Peptide-Induced Pore Formation

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TABLE 1: Rate Constants of the Magainin 2-Induced Leakage of Various Fluorescent Probes and the Effective Cross-Sectional Area of the Magainin 2-Induced Pores in Single 50% DOPG/DOPC-GUVs Whose Radius Was 5 ( 1 µm at 25 °C: (A) the Initial Stage, and (B) the Final Steady Stage (A) The Initial Stagea fluorescent probe/RSE (nm) calcein/0.74 initial -1 kleak (s ) Sp (nm2) n rlp (nm) TRD-3k/1.4 initial -1 kleak (s ) Sp (nm2) n rlp (nm) TRD-10k/2.7 initial -1 kleak (s ) Sp (nm2) n rlp (nm) AF-SBTI/2.8 initial -1 kleak (s ) Sp (nm2) n rlp (nm) TRD-40k/5.0 initial -1 kleak (s ) Sp (nm2) n rlp (nm) FITC-BSA/3.6 initial -1 kleak (s ) Sp (nm2) n rlp (nm)

4 µM magainin 2

7 µM magainin 2

15 µM magainin 2

(3.6 ( 1.0) × 10-1 (1.9 ( 0.4) × 103 7 24 ( 2

N.D.c

N.D.

(1.2 ( 0.1) × 10-1 (1.0 ( 0.1) × 103 9 18 ( 1

(1.9 ( 0.1) × 10-1 (2.1 ( 0.1) × 103 29 26 ( 1

(7.2 ( 0.5) × 10-1 (6.6 ( 0.5) × 103 12 46 ( 2

(5.5 ( 0.5) × 10-2 (1.2 ( 0.1) × 103 11 20 ( 1

(8.2 ( 0.8) × 10-2 (2.0 ( 0.1) × 103 12 25 ( 1

(2.4 ( 0.3) × 10-1 (4.9 ( 0.7) × 103 12 40 ( 3

(3.6 ( 0.7) × 10-2 (8 ( 2) × 102 6 16 ( 3

(1.2 ( 0.1) × 10-1 (2.2 ( 0.3) × 103 14 26 ( 2

(2.5 ( 0.2) × 10-1 (4.7 ( 0.4) × 103 17 39 ( 2

N.D.

(4.0 ( 0.3) × 10-2 (1.8 ( 0.3) × 103 10 24 ( 2

(1.2 ( 0.1) × 10-1 (4.6 ( 0.4) × 103 17 38 ( 2

N.D.

4.8 ( 0.3) × 10-2 (1.3 ( 0.1) × 103 9 20 ( 1

(1.9 ( 0.2) × 10-1 (6 ( 1) × 103 7 44 ( 4

(B) The Final Steady Stageb 2

-1

fluorescent probe/D (m s )

4 µM magainin 2

7 µM magainin 2

15 µM magainin 2

(5.6 ( 2.1) × 10-2 (3.3 ( 1.2) × 102 7

N.D.c

N.D.

(4.5 ( 0.4) × 10-3 (3.8 ( 0.5) × 101 9

(1.0 ( 0.1) × 10-2 (1.1 ( 0.1) × 102 28

(2.0 ( 0.6) × 10-2 (1.9 ( 0.5) × 102 12

(2.2 ( 0.5) × 10-3 (5 ( 1) × 101 11

(3.3 ( 0.4) × 10-3 (7.7 ( 0.7) × 101 12

(7.6 ( 0.9) × 10-3 (1.7 ( 0.3) × 102 12

no leakage

no leakage

6

12

(2.6 ( 0.4) × 10-3 (6 ( 1) × 101 16

N.D.

no leakage

no leakage

10

17

no leakage

no leakage

9

7

-10

calcein/3.3 × 10 steady -1 kleak (s ) Sp (nm2) n TRD-3k/1.7 × 10-10 steady -1 kleak (s ) Sp (nm2) n TRD-10k/9.1 × 10-11 steady -1 kleak (s ) Sp (nm2) n AF-SBTI/8.8 × 10-11 steady -1 kleak (s ) Sp (nm2) n TRD-40k/4.9 × 10-11 steady -1 kleak (s ) Sp (nm2) n FITC-BSA/6.8 × 10-11 steady -1 kleak (s ) Sp (nm2) n a

initial kleak

-1

N.D.

2

(s ): the rate constants of the leakage. Sp (nm ): the effective cross-sectional area of the pores. n: the number of GUVs examined. r1p steady (nm): the radius of the pore. b kleak (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.

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

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

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J. Phys. Chem. B, Vol. 114, No. 37, 2010

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

change in Sp or rp of the magainin 2-induced pore with time. This method can provide only qualitative information of the time course of Sp or rp of the pore, which is, however, the first information of the kinetic pathway of the antimicrobial peptideinduced pore formation in the lipid membranes. On the basis of a combination of the above data with previous published data, we propose a hypothesis on the following mechanism for magainin 2-induced pore formation in lipid membranes (Figure 5). First, magainin 2 in aqueous solution binds to the membrane interface of the external monolayer of a GUV. This binding rapidly achieves equilibrium, and we call this state the Bex state. In the Bex state, magainin 2 forms an R-helix that lies parallel to the membrane interface12 and inserts deeply into the membrane interface due to high interfacial hydrophobicity.17 This binding compresses the external monolayer, inducing some negative internal tension in the external monolayer σex (0), which tends to decrease the area of this monolayer and counterbalances with the external tension. Our previously published experimental results described this increase in area and in tension following the magainin 2 binding. For example, the binding of magainin 2 molecules induced a shape change in a DOPG/DOPC-GUV from a prolate to a shape composed of two spheres connected by a narrow neck, indicating that the area of the GUV membrane had been increased following the binding of magainin 2 to the membrane.17 We

Tamba et al. also previously observed that, in the interaction of magainin 2 with a single spherical GUV, the binding of magainin 2 suppressed the undulating motion of the GUV membrane, indicating an increase in the tension of the membrane. It is wellknown that the tension due to an external force induces the formation of a pore in the lipid membranes of GUVs as a result of thermal fluctuation of the lipid membrane lateral density.27-29 The rate of the pore formation increases as the tension increases.29 As we discussed above, in the interaction of magainin 2 with lipid membranes, the binding of magainin 2 induces an increase in external mechanical tension in the internal monolayer, which stretches the internal monolayer, raising the probability of pore formation.18 As a result, a pore forms in the membrane stochastically17,18 (step (ii)) and the rate constant of the pore formation increases with Xex. We can consider the change of the pore size over time as follows. The formation of a transmembrane pore decreases the stretch of the internal monolayer (resulting in a decrease in σin) and also induces a compression of the external monolayer (thereby increasing |σex|). Initially, σin > |σex| and thereby the radius of the pore increases with time, which decreases σin and at the same time increases |σex|, and finally at σin ) |σex| the pore growth stops. The unbalance of the tension in both the monolayers may induce the transfer of lipid molecules from the external to the internal monolayers through the rim of the pore, which decreases the difference in the absolute value of the tension of these monolayers to zero. Next, magainin 2 molecules in the external monolayer also transfer into the internal monolayer through the rim of the pore (steps (iii) and (iv)). It increases the surface concentration of magainin 2 in the internal monolayer, Xin, which changes the balance of tensions of both the monolayers. It increases the area of the internal monolayer, inducing the decrease in the pore radius. Hence, as the difference in surface concentration of magainin 2 in both the monolayers, Xex - Xin, decreases, the pore radius decreases; i.e., Xex - Xin is a key factor in determination of the pore radius. During this step, the large pore may transform to several smaller stable pores by the rearrangement of magainin 2 molecules in the rim of the large pore (step (v)). The stability of these final pores may be determined by the interaction free energy between magainin 2 molecules and the total free energy of the lipid membranes containing the pores. However, at present, we do not know its mechanism in detail. It is reported that the transfer (i.e., flipflop) of lipid molecules and the translocation of magainin 2 molecule from the outside to the inside of vesicles occurred in the magainin 2-induced pore formation,6 which supports our hypothesis. In the above scenario, the magainin 2-induced pore in the lipid membrane was produced because of the thermal fluctuation of the lipid membrane in the presence of tension, and thereby the pore was produced stochastically, which agrees well with our experimental results.17,18 On the basis of this scenario, only one large pore is formed in the initial stage of the leakage, because once a pore is formed the tension of the lipid membrane immediately decreases and this means that the possibility of the occurrence of other pores becomes very low. Assuming that this is true, we determined the radius of the large pore at the initial 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 are similar for the same magainin 2 concentration. Moreover, as the magainin 2 concentration is increased, the value of rlp also increases (Table 1A). With an increase in magainin 2 concentration in solution, its concentration in the external monolayer Xex

Antimicrobial-Peptide-Induced Pore Formation

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

increases. As described above, the higher the Xex value and the greater 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 of the GUV, R, in the same magainin 2 concentration (Figure 6). The values of the radius of the pore including rlp are determined by the rate constants of the transfer of lipid molecule and magainin 2 from the external to the internal monolayers, and thereby we need more experimental data to analyze the results of Figure 6 quantitatively. Here, it is noted that the radius of bacterial cells is much smaller than that of a GUV (e.g., the radius of most bacteria is ∼0.5 µm), and therefore, the radius of the transient pore in bacterial membrane produced by magainin 2 is much smaller than that observed in the 50% DOPG/DOPC-GUVs. The hypothesis on the mechanism of the magainin 2-induced pore formation described above can provide a rational, qualitative explanation of the obtained experimental results, although we need more detailed experimental evidence to prove this hypothesis. We are now developing a quantitative theory to describe this model to analyze the experimental results in more detail. On the basis of the hypothesis on the mechanism of pore formation described in this paper, magainin 2 induces pore formation in lipid membranes by using the intrinsic property of thermal fluctuation of lipid membranes. Formation of a pore by such a mechanism can provide a rational explanation as to how toroidal pores are formed in lipid membranes. Moreover, the following consideration of the surface concentration of antimicrobial peptides required for their pore formation in lipid membranes would support the hypothesis. For the magainin 2-induced pore formation, high surface concentrations of magainin 2 molecules at and above Xex ) 60 mmol/mol (molar ratio of the magainin 2 to lipid in the membrane surface) were required.18 It is also reported that in other antimicrobial peptides very high surface concentrations of peptides in lipid membranes or biomembranes were required for their pore formation and their activity to kill bacteria (represented by the minimum inhibitory concentration, MIC).12 If the antimicrobial peptides induce the pore in the lipid membranes by a specific manner such as ionic channel and pore-forming toxin proteins, high surface concentrations of peptides are not necessary for the pore formation. In contrast, the mechanism we proposed above is a physical model for the pore formation, which can reasonably explain why high surface concentrations are required for the pore formation. It is likely that, apart from magainin 2, other peptides and proteins can use a similar strategy to induce the formation of a toroidal pore.14 5. Conclusion To our knowledge, this is for the first time that the change in Sp or rp of the antimicrobial peptide-induced pores in the lipid

J. Phys. Chem. B, Vol. 114, No. 37, 2010 12025 membranes with time has been monitored. We demonstrated the new physicochemical method for this purpose. This method can provide only qualitative information of the time course of Sp or rp of the pore, which will be valuable and helpful to elucidate the mechanism of the magainin 2-induced pore formation. 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, stable pores at the final stage. Theoretical analysis of the leakage rate at the initial stage provided values for the radius of the pore. This radius increases with magainin 2 concentration and also increases with the radius of the GUV. These results provide a rational explanation for the mechanism of magainin 2-induced pore formation that we have proposed. We estimated the radius of the pores at the final steady stage, which increases with magainin 2 concentration. These data provide the first information concerning the kinetic pathway of magainin 2-induced pore formation in lipid membranes. Acknowledgment. This work was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 21310080) from the Japan Society for the Promotion of Science (JSPS), by a Grant-in-Aid for Scientific Research on Priority Areas (System Cell Engineering by Multiscale Manipulation) (No.20034023), and also by a Grant-in-Aid for Scientific Research on Priority Areas (Soft Matter Physics) (No. 21015009) from the Ministry of 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 using instruments at the center for Instrumental Analysis of Shizuoka University. References and Notes (1) Zasloff, M. Nature 2002, 415, 389. (2) Hwang, P. M.; Vogel, H. J. Biochem. Cell Biol. 1998, 76, 235. (3) Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5449. (4) Zasloff, M.; B. Martin, B.; Chen, H.-C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 910. (5) Wade, D.; Boman, A.; Wahlin, B.; Drain, C. M.; Andreu, D.; Boman, H. G.; Merrifield, R. B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 4761. (6) Matsuzaki, K.; Murase, K.; Fujii, N.; Miyajima, K. Biochemistry 1995, 34, 6521. (7) Matsuzaki, K.; Sugishita, K.; Ishibe, N.; Ueha, M.; S. Nakata, S.; K. Miyajima, K.; Epand, R. M. Biochemistry 1998, 37, 11856. (8) Boggs, J. M.; Euijung, J.; Polozov, I. V.; Epand, R. F.; Anantharamaiah, G. M.; Blazyk, J.; Epand, R. M. Biochim. Biophys. Acta 2001, 1511, 28. (9) Gregory, S. M.; Pokorny, A.; Almeida, P. F. F. Biophys. J. 2009, 96, 116. (10) Ludtke, S. J.; He, K.; Heller, K. H.; Harroun, T. A.; Yang, L.; Huang, H. W. Biochemistry 1996, 35, 13723. (11) Yang, L. T.; Weiss, M.; Lehrer, R. I.; Huang, H. W. Biophys. J. 2000, 79, 2002. (12) Melo, M. N.; Ferre, R.; Castanho, M. A. R. B. Nat. ReV. Bacteriol. 2009, 8, 1. (13) Leontiadou, H.; Mark, A. E.; Marrink, S. J. J. Am. Chem. Soc. 2006, 128, 12156. (14) Qian, S.; Wang, W.; Yang, L.; Huang, H. W. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17379. (15) Yamazaki, M. In AdVances in Planar Lipid Bilayers and Liposomes; Leitmannova, A. L. Ed.; Elsevier/Academic Press: London, 2008; Vol. 7, p 121. (16) Tamba, Y.; Ohba, S.; Kubota, M.; Yoshioka, H.; Yoshioka, H.; Yamazaki, M. Biophys. J. 2007, 92, 3176. (17) Tamba, Y.; Yamazaki, M. Biochemistry 2005, 44, 15823. (18) Tamba, Y.; Yamazaki, M. J. Phys. Chem. B 2009, 113, 4846. (19) Ariyama, H.; Tamba, Y.; Levadny, V.; Yamazaki, M. Proc. IEEE Int. Symp. Micro-NanoMechatronics Human Sci. 2009, 208. (20) Needham, D.; Nunn, R S. Biophys. J. 1990, 58, 997. (21) Goins, A. B.; Sanabria, H.; Waxham, M. N. Biophys. J. 2008, 95, 5362.

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(22) Venturoli, D.; Rippe, B. Am. J. Physiol. Renal. Physiol. 2005, 288, F605. (23) Salmon, E. D.; Saxton, W. M.; Leslie, R. J.; Karow, M. L.; McIntosh, J. R. J. Cell Biol. 1984, 99, 2157. (24) Gribbon, P.; Hardingham, T. E. Biophys. J. 1998, 75, 1032. (25) Schultz, S. G. Basic Principles of membrane transport; Cambridge University Press: Cambridge, UK, 1980. (26) Yoshida, N.; Tamura, M.; Kinjo, M. Single Mol. 2000, 1, 279.

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