Structure–Activity Relationship of the Antimicrobial Peptide Gomesin

Article pubs.acs.org/Langmuir

Structure−Activity Relationship of the Antimicrobial Peptide Gomesin: The Role of Peptide Hydrophobicity in Its Interaction with Model Membranes Bruno Mattei, Antonio Miranda, Katia R. Perez, and Karin A. Riske* Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil

ABSTRACT: Antimicrobial peptides are part of the innate immune system of animals and plants. Their lytic activity against microorganisms generally depends on their ability to disrupt and permeabilize membranes. Here we study the structure−activity relationship of the antimicrobial peptide gomesin (Gm), from the spider Acanthoscurria gomesiana, with large unilamellar vesicles (LUVs) composed of 3:7 palmitoyloleoyl phosphatidylglycerol: palmitoyloleoyl phosphatidylcholine. Several synthetic analogues of Gm were designed to alter the hydrophobicity/charge of the molecule, whereby selected amino acid residues were replaced by alanine. Isothermal titration calorimetry (ITC) was used to assess the thermodynamic parameters of peptide binding to LUVs and light scattering measurements were made to evaluated peptide-induced vesicle aggregation. The ability of the peptides to permeabilize vesicles was quantified through the leakage of an entrapped fluorescent probe. The activity of peptides could be quantified in terms of the leakage extent induced and their affinity to the membrane, which was largely dictated by the exothermic enthalpy change. The results show that analogues more hydrophobic than Gm display higher activity, whereas peptides more hydrophilic than Gm have their activity almost abolished. Vesicle aggregation, on the other hand, largely increases with peptide charge. We conclude that interaction of Gm with membranes depends on an interplay between surface electrostatic interactions, which drive anchoring to the membrane surface and vesicle aggregation, and insertion of the hydrophobic portion into the membrane core, responsible for causing membrane rupture/permeabilization.

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INTRODUCTION Antimicrobial peptides are defense molecules of the innate immune system of animals and plants effective against several microorganisms.1,2 Their main mechanism of action relies on the ability to induce membrane permeabilization with subsequent death of the pathogen. Even though antimicrobial peptides display a large diversity of composition and structure, most of them share some essential characteristics to provide a nonspecific interaction with the membranes of microorganisms: they are often cationic and amphipathic.1−4 The interest in antimicrobial peptides increases continuously because of their potential use as antibiotics. In particular, knowledge on the structure−activity relationship can help design new molecules, which are more effective against pathogens and less harmful to the host organism. Such studies have often been performed on model membranes, which, depending on lipid composition, can mimic either the membrane of microorganisms or the mammalian plasma membrane. © 2014 American Chemical Society

Gomesin (Gm) is an antimicrobial peptide originally isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana.5 Gm consists of 18 amino acid residues (Z-C-R-RL-C-Y-K-Q-R-C-V-T-Y-C-R-G-R-NH2, where Z is the pyroglutamic acid), comprising six positive charges and four cysteine residues attached by two disulfide bridges (Cys2,15 and Cys6,11), which stabilize a tridimensional β-hairpin structure, even in solution.6 Previous studies have shown that Gm displays a wide antimicrobial action spectrum, exhibiting activity against Grampositive and Gram-negative bacteria, fungi and yeasts.5 Gm also displays antiparasitic activity7 and antitumoral activity against murine melanoma cells.8,9 In a previous work,10 we showed with optical microscopy that Gm ultimately caused burst of giant unilamellar vesicles Received: January 14, 2014 Revised: February 26, 2014 Published: March 7, 2014 3513

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Table 1. List of Gm Analogues with Primary Sequence, Electric Charge (z), Hydrophobicity (ΔGw→b)a and Relative Hydrophobicity (ΔΔGw→b) with Respect to Gm peptide

sequence

z

ΔGw→b (kcal/mol)

ΔΔGw→b (kcal/mol)

Gm Gm-A1 Gm-A3 Gm-A5 Gm-A7 Gm-A9 Gm-A10 Gm-A12 Gm-A14

Z-C-R-R-L-C-Y-K-Q-R-C-V-T-Y-C-R-G-R-NH2 A-C-R-R-L-C-Y-K-Q-R-C-V-T-Y-C-R-G-R-NH2 Z-C-A-R-L-C-Y-K-Q-R-C-V-T-Y-C-R-G-R-NH2 Z-C-R-R-A−C−Y-K-Q-R−C-V-T−Y-C-R-G-R-NH2 Z-C-R-R-L-C-A-K-Q-R-C-V-T-Y-C-R-G-R-NH2 Z-C-R-R-L-C-Y-K-A-R-C-V-T-Y-C-R-G-R-NH2 Z-C-R-R-L-C-Y-K-Q-A-C-V-T-Y-C-R-G-R-NH2 Z-C-R-R-L-C-Y-K-Q-R-C-A-T-Y-C-R-G-R-NH2 Z-C-R-R-L-C-Y-K-Q-R-C-V-T-A-C-R-G-R-NH2

+6 +7 +5 +6 +6 +6 +5 +6 +6

2.45 3.42 1.81 3.18 3.56 2.04 1.81 2.55 3.56

0 0.97 −0.64 0.73 1.11 −0.41 −0.64 0.10 1.11

a

The hydrophobicity of each peptide was calculated based on the Wimley-White hydrophobicity scale.18,19 The pyroglutamic acid received the value of 0.01 kcal/mol and its substitution for alanine (which results in an N-terminal positive charge) received the value of +0.97 kcal/mol, which is consistent with the value of an arbitrary positive charge (presence of an arginine or the difference between a protonated and deprotonated histidine) added to the value of alanine. ΔΔGw→b is the difference between the hydrophobicity of the peptide and that of Gm.

the leakage of an entrapped fluorescent molecule, carboxyfluorescein (CF).

(GUVs) composed of mixtures of zwitterionic and anionic lipids (respectively POPC, palmitoyloleoyl phosphatidylcholine, and POPG, palmitoyloleoyl phosphatidylglycerol). The bursting ability of Gm increased with the molar fraction of POPG in the membrane. These studies pointed to a carpet-like mode of action. In a following work, the interaction of Gm with large unilamellar vesicles (LUVs) composed of 1:1 POPC:POPG was investigated with isothermal titration calorimetry (ITC), light scattering, and leakage of an entrapped fluorescent probe.11 There it was shown that the interaction between Gm and anionic membranes is an exothermic process and results in vesicle aggregation and permeabilization. We concluded that peptide−lipid interaction and vesicle aggregation are related but not mutually dependent processes. The interaction of Gm with model and biological membranes is certainly modulated by electrostatics and by the amphiphilic character of Gm and lipids. However, in spite of the knowledge that the amphiphilic nature of antimicrobial peptides is essential for their lytic activity, few studies have specifically related these phenomena with a hydrophobicity scale.12−15 In the present work, we explore the structure−activity relationship of Gm when interacting with LUVs of lipid composition established to mimic bacterial membranes (POPG:POPC 3:7, mol:mol) from a physical-chemical point of view. For that purpose, several analogues of Gm were synthesized. Specific amino acid residues of the Gm sequence were replaced by alanine, an amino acid with a methyl group as side chain, which maintains the spatial isomerism, but does not exhibit significant physicochemical characteristics. The replaced amino acid residues were pGlu1, Arg3, Leu5, Tyr7, Gln9, Arg10, Val12 e Tyr14. The analogues are referred to as Gm-Ax, where x indicates the position of the amino acid residue replaced by alanine. These substitutions lead to peptides with different hydrophobic character, some more and some less hydrophobic than Gm. In addition, some analogues have a different number of charges than Gm (z = +6): Gm-A3 and Gm-A10 have only five charges, whereas Gm-A1 has seven. These substitutions were made to explore the influence of the hydrophobicity/ charge of Gm on its interaction with biomimetic systems. Different experimental approaches are used here to explore the structure−activity relationship of Gm and its analogues. The affinity and interaction of Gm analogues with membranes is investigated by ITC, the extent of vesicle aggregation is evaluated by light scattering, and the ability of the peptides to induce membrane permeabilization is assessed by monitoring

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MATERIALS AND METHODS

Materials. The phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol) (sodium salt) (POPG), were purchased from Avanti Polar Lipids (Birmingham, AL). The fluorescent probe 5(6)carboxyfluorescein (CF), the detergent Triton X-100 and others reagents were purchased from Sigma-Aldrich (St. Louis, MN, USA). CF was purified prior to use as described in Ralston et al.16 Peptides Synthesis and Characterization. Gm and its analogues (listed in Table 1) were synthesized and purified as previously described in detail elsewhere.10,11 Briefly, the synthesis was performed by the solid phase method, using the metilbenzidrilamine resin and the t-Boc strategy. Formation of the disulfide bridges was achieved immediately after cleavage. Lyophilized crude peptides were purified by high performance liquid chromatography (HPLC) in a C18 column until complete purification of the fractions. The efficiency of the purification process was then monitored by HPLC coupled to a mass spectrometer. The concentration of peptides stock solutions was measured by their absorbance at λ = 280 nm, according to the Lambert−Beer Law and ref 17. The peptides were classified according to their hydrophobicity. In this work, we used an experimentally obtained scale that gives the free energy of transfer of each amino acid residue from water to the bilayer interface.18,19 By summing the individual contribution of each residue, the total free energy of transfer of the peptide from water to the bilayer interface, ΔGw→b can be estimated. This hydrophobicity scale is adequate to describe antimicrobial peptides with transmembrane action.18 The ΔGw→b values calculated for Gm and for each analogue are listed in Table 1. For clarity, we define the term ΔΔGw→b, which is the difference between the hydrophobicity of the analogues and that of Gm. Therefore, positive values of ΔΔGw→b indicate peptides more hydrophilic than Gm, and negative values indicate peptides more hydrophobic than Gm. Preparation of Large Unilamellar Vesicles (LUVs). A lipid film was formed on the walls of a test tube from a solution of lipids (POPG:POPC 3:7, mol:mol) in chloroform, dried with a stream of N2 and left in vacuum for 2 h. A buffer solution containing 30 mM HEPES, pH 7.4, with 100 mM NaCl was added, and multilamellar vesicles (MLVs) were formed by mechanical agitation. Subsequently, to obtain LUVs, this lipid dispersion was extruded at least 11 times through polycarbonate membranes with a pore size of 100 nm. In all experiments, the phospholipid concentration was measured by indirect determination of phosphorus content, according to the methodology described by Rouser.20 Isothermal Titration Calorimetry. The isothermal titration calorimetry (ITC) measurements were performed with a Microcal VP-ITC from Microcal (Northampton, MA). The reference cell was 3514

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Figure 1. ITC data obtained from titrations of Gm and its analogues (see legend) with LUVs composed of POPG:POPC 3:7. (a,b) heat per mole of injectant as a function of the lipid-to-peptide molar ratio. The data are separated with respect to the hydrophobicity of the analogues in respect to Gm (see Table 1). Each injection corresponds to 5 μL. (c) Binding isotherms obtained for Gm and the analogues that display a significant ΔH of interaction (ΔH < −5 kcal/mol; see Table 2). The peptide concentration in the cell was 13.5 μM for most analogues (except for Gm-A7 and GmA14, which were 16 μM) and the lipid concentration in the syringe was 12 mM. Temperature was 25 °C. The lines represent the data obtained from the surface partition model (the parameters are listed in Table 2). filled with water and the reaction cell with the peptide solution (∼ 15 μM). The volume of the cells was 1.46 mL. The syringe was loaded with a suspension of LUVs (12 mM POPG:POPC 3:7 mol:mol). The titrations consisted of 5 μL injections made every 10 min. A first 0.5 μL injection was always made and discarded in the analysis. All measurements were done at 25 °C. The enthalpy variation of the reaction, ΔH, is given by

ΔH =

at λ = 580 nm was followed for 10 min. The measurements were performed at room temperature (∼ 23 °C) and under stirring. Entrapment of Carboxyfluorescein (CF) in LUVs and Leakage Assay. LUVs were prepared as described above with a buffer solution of 30 mM HEPES, pH 7.4, with 50 mM CF and 85 mM glucose, added to adjust the osmolarity of the solution. To remove the free CF outside vesicles, the suspension of LUVs was eluted with 30 mM HEPES buffer, pH 7.4, with 100 mM NaCl through a Sephadex resin G-25 Medium column where vesicles with entrapped CF (CF-LUVs) were collected in the void volume of the column. An aliquot of CF-LUVs was diluted to yield ∼50 μM final lipid concentration (the exact value was determined later by determination of the phosphorus content) in a cuvette. The fluorescence emission of CF fluorescence was monitored at λ = 520 nm using λ = 490 nm as excitation wavelength with a spectrofluorimeter F-2500 from Hitachi (Washington, DC). Different concentrations of peptide (1, 2.5, 5, and 10 μM) were added to the LUVs suspension. At the end of each experiment, Triton X-100 was added to promote full CF leakage. The percentage of CF leakage was given by 100(Ft − Fo)/(Fmax − Fo), where Ft is the fluorescence at a selected time, Fo is the initial fluorescence (before addition of peptide), and Fmax is the maximum fluorescence obtained after addition of Triton X-100.24

∑i δhi o c pep Vcell

(1)

is the initial where δhi is the heat of the ith injection, and concentration of peptides in the cell, with volume Vcell. The molar fraction of bound peptide per mole of lipid, Xb(i), is given by copep

i

Xb(i) =

∑k = 1 δhk ΔHiVinjc Lo

(2)

where δhk is the heat of the kth injection, Vinj is the volume of each injection, and coL is the concentration of the lipid dispersion in the syringe. The binding of Gm and its analogues to the bilayer was described with a surface partition model combined with the Gouy−Chapman theory, as previously presented and discussed in detail.11,21−23 In this model, Xb is given as Xb = KcM, where K is the intrinsic binding constant and cM is the concentration of peptide close to the membrane surface, given by the Boltzmann distribution considering the charge of the peptide, z, the membrane surface potential, which changes along the titration, and the temperature. A numerical solution for K, the surface potential, and cM was found for each experimental data pair of Xb and ceq, the concentration of free peptide. The variation of the Gibbs free energy of the interaction, ΔG, was obtained as ΔG = −RT ln 55.5K, where the term 55.5 corrects the unit of K to molar fraction. The entropy change, ΔS, was calculated from the thermodynamic relation ΔG = ΔH − TΔS. Light Scattering Measurements. The measurements of 90° light scattering were done in a spectrofluorimeter F-2500 from Hitachi (Washington, DC). A peptide solution (1.5 mL at ∼15 μM) was placed in a quartz cuvette of 1 cm light path and was titrated with a suspension of LUVs (5 μL injections of 12 mM lipid every 10 min), mimicking the titration experiment performed with ITC, described above. Immediately after each injection, the intensity of light scattering

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RESULTS Different analogues of Gm were synthesized yielding peptides more hydrophobic (Gm-A3, Gm-A9, Gm-A10) or more hydrophilic (Gm-A1, Gm-A5, Gm-A7, Gm-A12, Gm-A14) than Gm. Also, some of these analogues display one charge less (Gm-A3, Gm-A10) or more (Gm-A1) than Gm. The peptides and their hydrophobicity, given by ΔGw→b, which also accounts for peptide charge, are listed in Table 1 (see Materials and Methods section for the discussion on the hydrophobicity scale used). The interaction of Gm and its analogues with LUVs composed of POPG:POPC 3:7 (mol:mol), which mimic the composition of bacterial membranes, were investigated with isothermal titration calorimetry (ITC), light scattering, and a leakage assay. Isothermal Titration Calorimetry (ITC). Titrations of Gm and its analogues with LUVs were performed with ITC. Each 3515

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Table 2. List of Gm Analogues with Electric Charge (z), Hydrophobicity (ΔΔGw→b) and Thermodynamic Parameters of the Peptide−Lipid Interaction peptide

aa replaced

z

ΔΔGw→b (kcal/mol)

ΔH (kcal/mol)

K (M−1)

ΔG (kcal/mol)

TΔS (kcal/mol)

Gm-A3 Gm-A10 Gm-A9 Gm Gm-A12 Gm-A5 Gm-A1 Gm-A7 Gm-A14

Arg Arg Gln Val Leu pGlu Tyr Tyr

+5 +5 +6 +6 +6 +6 +7 +6 +6

−0.64 −0.64 −0.41 0 0.10 0.78 0.97 1.11 1.11

−5.8 −9.6 −9.0 −7.9 −1.6 −6.4 −0.3 −1.6 −1.0

50.000 8.000 5.000 125 50 -

−8.8 −7.7 −7.4 −5.2 −4.7 -

+3.0 −1.9 −1.6 −2.7 −1.7 -

effective peptide charge used in the model was the same as the total peptide charge. In our previous study, the interaction of Gm with LUVs composed of 1:1 POPG:POPC resulted in a similar value of ΔH, but the intrinsic binding constant found before was higher than the one reported here. It should be mentioned that the POPG fraction is different and that the model used is probably not perfect in describing Gm−lipid interaction for various reasons, including influence of aggregation, limitations of the Gouy−Chapman theory, nonideal mixing, as already discussed.11 However, the main aspects of the Gm-lipid interaction are always preserved: binding is mainly driven by a large negative enthalpic contribution and the stoichiometry of the interaction is mainly given by one POPG per charge of Gm. More importantly, the magnitude of ΔH is extracted directly from the ITC results and is modelindependent and our main goal here is to compare the results obtained with Gm and its analogues. Comparison of the results shown in Table 2 shows that the enthalpic term accounts for most of the magnitude of ΔG and that the values of K, and consequently ΔG, follow the hydrophobicity scale, ΔΔGw→b: The affinity of the peptide to the membrane is higher for more hydrophobic peptides. Note that the higher values of K obtained for Gm-A3, Gm-A9, and Gm-A10 are consistent with the steeper variation of the heat/mol of injectant curve of these analogues (see Figure 1a). The values of TΔS do not seem to correlate with peptide hydrophobicity; however, the interaction of Gm with membranes is mainly enthalpy-driven, and the entropic contribution does not play a significant role. Light Scattering Measurements. Previous studies from our group have shown that Gm is capable to induce extensive aggregation of LUVs composed of POPG:POPC 1:1 in the same lipid-to-peptide range for which exothermic peaks were detected with ITC.11 Static light scattering measurements were used to evaluate vesicle aggregation induced by Gm and its analogues in the conditions explored here. Figure 2 shows the results obtained for Gm and two selected analogues (Gm-A1 and Gm-A10). Light scattering measurements were performed in the same way as those of ITC, by titration of a solution of peptide with a suspension of liposomes. The control was performed by injecting a suspension of liposomes into buffer and is represented as a dashed line in Figure 2. For the first injections of LUVs into a peptide solution, a fast increase in light scattering is observed, followed by a slower decrease, which was attributed to precipitation of the aggregates formed, whose size was so big that continuous stirring was not enough to prevent their precipitation. Indeed, observations with optical microscopy revealed that mixtures of Gm or its analogues with the LUVs composition explored here also lead to formation of micrometer-sized aggregates (not shown), similar to that

injection of liposome suspension into a peptide solution results in a heat flow that can be integrated to give values of heat per injection. Figure 1 shows the heat per mol of injectant as a function of the lipid-to-peptide molar ratio obtained from such titration experiments. Figure 1a shows the results for Gm and the analogues more hydrophobic than Gm and Figure 1b shows the data for Gm and the analogues more hydrophilic than Gm (see Table 1 for peptide hydrophobicity and charge). The interaction of Gm and its analogues with LUVs is an exothermic process, in accordance with previous results obtained for Gm.11 However, the profile and magnitude of the curves varies considerably for each analogue. The total enthalpy change of the peptide−lipid interaction, ΔH, can be calculated as the sum of the heats given off along the titration divided by the number of moles of peptide in the calorimeter cell (see eq 1). These values are given for all peptides in Table 2 and are shown to vary considerably among the analogues. Overall, the magnitude of ΔH decreases as the peptides become more hydrophilic, with few exceptions, which show that the position of the amino acid modification is also relevant. The calorimetric data can also be displayed as binding isotherms, which shows the extent of peptide binding per mole of lipid as a function of free peptide available (Xb as a function of ceq; see Materials and Methods section), and thus express an affinity of the peptides for the membrane. The binding isotherm can only be reliably obtained for titrations which displayed a considerable heat of interaction (ΔH < −5 kcal/ mol in our case), and those are shown in Figure 1c. The binding isotherm data show that the extent of peptide binding increases with the hydrophobicity of the peptide. The ITC data can be further explored to extract other thermodynamic parameters, such as the intrinsic binding constant K, and consequently the free energy and entropy variations, ΔG and ΔS. In a previous work we have shown that the interaction of Gm with charged LUVs could be described with a surface partition model combined with the Gouy− Chapman theory.11 Such approach mainly separates the affinity of the peptide to the membrane in two terms, one electrostatic, which regulates the concentration of peptide close to the membrane surface, and a surface partition, which accounts for the adsorption/insertion of the peptide into the membrane (see Materials and Methods for a brief discussion of the model). This model was put forward here to extract the intrinsic binding constant K of Gm and its analogues with LUVs. The modeling of the experimental data could only be performed for the analogues that exhibited a considerable magnitude of ΔH. The theoretical curves obtained from the model are shown in Figure 1 as lines, and they could reasonably well fit the data. The respective parameters are also listed in Table 2. The 3516

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exothermic interaction with liposomes could induce a large extent of aggregation, as is the case of Gm-A1, which showed ΔH = −0.3 kcal/mol and was the analogue that exhibited a larger extent of aggregation. This result confirms that, even if aggregation is coupled with the exothermic process detected with ITC, the magnitude of the exothermic signal does not directly correlate with the extent of vesicle aggregation induced by the peptide. The vesicle aggregation of Gm can be understood as an interaction between Gm and two (or more) adjacent membranes. CF Leakage Assay. The ability of Gm and its analogues to induce membrane permeabilization was measured with fluorescence spectroscopy through experiments of leakage of carboxyfluorescein (CF) encapsulated in LUVs. The leakage kinetics were performed at different peptide-to-lipid molar ratios (0.01, 0.03, 0.12 and 0.21) for all peptides. Figure 3a shows the kinetics of CF leakage induced by Gm and its analogues for one chosen molar ratio, 0.12 peptide/lipid. Similar kinetic curves were obtained for the other peptide-to-

Figure 2. Results of 90° light scattering (λ = 580 nm) obtained from titrations of Gm and the analogues Gm-A1 and Gm-A10 with LUVs composed of POPG: POPC 3:7. The concentration of Gm and the analogues was 13.5 μM and injections were 5 μL of a suspension of 12 mM lipid. After each injection, the light scattering intensity was recorded for 10 min. The dashed line represents the average of light scattering obtained for injections of LUVs into buffer only. Each injection of LUVs into buffer results in a roughly constant light scattering intensity, which linearly increases with the lipid concentration. The measurements were done at room temperature (∼23 °C) under magnetic stirring.

previously reported for Gm interacting with POPG:POPC 1:1.11 Further injections of LUVs into the peptide solution result in a constant light scattering value, which then linearly increases with the concentration of lipid added, showing that no further aggregation is induced above a certain lipid-topeptide molar ratio, which coincides with the end of the exothermic signal detected with ITC (this correlation cannot be done for Gm-A1, because the exothermic signal detected was always too low). The light scattering profile obtained for each peptide is different, showing that the extent of aggregation depends on peptide hydrophobicity. Analogues with a different charge than Gm exhibit a quite different aggregation profile, whereas those with the same charge of Gm show an overall aggregation profile quite similar to that displayed by Gm. Therefore, peptide charge is the key factor influencing the extent of aggregation induced. Since the extent of peptide-induced aggregation is not straightforward to quantify, due to precipitation of aggregates formed, we chose to show only the comparison between the aggregation induced by Gm and two analogues, one more charged (Gm-A1, z = +7) and one less charged (Gm-A10, z = +5) than Gm. Figure 2 shows that Gm-A1 induces a larger extent of vesicle aggregation than Gm, visualized as a larger contribution of precipitation of the aggregates formed. On the other hand, less aggregation is induced by Gm-A10, since the effect of precipitation of aggregates is less pronounced. These results show that vesicle aggregation is mainly driven by the electrostatic interaction between the positive charges of the peptide and the negative charges of the membranes. Analogues more hydrophilic but with the same charge as Gm exhibit an aggregation profile quite similar to that of Gm, although with a somewhat higher extent of aggregation (data not shown). It is important to mention that even analogues with a very small

Figure 3. (a) Kinetics of CF leakage encapsulated in LUVs composed of POPG:POPC 3:7 in response to addition of Gm and its analogues, at a molar ratio of 0.12 peptide/lipid. The peptides were injected 100 s after the beginning of the kinetics. After ∼53 min, Triton X-100 was added to induce complete leakage of CF. The measurements were done at room temperature (∼23 °C) under magnetic stirring. The symbols were added to the curves to discriminate them. Dashed lines show the fit curves using eq 3. (b) Leakage percentages of process 1 (A1, obtained from fits of the experimental curves with eq 3) as a function of the peptide-to-lipid molar ratio. 3517

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lipid ratios (not shown). The low fluorescence intensity recorded before addition of the peptides is due to the selfquenching of CF and represents 0% leakage. Addition of the peptides induces a fast increase in fluorescence intensity. At the end of the experiment, Triton X-100 is added to induce complete CF leakage (100% leakage). The leakage kinetics data of the analogue Gm-A1 will not be presented because of the high extent of aggregation induced, which was shown to interfere with the fluorescence intensity measurements for certain conditions. Peptide hydrophobicity directly influences the extent of membrane permeabilization (Figure 3a). Analogues more hydrophobic than Gm induce a higher leakage percentage, whereas analogues more hydrophilic induce a lower leakage percentage as compared with Gm. From the curve shape, it is possible to see that leakage kinetics exhibits an initial and fast fluorescence increase (on the order of seconds) followed by a much slower kinetics (on the order of several minutes). This was attributed to CF leakage arising from different sizes of vesicle aggregates. It is reasonable to expect that CF leaks faster from individual vesicles, and that this process becomes progressively slower as the number of vesicles in an aggregate increase, because diffusion of the fluorescent probe from a large aggregate will be significantly delayed. Since formation of large aggregates occurs within the first seconds, as seen by light scattering experiments (see Figure 2), we suggest that the leakage kinetics can be separated in two main processes: a fast one, which we call process 1, related to the peptide-induced leakage from individual vesicles and/or from small aggregates, and a much slower process 2, associated with the leakage observed when the vesicles are largely aggregated. Therefore, a biexponential behavior can be used to describe the leakage kinetics: Leakage (%) = A1(1 − e−t / t1) + A 2(1 − e−t / t2)

Figure 4. Percentage of total leakage (A) and the corresponding percentage of leakage from process 1 (A1) and process 2 (A2) as a function of the peptide hydrophobicity. The results shown were obtained from fits with eq 3 to the experimental data obtained at peptide-to-lipid molar ratios of 0.12 and 0.21.

(0.12 and 0.21), which were shown to represent saturation values of leakage percentage (see Figure 3b). Figure 4 shows that peptide hydrophobicity has opposite effects on A1 and A2: A1 mainly decreases, whereas A2 essentially increases with the hydrophobicity of the peptide. As expected, the correlation is not perfect, and the position of the amino acid residue substitution also plays an important role. The total percentage of leakage, A, is primarily dictated by the leakage through process 1. Light scattering and optical microscopy experiments showed that all peptides studied here induced formation of micrometersized aggregates within seconds. Therefore, we expect that, given enough time, the kinetics of CF leakage induced by all peptides will be mainly governed by process 2 (leakage from large aggregates). However, the peptides which show a strong interaction with the membrane (Gm and its more hydrophobic analogues) exhibit a high degree of CF leakage within seconds, via process 1, and only a small percentage of CF remains to leak through process 2, which will be therefore less important. On the other hand, the more hydrophilic peptides cause a milder leakage in the first seconds, and leakage from process 2, after aggregates have reached large sizes, will become more significant.

(3)

where A1 and A2 are the percentage of leakage at the end of the processes 1 and 2, which decay with the characteristic times t1 and t2. The total percentage of leakage induced by the peptide is then given by A = A1 + A2. The experimental leakage kinetics data obtained for all peptides at different peptide-to-lipid molar ratio were fit with eq 3. Overall, the model used could reasonably well fit the data. The fits of the curves obtained at 0.12 peptide/lipid are shown as dashed lines in Figure 3a. The CF leakage percentage was found to be sensitive to the peptide hydrophobicity, whereas the characteristic times were roughly the same for all peptides: t1 ∼1−10 s and t2 ∼ 15 min. According to our interpretation, process 1 is related mainly with the process of membrane permeabilization, whereas process 2 describes the CF release from large aggregates. Indeed, the typical leakage characteristic time reported for other antimicrobial peptides are comparable to t1,14,25−27 supporting the hypothesis that this fast time better represents the real leakage characteristic time of Gm and its analogues. Figure 3b shows the fit parameter A1 as a function of the peptide-to-lipid molar ratio for all peptides. Clearly, peptide hydrophobicity has a direct effect on A1, which is higher for more hydrophobic peptides. To better visualize the role of peptide hydrophobicity on the CF leakage process, Figure 4 shows the fit parameters A, A1, and A2 as a function of ΔΔGw→b, which sets the hydrophobicity scale. The results shown are the average values of the data obtained for the two highest peptide-to-lipid molar ratio used

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DISCUSSION In this work we explored the structure−activity relationship of the antimicrobial peptide Gm when interacting with charged LUVs. This was accomplished by comparing the activity of Gm and eight synthetic analogues in which specific residues were replaced by alanine, thus rendering peptides with different hydrophobicity/charge. Peptide−lipid interaction was assessed in several ways and was shown to be highly sensitive to peptide hydrophobicity/charge. ITC results showed that peptide association to the membrane is mainly driven by a negative enthalpic contribution. In parallel, light scattering revealed that peptide binding is accompanied by vesicle aggregation and CF leakage experiments showed that all peptides were able to permeabilize LUVs. Peptides which exhibited a higher magnitude of ΔG and/or ΔH also induced a higher leakage percentage (especially from process 1, A1, which better 3518

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analogues, which exhibit a quite stable β-hairpin structure even in solution.6 Nonetheless, the ΔH found for Gm is similar to the ΔH of magainin assigned to the nonclassical hydrophobic effect, i.e., interaction of hydrophobic residues with the membrane core. Therefore, we can propose that the origin of the enthalpic contribution of Gm is related to the extent of hydrophobic interactions made upon insertion of the hydrophobic residues into the membrane core, which disturbs the bilayer structure and results in increased membrane permeability. The magnitude of ΔH would then reflect the size and/ or penetration depth of the hydrophobic portion of Gm, which changes in the analogues investigated here, as will be discussed later. The extent of peptide-induced vesicle aggregation does not match the same trend found for the leakage percentage and ΔG and/or ΔH. On the other hand, the extent of aggregation is mainly related with the charge of the analogues, showing that vesicle aggregation is associated with the superficial anchoring of the peptides onto the membrane surface. The tridimensional structure of Gm in water was determined by nuclear magnetic resonance6,28 and is represented in Figure 6. The two disulfide bonds help maintain a quite stable βhairpin structure.6 Gm has an amphipathic character with a relatively large hydrophilic surface and a small hydrophobic face mainly formed by the residues Leu5, Tyr7, Val12 and Tyr14 (shown in brown in Figure 6). The latter is the most important region of the molecule to interact with the membrane core. The charges of Gm, represented in blue in Figure 6, are almost equally distributed throughout the amphiphilic surface of Gm: two charges next to the N-terminal (Arg3,4), two in the turn region (Lys8, Arg10) and two close to the C-terminal (Arg16,18). The positive charges and other polar residues possibly act as an anchor to the membrane surface. The Gm analogues investigated here were designed to specifically alter the hydrophobic/hydrophilic balance of the native Gm structure. In the following, we will correlate the activity of the analogues with their structure. Three of the analogues studied had polar or charged residues replaced by alanine, yielding peptides more hydrophobic than Gm. These analogues displayed higher affinity for the membrane and induced higher leakage percentage than Gm. However, only Gm-A9 and Gm-A10 exhibited an increased

represents the permeabilization ability of the peptides), and this activity showed an overall increase with the peptide hydrophobicity. In fact, previous studies showed that the leakage extent can be directly associated with the affinity of the peptide to the membrane.13,14 To better illustrate this behavior, Figure 5 shows that A1 is indeed highly correlated with ΔG. However,

Figure 5. Dependence of the leakage percentage through process 1, A1, with ΔH (black squares) and ΔG (red circles) obtained from ITC measurements (Table 2).

ΔG could not be obtained for the peptides that yielded a low interaction heat (see Table 2). Since ΔG is mainly governed by ΔH, Figure 5 also shows the dependence of A1 with ΔH, obtained for all peptides. The same trend is observed, except for Gm-A3 (shown as an open square), which exhibited high leakage percentage, high ΔG, but relatively low ΔH, compared with Gm. The interaction of other cationic amphipathic antimicrobial peptides, such as magainin, with model membranes was also investigated with ITC and shown to be characterized by a large negative enthalpy.22 In that case, however, a significant contribution to the total ΔH came from α-helix formation upon binding,22 which is not expected for Gm and its

Figure 6. Structure of Gm as described in ref 6. The structure was taken from PDB (ID: 1kfp)28 and drawn as molecule surface with Python Molecular Viewer29 in four different views. The hydrophobic residues Leu5, Tyr7, Val12, and Tyr14 are shown in brown and the charges in blue. The residues replaced by alanine to generate the different analogues are explicitly indicated. 3519

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magnitude of ΔH; Gm-A3 displayed a lower ΔH (see Table 2). These differences can be explained based on the positions of the substitutions. Changes in the turn region (Gln9 and Arg10) ensures that the original hydrophobic face of Gm becomes larger and can therefore insert deeper into the membrane core, resulting in more hydrophobic interactions made and higher perturbation of the membrane structure (higher ΔH, K, and leakage percentage). On the other hand, the acquired neutral region in position 3 (Gm-A3), which lies further away from the hydrophobic face, almost facing the opposite side of the peptide, cannot contribute to a deeper penetration of the original hydrophobic face of Gm (shallower penetration, lower ΔH). The five other substitutions made decreased the hydrophobicity of Gm, either by substituting each of the four hydrophobic residues that form the hydrophobic face of Gm, or the N-terminal residue in order to increase the electric charge of Gm (addition of the N-terminal positive charge). Replacement of any residue from the hydrophobic face resulted in peptides which exhibited a hardly detectable ΔH and a significantly lower leakage percentage. However, the interaction of Gm-A5 was not as weak as that of the other analogues (GmA7, Gm-A12, and Gm-A14). We hypothesize that the hydrophobic face of Gm that inserts into the membrane can be slightly bent toward the Val12 residue. The increase in electric charge of Gm by substitution of pGlu1 for alanine (GmA1) almost completely abolished the ITC signal and the extent of CF leakage via process 1. This effect may be related with a greater anchoring of the peptide to the bilayer surface, which decreases the degree of freedom of the peptide, thus hindering insertion of its hydrophobic face into the membrane core. All peptides induced a large extent of vesicle aggregation. The extension of aggregation increased considerably with the peptide charge (and mildly with peptide hydrophilicity for peptides bearing the same charge). Therefore, aggregation is a direct result of surface electrostatic interactions between the positive charges of Gm and the negatively charged POPG lipids. In fact, ITC revealed that the stoichiometry of the interaction is mainly given by one POPG per Gm charge (Table 2 and ref 11). The charges of Gm are well distributed along the peptide hydrophilic surface (see Figure 6), suggesting the existence of several anchoring regions of the peptide onto the membrane surface. More importantly, if all charges are expected to bind to lipids, this cannot be accomplished with a single flat bilayer. Consequently, binding of Gm is accompanied by vesicle aggregation, because the distribution of charges along the Gm surface attracts lipid headgroups all around it. In LUVs this interaction leads to extensive aggregation. On the other hand, optical microscopy of GUVs did not show any vesicle aggregation.10 In fact, GUVs are large and heavy and typically do not touch each other. However, dense regions on the surface of GUVs were often observed prior to vesicle burst. These dense regions of lipids indicate a local membrane bending mediated by the peptide, and this effect was also reported for other antimicrobial peptides.30 Therefore, the origin of both aggregation of LUVs and formation of dense peptide−lipid regions in GUVs is probably the even distribution of charges along the hydrophilic surface of Gm and its analogues. Formation of large aggregates is also characteristic for βamyloid peptides, which in Alzheimer’s disease sediment as amyloid plaques in the extracellular space. However, the molecular origin of the formation of β-amyloid fibrils is the aggregation between adjacent peptides to form an extended β-

sheet structure.31 The role of the lipid membrane in this process is to provide a platform that concentrates and aligns the peptides thus facilitating their extensive aggregation.32,33 The present study of the structure−activity relationship of gomesin was performed with a membrane model, with the aim of better understanding how gomesin interacts and disturbs lipid membranes. The lytic mechanism of gomesin and other antimicrobial peptides can be more complex, as the lipid membrane is not the only barrier to be transposed and/or perturbed to induce the death of pathogens. For a complete picture of the action of gomesin in vivo, comparison between the results shown here with the biological activity of gomesin and its analogues is crucial. A following study of the antimicrobial activities of gomesin and its alanine-substituted analogues is currently being finalized and can be partly found in ref 34.

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SUMMARY This work provides a thorough investigation of the structure− activity relationship of the antimicrobial peptide Gm upon interaction with model membranes. Several synthetic analogues of Gm were designed to yield peptides with different hydrophobicity/charge. The interaction of these peptides with liposomes is an enthalpy-driven process accompanied by vesicle aggregation and membrane permeabilization. Our results show that peptide hydrophobicity plays a key role in the activity of the peptides: more hydrophobic peptides generally exhibit higher affinity to the membrane, interaction enthalpy, and percentage of leakage. Notably, the ability to permeabilize vesicles was directly correlated with the peptide affinity to the membrane. Vesicle aggregation, on the other hand, was mainly governed by peptide charge: more charged peptides were able to induce higher extents of aggregation. Based on the position and character of the substitutions made and on the corresponding changes in peptide activity, we can propose a mechanism of action for Gm. The hydrophobic portion of Gm covers a relatively small fraction of the peptide surface, closer to the turn region. On the other hand, the positive charges are well distributed along the hydrophilic surface of Gm. The charges of Gm provide anchoring points onto the membrane, and their even distribution on the surface of Gm explains the high extent of LUVs aggregation induced or formation of dense regions on GUVs. The ability of Gm to disturb and permeabilize the membrane is mainly connected with the insertion of its hydrophobic portion into the membrane core. We conclude that the action of Gm can be described with the interfacial activity model,4 may be better assigned to the carpet mode of action, and that the interaction of Gm with model membranes arises from the balance between surface electrostatic interactions and insertion of the hydrophobic portion into the membrane core.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Departamento de Biofı ́sica, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Pedro de Toledo, 669, L9D, CEP 04039-032, São Paulo, Brazil. Tel. +5511-55764967; E-mail: [email protected]. Notes

The authors declare no competing financial interest. 3520

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ACKNOWLEDGMENTS The financial support from FAPESP, CNPq, INCT-FCx, and CAPES are acknowledged. We are thankful to Dr. Joachim Seelig for allowing us to use the ITC analysis program developed by his group, and to Wagner Torres Lamas for synthesizing the analogues of Gm.

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