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Interaction of the antimicrobial peptide gomesin with model membranes: a calorimetric study Tatiana M Domingues, Bruno Mattei, Joachim Seelig, Katia R Perez, Antonio Miranda, and Karin A. Riske Langmuir, Just Accepted Manuscript • DOI: 10.1021/la401596s • Publication Date (Web): 11 Jun 2013 Downloaded from http://pubs.acs.org on June 17, 2013

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Interaction of the antimicrobial peptide gomesin with model membranes: a calorimetric study Tatiana M. Domingues1,†, Bruno Mattei1,†, Joachim Seelig2, Katia R. Perez1, Antonio Miranda1, Karin A. Riske1,*

1

2

Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil

Biozentrum, University of Basel, Div. of Biophysical Chemistry, Basel, Switzerland

KEYWORDS isothermal titration calorimetry, antimicrobial peptides, fluorescence leakage assay, vesicle aggregation.

* Corresponding author, [email protected], Tel. + 55-11-5576 4967 †

These authors contributed equally.

ACS Paragon Plus Environment 1

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ABSTRACT

Gomesin is a potent cationic antimicrobial peptide (z = +6) isolated from the Brazilian spider Acanthoscurria gomesiana. The interaction of gomesin with large unilamellar vesicles (LUVs) composed of a 1:1 mixture of zwitterionic (POPC) and anionic (POPG) phospholipids is studied with isothermal titration calorimetry (ITC). In parallel, light scattering and optical microscopy are used to assess peptide-induced vesicle aggregation. The ability of gomesin to permeabilize the membrane is examined with fluorescence spectroscopy of the leakage of carboxyfluorescein (CF). Vesicles coated with 3 mol% PE-PEG lipids are also investigated to assess the influence of peptide-induced vesicle aggregation in the activity of gomesin. The ITC and light scattering titrations are done in two ways: lipid into peptide and peptide into lipid injections. Although some differences arise between the two setups, the basic interaction of gomesin with anionic vesicles is preserved. A surface partition model combined with the Gouy-Chapman theory is put forward to fit the ITC results. The intrinsic binding constant of gomesin is found to be K ~ 103 M-1. The interaction of gomesin with anionic membranes is highly exothermic and enthalpydriven. Binding of gomesin is virtually always accompanied by vesicle aggregation and changes in membrane permeability leading to CF leakage. Addition of PE-PEG to the membrane strongly attenuates vesicle aggregation but does not change significantly the mode of action of gomesin. The results point to a strong interaction of gomesin with the membrane surface, causing membrane rupture without a deep penetration into the bilayer core.


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INTRODUCTION

Antimicrobial peptides are part of the innate immune defense system of animals and plants against bacteria, fungi and other pathogenic agents (1-4). Because of that, this class of molecules presents an alternative to conventional antibiotics. The capacity of antimicrobial peptides to kill microorganisms generally relies on their ability to induce membrane rupture/permeabilization. Most antimicrobial peptides are cationic and have an amphipathic character, features which are essential to provide an unspecific interaction with the negatively charged membranes of microorganisms. The mechanism of action of antimicrobial peptides has been extensively studied with model membranes, such as liposomes (5-10). Different mechanisms have been described in an attempt to classify the lytic action of antimicrobial peptides. Among them, the most accepted and discussed are the carpet and the toroidal pore mechanisms (2). In both cases, the peptides initially accumulate at the membrane surface. Then, above a threshold, toroidal pores are formed by peptides aligned perpendicular to the membrane plane together with phospholipid molecules (11,12). Alternatively, the peptides cover the membrane surface like a carpet and ultimately disrupt the bilayer (13,14). At high peptide/lipid ratios, a detergent-like mechanism is proposed, in which bilayer patches are solubilized into micelle-like structures (4,14). Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana (15,16) and has proven to be very effective against a wide spectrum of bacteria, fungi and parasites (15-19). Moreover, gomesin showed antitumor activity in vitro and in vivo (20). Gomesin contains 18 aminoacid residues (ZCRRLCYKQRCVTYCAGR-NH2,


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where Z represents the modified aminoacid pyroglutamic acid and NH2 indicates amidation of the carboxy-terminal group), including four cysteines that form two disulfide bridges, and six cationic residues. Gomesin adopts a quite stable β-hairpin-like structure both in aqueous medium (21) and at amphiphilic surfaces (22). Although gomesin exhibits hemolytic activity against human erythrocytes, this activity could be decreased or almost completely abolished through chemical modifications of the gomesin molecule (22). In a recent work, we used optical microscopy to follow the mode of action of gomesin against giant unilamellar vesicles (GUVs) composed of mixtures of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine

(POPC)

and

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-

glycerol) (POPG) (23). We showed that gomesin often induced formation of dense domains at the vesicle surface and ultimately caused vesicle burst. A stable vesicle with a permeable membrane was never observed. The lytic activity of gomesin was measured and shown to increase with the molar fraction of the negatively charged lipid POPG in the membrane. Here we investigate the interaction of gomesin with large unilamellar vesicles (LUVs) composed of 1:1 POPC:POPG using mainly isothermal titration calorimetry (ITC), a powerful technique that allows the characterization of the thermodynamics of peptide-lipid interaction (for reviews see 24,25). This knowledge can be related to structural details of the process, such as formation/breaking of non-covalent bonds (enthalpic contribution), changes in the degrees of freedom of the system (entropic contribution) and peptide-membrane affinity (binding constant). We show that binding of gomesin to charged LUVs is mainly an exothermic process. In parallel, light scattering is used to assess vesicle aggregation induced by gomesin. In order to reduce peptide-induced

vesicle

aggregation,

3

mol%

of

1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG), a lipid with a bulky


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PEG moiety attached to its headgroup, was added to the bilayer. Grafting the membrane surface with bulky polymers has been extensively used as steric barriers to prevent adhesion between vesicles and to extend the lifetime of drug carrier liposomes in the organism (26,27). The molar fraction of PE-PEG added in this work ensures an almost complete coverage of the membrane surface, without however significantly altering bilayer properties (28,29). The ability of gomesin to alter membrane permeability is evaluated via the leakage of the fluorescence probe carboxyfluorescein (CF) entrapped in LUVs. A surface partition model combined with the GouyChapman theory is put forward to extract the intrinsic binding constant of gomesin to model membranes.

MATERIALS AND METHODS

Materials. The phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene

glycol)-2000]

(ammonium salt) (PE-PEG) were from Avanti Polar Lipids (Alabaster, AL) and the fluorescent probe 5,6-carboxyfluorescein (CF) was from Sigma (St. Louis, MO) and previously purified as described in (30). Peptide synthesis. Peptides were synthesized manually by the solid-phase method using the tBoc strategy as described in (31). Briefly, the synthesis was performed on a 4methylbenzhydrylamine-resin (0.8 mmol/g). Full deprotection and cleavage of the peptide from


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the resin were carried out using anhydrous HF treatment with anisole and dimethyl sulfide as scavengers at 0 °C for 1.5 h. Formation of disulfide bridges was achieved immediately after the HF cleavage and extraction of the crude peptide. The resulting peptide solution was kept at pH 6.8–7.0 and 5 °C for 72 h. Cyclization reactions were monitored by reverse-phase liquid chromatography coupled to an electrospray ionization mass spectrometer (LC/ESI-MS). Lyophilized crude peptides were purified by preparative RP-HPLC on Jupiter C18 column (22.1×250 mm, 300 Å pore size, 15 µm particle size) in two steps. The first was performed by using Triethylammonium phosphate pH 2.25 as solvent A and 60% acetonitrile (ACN) in A as solvent B. The second step was carried out using 0.1% trifluoroacetic acid/H2O as solvent A and 60% ACN in A as solvent B. Pure peptides were characterized by amino acid analysis and by LC/ESI-MS. The concentration of the gomesin stock solution was determined from optical absorbance at 280 nm. Preparation of Large Unilamellar Vesicles (LUVs). A lipid film was formed from a lipid chloroform solution of POPC:POPG 1:1 (mol/mol) with or without 3 mol% PE-PEG, dried under a stream of N2 and left under reduced pressure for about 2 h, to remove the residual organic solvent. Vesicles were prepared by addition of 30 mM HEPES pH 7.4 with 100 mM NaCl (ITC, optical microscopy and light scattering experiments) or 86 mM glucose with 50 mM CF (leakage experiments), followed by vigorous vortexing. The lipid dispersion was extruded at least 11x through polycarbonate filters with pores of 100 nm to yield large unilamellar vesicles. The phospholipid content of all lipid dispersions used throughout was determined by phosphorous assay as described in (32). Isothermal

titration

calorimetry

(ITC).

A

VP-ITC

(Microcal,

Northampton,

USA)

microcalorimeter was used. The calorimeter cell (1.46 mL) was filled with the peptide solution


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(15 µM) or with 0.1 mM lipid dispersion and the syringe was loaded with a dispersion of LUVs (6 mM lipid) or with peptide solution (300 µM). In lipid-into-peptide titrations 5 µL injections were made every 10 minutes. In peptide-into-lipid titrations the injection series was 5×2 µL, 13×5 µL, every 10 minutes. A first 0.5 µL injection was always made and discarded in the analysis. The temperature was set to 25 oC. Light scattering measurements. 90o light scattering was measured at λ = 600 nm with a Spectrofluorimeter Hitachi F-2500 (Hitachi, Tokyo, Japan). The titrations (lipid into peptide and peptide into lipid) were done in the same conditions as described for the ITC experiments, using a 1×1 cm cuvette filled with 1.46 mL solution. The measurements were performed at room temperature (~23 oC) and under magnetic stirring. Determination of vesicle size was done with dynamic light scattering (DLS) using the Zetasizer Nano ZS (Malvern, Worcestershire, UK). Optical microscopy. Optical microscopy images were obtained with an inverted Zeiss Axiovert 200 microscope equipped with a 63x Ph2 objective and a Zeiss AxioCam HSm (Zeiss, Jena, Germany). Dispersions of 37 µM LUVs composed of 1:1 POPC:POPG with 0 and 3 mol% PEPEG in the presence of 15 µM gomesin were left under magnetic stirring in a glass vial and small aliquots were taken every minute, placed in an observation chamber and observed under phase contrast optical microscopy. Entrapment of carboxyfluorescein (CF) in LUVs and leakage measurements. LUVs with entrapped CF were prepared by hydrating the lipid film with 30 mM HEPES, pH 7.4 containing 86 mM glucose and 50 mM CF. At this concentration the fluorescence of CF is self-quenched. Free CF was removed by passing the extruded LUVs through a Sephadex-G25 Medium column (1.2×20 cm) eluted with 30 mM HEPES, pH 7.4 containing 100 mM NaCl. This ensured that the


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inner and outer vesicle solutions had the same osmolarity. The LUVs were collected at the void volume. The leakage kinetics were followed by the fluorescence intensity increase due to CF release from LUVs after addition of gomesin. The increase in CF fluorescence as a function of time at 25 ºC was recorded continuously with a Hitachi F-2500 Fluorescence Spectrophotometer (λex = 490 nm and λem = 520 nm). At the end of each experiment, total CF fluorescence intensity was determined by the addition of Triton X-100 (at a final concentration of 2% w/w). The percentage of CF leakage, % leakage, was determined by Leakage % = (I(t) – Io) / (IT – Io) x 100

(eq. 1)

where I(t) is the fluorescence intensity at time (t), Io is the fluorescence intensity before addition of peptide and IT is the fluorescence intensity after addition of Triton X-100 (33). Surface partition binding model. The binding of gomesin to the membrane surface was modeled with a surface partition binding model combined with the Gouy-Chapman theory, as previously described for other peptides, such as magainin 2 (34,35). A simple partition model assumes that the extent of peptide binding per mole of lipid, Xb, is related to the concentration of free peptide, cf, through: Xb = Kapp cf

(eq. 2)

Since the peptide is charged, its affinity to the membrane surface decreases as more peptides adsorb to the membrane due to electrostatic repulsion. Therefore, the binding constant in eq. 2, Kapp, is not constant and changes with cf. The model used here assumes that Xb is directly proportional to the surface concentration of peptide, cM, and not to the bulk concentration, cf: Xb = K cM

(eq. 3)


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In eq. 3, K is the intrinsic binding constant, which is constant throughout the experiment and gives the non-electrostatic contribution to the affinity of the peptide. The surface concentration of peptide is given by the Boltzmann distribution: cM = cf e

− z p F Ψo / RT

(eq. 4)

where zp is the effective peptide charge (the nominal charge of gomesin is + 6, which sets the upper limit of the effective charge zp), F is the Faraday constant, Ψo is the surface potential, R the gas constant and T the absolute temperature. The surface potential is related to the surface charge density σ as described by the Gouy-Chapman theory (36). On the other hand, σ is directly related to the extent of peptide binding to the charged surface by:

σ=

e o − X PG + z p X b A L 1 + X b (A P / A L )

(eq. 5)

where eo is the electron charge, XPG the fraction of the negatively charged lipid, AL the area per lipid and Ap the area occupied by one adsorbed peptide. In the present analysis, eq. 5 was extended to include Na+ binding to POPG in terms of a Langmuir adsorption isotherm (37). The Na+ binding constant was 0.6 M-1. From the ITC experiments (lipid-into-peptide, L-P) the extent of peptide binding, Xb, and the enthalpy variation per mole of peptide, ∆Hpep, can be directly measured. The total enthalpy change, ∆Hpep, is obtained from the sum over all injections of the heat per injection i, hi, divided by the number of moles of peptide in the calorimeter cell:

∆H pep =

∑h

i

i o pep

c Vcell

(eq. 6)


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where copep is the initial concentration of peptide in the calorimeter cell and Vcell is the volume of the cell. In the peptide-into-lipid titrations, the heat of reaction per mole of lipid, ∆Hlip, can be extracted in the same way as shown in eq. 6, by substituting copep by colip, the initial concentration of lipid in the calorimeter cell. The extent of peptide binding per mole of lipid after each injection i, Xib, can be obtained as follows: i

X ib =

∑h

k

(eq. 7)

k =1

∆H iVinjc lip

where hk is the heat of injection k, ∆H is given in eq. 6, and Vinj is the injected volume of lipid dispersion with concentration clip. The binding isotherm is obtained from a plot of Xb against the concentration of free peptide, cf, which can be obtained from the experiments as follows: cif = copep(1 − Xip )

(eq. 8)

where Xp is the fraction of peptide bound, given by: i

Xip =

∑h

k

k =1

∆H Vcellcopep

(eq. 9)

The experimental binding isotherms were compared with theoretical simulations and a numerical solution for K, Ψo, and cM was found for each experimental data pair of Xb and cf.


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RESULTS AND DISCUSSION

ITC data The binding of cationic antimicrobial peptides to anionic vesicles is often accompanied by vesicle aggregation. Gomesin induces a large extent of vesicle aggregation, which can be perceived even with naked eyes. A usual way to hinder vesicle aggregation is to add to the bilayer a small fraction of lipids with bulky PEG moieties attached to their headgroups, which act as steric barriers (28,38). Therefore, the interaction of gomesin with LUVs composed of 1:1 POPC:POPG (mol/mol) was studied in the absence (bare LUVs) and presence (coated LUVs) of 3 mol% PE-PEG. The heat of gomesin binding to LUVs with and without 3 mol% PE-PEG was measured with isothermal titration calorimetry (ITC). The thermodynamics of gomesin binding was assessed with ITC by either lipid into peptide (L-P) or peptide into lipid titrations (P-L). The results are shown in Figure 1. Figure 1a shows the titration of a solution of 15 µM gomesin with 5 µL aliquots of a 6 mM lipid suspension of bare LUVs (L-P titration). The interaction of gomesin with bare LUVs is an exothermic process. Each injection produces a heat of reaction, which is shown in Figure 1c as heat per mole of lipid injected. The magnitude of the heat per injection decreases as the lipidto-peptide molar ratio increases because less peptide is available for binding after each injection. The reaction comes to an end after 10 injections when all gomesin is bound to the injected lipid. The last injections correspond to the heat of dilution of LUVs, and this contribution was subtracted from all injections in Figure 1c. The molar heat of reaction is then given as the total


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heat released divided by the molar amount of gomesin in the calorimeter cell (see eq. 6). For the example given in Figure 1a the exothermic reaction enthalpy is ∆Hpep = – 7.8 kcal/mol. Figure 1e shows the results of the inverse peptide-into-lipid titration (P-L). The calorimeter cell contains a lipid suspension of concentration 100 µM and different volumes (5×2µL, 13×5µL) of a 300 µM gomesin solution are injected. The corresponding heat per mole of peptide injected is shown in Figure 1g. The first injections show an increase in the magnitude of the heat per injection, after which the reaction comes to an end when all lipids are bound. The heat of reaction referred to the total lipid, ∆Hlip, is given as the sum over the heats per injection divided by the number of moles of lipid in the calorimeter cell (eq. 6). The reaction enthalpy per mole of lipid is ∆Hlip = – 0.68 kcal/mol. The ratio ∆Hpep/∆Hlip = 11.5 suggests that 1 molecule of gomesin interacts with 11-12 lipids, provided no other reactions interfere. Since 50% of the total lipid is POPG and if one assumes that gomesin interacts preferentially with POPG, then 1 gomesin binds 5-6 POPG lipids. This stoichiometry agrees well with the maximum charge of gomesin of zp = 6. Analogous experiments were done with coated LUVs containing 3 mol% PE-PEG to minimize vesicle aggregation. The L-P titration is shown in Figure 1b together with the integrated heats per mol of injectant (Figure 1d). The interaction of gomesin with coated LUVs is mainly exothermic, but comprises a small endothermic contribution (see injections 8-15 in Figure 1b), not detected in the absence of PE-PEG. The origin of this endothermic contribution is unclear. The last injections, also endothermic, correspond to the heat of dilution of LUVs only. The reverse P-L titration is shown in Figure 1f, with the corresponding heats per injection below (Figure 1h). Here again the first injections lead to an unexpected increase in the magnitude of


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heat per mol of injectant. The total heat of reaction was calculated in the same way as presented above for bare LUVs. The results obtained were ∆Hpep = – 7.0 kcal/mol and ∆Hlip = – 0.73 kcal/mol. The value of ∆Hpep obtained with coated LUVs is slightly lower than that obtained with bare LUVs, probably because of the presence of the endothermic contribution. The ratio ∆Hpep/∆Hlip = 10 agrees with the results obtained for bare LUVs, although here the small contribution of the endothermic component in ∆Hpep lowers this ratio. Still, 1 gomesin binds ~5 POPGs.

Peptide-induced vesicle aggregation The extent of vesicle aggregation induced by gomesin was assessed by optical microscopy and by 90o light scattering measurements. Optical microscopy was used to characterize the aggregates formed. Figure 2 shows optical micrographs of dispersions of LUVs containing 0 and 3 mol% PE-PEG obtained at different incubation times with gomesin at high peptide-to-lipid molar ratio (0.4 P/L). The micrographs clearly show that gomesin induces a huge extent of vesicle aggregation of bare LUVs. In the first minutes, micrometer-sized aggregates are formed and reach sizes as big as ~100 µm within 10 min. Coating of the vesicle surface with PEPEG drastically reduces peptide-induced vesicle aggregation. Within the optical resolution of the objective (~ 0.5 µm), no aggregates can be seen in the dispersion of coated LUVs. Light scattering data (both P-L and L-P titrations) were obtained in the same conditions as the ITC experiments. The results are shown in Figure 3. In the L-P titrations, the light scattering data obtained from injections of LUVs into buffer only are also shown in Figure 3a,b as data connected with a line. Each injection of LUVs (bare and coated) into buffer results in a


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linear intensity increase with the lipid concentration (see data connected with a line). On the other hand, injection of LUVs into a gomesin solution leads to different responses depending on the presence of surface coating. The first injections of coated LUVs into gomesin give rise to a small increase in light scattering as compared to injection of LUVs into buffer only. After about 7 injections, the light scattering intensity increased linearly with a slope close to that found for injections of LUVs into buffer. The endothermic peaks observed in the ITC experiment are not accompanied by an increase in light scattering. Figure 3a shows the light scattering results obtained with bare LUVs. Each injection of LUVs into a gomesin solution which gave rise to an ITC signal caused initially a large and fast increase in the light scattering intensity, followed by a slow and steady decrease. Optical micrographs revealed that micrometer-sized aggregates are formed within few minutes when bare LUVs are mixed with gomesin (see Figure 2). The magnetic stirring used in the light scattering measurements is not enough to prevent the precipitation of such huge aggregates formed. Therefore, the slow and steady decrease in light scattering observed is caused by the precipitation of such huge aggregates and light scattering is not able to quantify the extent of vesicle aggregation in that case. The last injections, obtained when all peptides are bound, show a roughly constant light scattering intensity with time, which increases linearly with each injection. Figures 3c,d show the light scattering results for injections of gomesin into a dispersion of LUVs (P-L experiments) in the absence and presence of 3 mol% PE-PEG as relative to the light scattering intensity of the lipid dispersion before addition of gomesin. An increase in light scattering is detected since the first injection (P/L = 0.004), showing that gomesin induces vesicle aggregation even with surface coating and at very low P/L. The light scattering of bare LUVs increases almost linearly with P/L until the 9th injection, when the aggregates become so


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big that they start to precipitate. Parallel measurements with dynamic light scattering (DLS) show that the size of the aggregates increases moderately between 100 and 200 nm up to P/L = 0.04 and reach micrometer sizes when precipitation starts to occur (data not shown). In the presence of surface coating, gomesin is still able to induce vesicle aggregation, but the increase in light scattering is much more modest and the aggregates never get big enough to precipitate. The light scattering results show that the binding of gomesin, which gives rise to the exothermic ITC signal, is virtually always accompanied by aggregation of anionic vesicles, in both titration directions (P-L and L-P) and even at very low P/L ratio. This agrees with the high charge of gomesin and points to a major interaction of the peptide with the membrane surface. Although coating of the LUVs with PE-PEG decreases substantially the extent of aggregation caused, a mild aggregation is always present.

Leakage experiments Most antimicrobial peptides cause drastic alterations in membrane permeability. A common method of quantifying the extent of membrane permeabilization caused by an antimicrobial peptide is via its ability to induce leakage of vesicle-entrapped molecules. Fluorescence spectroscopy was used to quantify the leakage of carboxyfluorescein (CF) from the inner volume of LUVs composed of 1:1 POPC:POPG with and without 3 mol% PE-PEG induced by different concentrations of gomesin. Figure 4 shows the leakage kinetics data obtained for two different P/L molar ratios measured. The low fluorescence intensity recorded before addition of gomesin refers to the intensity of the self-quenched CF entrapped in the inner volume of LUVs and denotes 0% leakage. Addition of gomesin causes a fast increase in fluorescence intensity, a direct evidence that gomesin is inducing leakage of CF from the LUVs.


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At the end of the experiment, a high concentration of the detergent Triton X-100 (0.2% w/w) is added to induce complete CF leakage (100% leakage). The insert in Figure 4b shows the percentage of CF leakage (after 20 min) obtained for 0 and 3 mol% PE-PEG at different P/L molar ratios. In the absence of PE-PEG, leakage was detected even at the lowest P/L assessed (4% leakage at P/L = 0.001). This indicates that binding of gomesin to bare LUVs is practically always accompanied by membrane permeabilization. On the other hand, significant leakage from coated LUVs was only detected at P/L = 0.01. Above that, the lytic activity of gomesin was always higher against coated than against bare LUVs. The curve showed in Figure 4b for bare LUVs exhibits a depression around 3 min. This arises from the marked increase in sample turbidity in the first minutes (see Figure 3a), which reduces the fluorescence intensity of CF (39). As the huge aggregates precipitate with time, the influence of light scattering in the fluorescence intensity measurement decreases. Since in the absence of PEPEG sample turbidity increases considerably (see Figure 3), it is not possible to assure that the lower percentage of leakage of bare LUVs above P/L = 0.01 is not actually an artifact coming from a decrease in the fluorescence intensity due to high sample turbidity.

Thermodynamic analysis of the interaction of gomesin with bilayers Binding of gomesin to LUVs composed of 1:1 POPC:POPG is mainly an exothermic process and is virtually always accompanied by vesicle aggregation and CF leakage. The ITC results suggest two different models for the thermodynamic analysis of the binding isotherm. The simpler model assumes a specific binding of gomesin to POPG lipids and can be formulated in terms of a Langmuir adsorption isotherm (40). The second model avoids a specific molecular


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interaction but considers binding as a combination of electrostatic attraction and surface partitioning. The Langmuir model postulates identical binding sites on the membrane surface composed of n lipids. As gomesin activity is always accompanied by changes in membrane permeability, as was shown with the leakage experiments, the total amount of lipid was used in the evaluation. Table 1 summarizes the results of the Langmuir adsorption isotherm for the different titrations shown in Figure 1 in terms of the number of POPG lipids involved n, the binding constant K, and the reaction enthalpy ∆Hpep. The differences between coated and bare LUVs are small and no systematic differences between the two groups were detected. A more detailed insight into the binding reaction is given by considering in detail the electrostatic attraction of gomesin to the anionic membrane surface followed by a chemical adsorption into the hydrophobic membrane. The concentration of gomesin, which carries 6 positive charges, is much higher in the vicinity of the anionic membrane than in bulk solution. We have described this equilibrium by calculating the electrostatic attraction via the GouyChapman theory and combining it with a surface partition equilibrium. Such approach has been previously described in detail in the literature and has proven to be a good means to analyze the interaction of different antimicrobial peptides with model membranes (34,35,41,42). A brief description of the model used is given in the Materials and Methods section. Figure 5 shows the results of this analysis as fits of the cumulant heat (the total heat summed up to injection i) obtained for the L-P and P-L titrations, of both bare and coated LUVs. It should be noted that the model is not expected to fit particular features of the experimental result, as the small endothermic component detected at the end of the L-P titration of coated LUVs and the initial dip in the P-L titrations, and therefore the aim was to fit the general behavior of the curves. A


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summary of the thermodynamic data derived with the surface partition model combined with the Gouy-Chapman theory is also given in Table 1. The surface partition model combined with the Gouy-Chapman theory can reasonably well fit the L-P and P-L data for bare and coated LUVs. As expected, the binding constants obtained with this model are considerably smaller compared to the binding constants obtained with the Langmuir model, as the contribution of electrostatic attraction was removed. Nevertheless, the model also predicts an effective peptide charge of zp = 5-6. The parameters obtained with the surface partition model differ by one order of magnitude between the L-P and P-L experiments. This difference could arise because of the influence of the aggregation process which depends on the experimental setup. Clearly, peptides and lipids engaged in forming huge aggregates cannot equilibrate with new peptides or lipids injected afterward. Therefore, the binding constants obtained could still be apparent ones, since vesicle aggregation brings non-equilibrium aspects to the peptide-lipid interaction. However, it is important to mention that the differences observed between the titration directions are maintained also for coated LUVs, for which the extension of aggregation is largely reduced. Furthermore, the binding constant obtained in both titrations for coated LUVs are one order of magnitude higher than the corresponding ones obtained for bare LUVs. This agrees with the fact that the lytic activity of gomesin seems to be stronger against coated LUVs (see Figure 4). This difference in the affinity between bare and coated membranes could also arise due to artifacts coming from vesicle aggregation, as this process in high degrees could reduce the concentration of free lipid and peptide available. It should be mentioned, however, that the endothermic contribution, clearly present in the L-P experiment of coated LUVs, cannot be fit with the model (see points above L/P = 10 in Figure 2a, which correspond to the injections giving rise to


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endothermic peaks), and can interfere with the analysis of the exothermic contribution which was attributed to the binding of gomesin to the membrane. The surface partition model is not perfect for a number of reasons. LUVs containing 50 mol% of charged lipids generate a very high surface potential. In an ionic media of more than 100 mM NaCl, the Gouy-Chapman theory comes to its limits. Also, the assumption of ideal mixing might not be fulfilled along the whole titration. For LUVs coated with 3 mol% PE-PEG, the size of the PEG brush is an unknown parameter and constitutes an additional problem. It may shield the membrane surface and the surface potential calculated with the Gouy-Chapman theory may not be fully active at the level of the PEG brush and might probably be overestimated in the analysis. Considering the points discussed above about the model and the variances in the thermodynamic parameters obtained from different experimental setups, a number of general conclusions can still be derived from the ITC data. Three different types of evaluation lead to the conclusion that the effective charge of gomesin is zp = 5-6. This is also close to the maximum charge possible for gomesin. The result is quite unexpected as in most other studies on highly charged peptides the effective charge is always much smaller than the nominal charge. A case in point is melittin which has a maximum charge of 5, but the effective charge in membranes studies is only 2 (41). With the knowledge of K and ∆Hpep, the other thermodynamic relevant variables, ∆G and ∆S, are easily obtained through the relations: ∆G = – RT ln 55.5 K = ∆H – T∆S

(eq. 10)


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where the term 55.5 M, the concentration of water, corrects the unit of K to molar fraction. For the binding constants K obtained from the surface partition model combined with the GouyChapman theory, K = (3-10)×103 M-1 and (2-50)×104 M-1 , for bare and coated vesicles, respectively (see Table 1), the corresponding ∆G values found were – (7.1-7.8) kcal/mol and – (8.2-10) kcal/mol, respectively. Since ∆Hpep is large and exothermic, it follows that the binding of gomesin is an enthalpy-driven process, and the entropic term (– T∆S) contributes to no more than 20% of the total free energy of interaction, mainly in the coated LUVs. This might arise because of changes in the PEG structure upon peptide binding. For the interaction of gomesin with charged membranes, entropic contributions appear to play no significant role at room temperature. The origin of the enthalpy change is still unclear. As suggested by ITC, light scattering and leakage experiments, binding of gomesin is practically always accompanied by vesicle aggregation and membrane rupture. Therefore, the exothermic enthalpy could entail the actual binding of gomesin to the membrane (including to adjacent layers causing aggregation) and the destabilization/rupture of the membrane structure. The binding itself could be attributed to a non-classical hydrophobic contribution, arising from interaction of hydrophobic residues with the membrane core, and/or a surface interaction with the lipid headgroups. Because the structure of gomesin remains basically the same upon interaction with amphiphilic surfaces due to the disulfide bridges (21), no contribution of ∆H arising from structuring of the peptide is expected, as observed with various linear peptides that acquire an α-helical structure upon binding (35). As a comparison, the interaction of magainin 2 with model membranes showed also a large negative enthalpy variation, which was associated to α-helix formation and to the non-classical hydrophobic effect (35). The latter contribution is of similar magnitude to the enthalpy variation measured for gomesin in this work.


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CONCLUSION

Taken together, the ITC, light scattering and leakage experiments show that the interaction of gomesin with anionic vesicles is enthalpy-driven and is virtually always accompanied by vesicle aggregation and membrane perturbation leading to leakage of internal vesicle contents. Coating of the vesicle surface with 3 mol% PE-PEG considerably reduces the extent of vesicle aggregation, although a mild degree of vesicle aggregation is always detected. Although surface coating changed somewhat the thermodynamic parameters and leakage response, the general mode of action of gomesin seem to be preserved. Also, it can be concluded that the activity of gomesin is not dependent on the extent of vesicle aggregation induced, as the exothermic signal measured with ITC and CF leakage were not decreased upon surface coating. The parallel ITC and light scattering results obtained from L-P and P-L titrations differ to some extent. Indeed, the huge capacity of gomesin to induce vesicle aggregation certainly contributes to obtaining somewhat different results depending on the experimental setup, as vesicle aggregation in large degrees is an irreversible process. However, it is important to stress that the basic interaction of gomesin with membranes is maintained in both titration directions and irrespective of the presence/absence of surface coating. Similar thermodynamic trends were extracted from both titrations, as shown in Table 1, and gomesin was shown to interact strongly with the charged POPG lipids, essentially in a one-to-one fashion, i. e., one gomesin charge per lipid charge. The results point to a strong interaction of gomesin with the membrane surface. According to NMR data (21), the structure of gomesin, which is stabilized by the disulfide bridges, exhibits an amphipathic character, with a relatively small hydrophobic face formed by


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Leu5, Tyr7, Tyr14 and Val12, and hydrophilic parts on both extremities (Arg3,4,16,18 close to the termini and Lys8, Gln9 and Arg10 in the turn). Therefore, it is reasonable to assume that gomesin will most probably lie anchored and parallel to the membrane surface, with a relatively shallow penetration of its hydrophobic face. This mode of action described above would also correlate with the observations made previously with optical microscopy of giant vesicles in the presence of gomesin, which suggested a carpet-like mode of action (23). There, it was shown that gomesin often caused formation of dense regions in the bilayer, followed by vesicle bursting. Vesicle aggregation was not observed, because giant vesicles typically do not touch each other frequently, as opposed to LUVs. However, the formation of the dense regions certainly points to a local membrane wrinkle, mediated by peptides, as observed also with other antimicrobial peptides, as mastoparan X (43). The sudden burst of GUVs was previously associated as indicative of a carpet-like mode of action (44-46), and possibly arises because of a surface stress caused by the binding of the peptides, which ultimately leads to disruption of the bilayer integrity. The mode of action of peptides has been mainly divided into two major classes (2-5, 47). In one mechanism, peptides form a pore across the bilayer, either with lipids bending to help form the pore walls, as in the toroidal model (12), or as an interruption of the bilayer, as in the barrel stave (2). Generally, formation of pores requires that few peptides assume a perpendicular orientation to the bilayer plane and form a bundle at the pore, and the membrane structure is roughly preserved away from the pore (12). However, it has been shown that peptide association to form a toroidal pore is not as ordered as previously imagined (48). On the other hand, other mechanisms rely on covering of the surface with peptides aligned along the membrane plane, in a carpet-like mode of action (13). Eventually, the membrane ruptures and bilayer fragments


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might be removed, resembling a solubilization process, in a detergent-like mechanism (49). The hydrophobic match between peptide and the bilayer thickness is an important parameter in determining the general mechanism of action (46, 50, 51), although other factors are also important. For some peptides, like the bee-venom melittin (52) and the designed peptide LAH4 (53), both pore-forming and membrane-rupturing mechanisms have been reported, depending on conditions. For instance, peptide alignment to the membrane surface was found to depend on peptide (53) or membrane charge (52), where a parallel orientation was generally found when either peptide or membrane charge was enhanced, whereas perpendicular insertion was observed for milder electrostatic interactions. Both mechanisms were found to induce leakage of entrapped molecules in model vesicles and to exhibit significant antimicrobial activity. The high charge of gomesin, together with the fact that the stoichiometry of the interaction indicates a one-to-one charge association indicate that gomesin would not cause opening of pores, but rather disrupt the membrane. The high ability of gomesin to induce lipid aggregation is also associated with its strong electrostatic interaction with the lipid headgroups. However, some penetration of the hydrophobic residues is certainly important for its activity, since leakage is observed even at very low peptide-to-lipid ratios. Therefore, the results shown here, together with previous observations, suggest that gomesin causes destabilization of the membrane structure as a result from the interplay between strong interaction with the membrane surface together with a shallow insertion of its hydrophobic face. Also, we want with this work to call the attention to peptideinduced vesicle aggregation, which is often neglected. Highly charged peptides interacting with charged membranes usually induce vesicle aggregation (54, 55), which might bring irreversible aspects to the lipid-peptide association and artifacts to the results obtained from several techniques, mainly optical ones.


Langmuir

FIGURES

Peptide into Lipid

Lipid into Peptide Time (min) 0

0.0

50

100

150

Time (min)

0

50

100

150

200

250

0

100

150

e

b

a

50

0

50

100

150

200

f

0.0

µcal/s

-0.2 -0.2 -0.4

-0.6

-0.4

3 mol% PE-PEG

0 mol% PE-PEG

0.0

kcal/mol injectant

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

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d

c

3 mol% PE-PEG

0 mol% PE-PEG 0

g

h

-2

-0.2

-4

-0.4

-6 -0.6 -8 -0.8 -10 -1.0 -12 0

5

10

15

20

0

5

10

15

20

25

30

35

0.00

0.05

0.10

Lipid/Peptide molar ratio

0.15

0.00

0.05

0.10

0.15

0.20

Peptide/Lipid molar ratio

Figure 1. Heat flow measured with ITC (above; a,b,e,f) and the corresponding integrated heat per mol of injectant (below; c,d,g,h) during lipid into peptide (left; a-d) and peptide into lipid (right; e-h) titrations. In the lipid into peptide experiments, 5 µL-aliquots of a dispersion of 6mM lipid were injected into 15 µM gomesin every 10 minutes. In the peptide into lipid experiments, aliquots of 300 µM gomesin (5×2 µL, 13×5 µL) were injected into 0.1 mM lipid every 10 minutes. The temperature in the ITC cell was set to 25 oC. The LUVs were composed of 1:1 POPC:POPG with and without 3 mol% PE-PEG and the buffer was 30 mM HEPES pH 7.4 with 100 mM NaCl.


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Figure 2. Phase contrast optical microscopy images of dispersions of LUVs (37 µM 1:1 POPC:POPG with 0 and 3 mol% PE-PEG) in the presence of 15 µM gomesin. This lipid-topeptide molar ratio corresponds roughly to the second injection in Figure 1a,b. The time on top of each snapshot indicates the reaction time.


Langmuir

Lipid into Peptide

Peptide into Lipid

L/P

4

5

10

15

20

25

a

P/L

0

5

10

15

20

0.00

25

b

c Relative Light Scattering

0

Light Scattering (a.u.)

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

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3 buffer buffer

2

1

50

100

150

0

50

100

150

200

0.15

d

3 4

2 1

3

2

3 mol% PE-PEG

0 mol% PE-PEG

3 mol% PE-PEG

0 mol% PE-PEG 0

0.10

4

5

1

0

0.05

0

50

100

150

0

50

100

150

200

Time (min)

Time (min)

Figure 3. 90o light scattering intensity (λ = 600 nm) measured during lipid into peptide (left; c,d) and peptide into lipid (right; a,b) titrations. The experimental conditions are the same as described for the ITC experiments (see caption of Figure 1). In the L-P light scattering results, injections of LUVs into buffer only are also shown (connected with a line). In the P-L experiments, light scattering is shown as the intensity relative to that of the lipid dispersion before injection of peptide. Small corrections due to sample dilution during titration were taken into account. The x-axis above a) and b) show the lipid/peptide molar ratio corresponding to each injection. The insert in the P/L side shows the relative light scattering intensity as a function of the peptide/lipid molar ratio obtained with bare (■) and coated (○) LUVs. Only the data that do not lead to precipitation are shown. Light scattering experiments were performed at room temperature and under magnetic stirring. The LUVs were composed of 1:1 POPC:POPG with and without 3 mol% PE-PEG and the buffer was 30 mM HEPES pH 7.4 with 100 mM NaCl.


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100

a) 0.02 P/L

% leakage

80

60

gomesin

40

TX-100

20

0mol% PEG 3mol% PEG

0 0 100

10

20

30

40

50

60

b) 0.20 P/L

80

100 60

% leakage (20 min)

% leakage

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

Langmuir

40

20

80 60 40 20 0 0.00

0.05

0.10

0

0.15

0.20

P/L 0

5

10

15

20

25

Time (minutes)

Figure 4. Kinetics of CF leakage. The percentage of CF leakage was calculated with eq. 1. The cuvette initially contained 50 µM 1:1 POPC:POPG LUVs with 0 and 3 mol% PE-PEG loaded with 50 mM CF (0% leakage). At time zero, a) 1 and b) 10 µM gomesin were injected into the cuvette (P/L = 0.02 and 0.2, respectively). After 23 or 55 min a high concentration of Triton X100 (0.2% w/w) was added to induce complete release of CF (100% leakage). The symbols in the leakage kinetics were added to discriminate between the leakage kinetics in the presence and absence of surface coating (see legend). Experiments were performed at room temperature and under magnetic stirring. Buffer: 30 mM HEPES pH 7.4 with 50 mM CF and 86 mM glucose (inside) and 30 mM Hepes pH 7.4 with 100 mM NaCl (outside). The insert shows the percentage of leakage after 20 minutes obtained from different P/L ratios.


Langmuir

0

cumulant heat (µcal)

a 0mol% PEG 3mol% PEG

-50

-100

-150

L-P -200 0

5

10

15

20

25

30

lipid/peptide 0

cumulant heat (µcal)

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

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b

-20 -40 -60 -80 -100

P-L -120 0.00

0.05

0.10

0.15

peptide/lipid

Figure 5. Cumulant heat obtained from a) lipid into peptide and b) peptide into lipid titrations. The points show the experimental results (see Figure 1) and the lines show the best fits obtained with a surface partition model combined with the Gouy-Chapman theory. The thermodynamic parameters obtained from the fits are shown in Table 1.


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TABLES.

Table 1 – Thermodynamic parameters obtained from two different models to the ITC data of both lipid into peptide (L-P) and peptide into lipid (P-L) titrations. ∆Hpep is the reaction heat per mole of gomesin, K is the binding constant, n is the number of POPG lipids involved in binding in the Langmuir adsorption model and zp the effective charge of gomesin in the surface partition model combined with the Gouy-Chapman theory. ∆Hpep

K (M-1)

n / zp

-8

5×106

5.5

-8

5×10

6

-9.8

1.0×106

7

-9.2

6

6

(kcal/mol) Langmuir Adsorption L-P 0 mol% PE-PEG 3 mol% PE-PEG

4

P-L 0 mol% PE-PEG 3 mol% PE-PEG

0.7×10

Surface Partition with Gouy-Chapman theory L-P 0 mol% PE-PEG 3 mol% PE-PEG

-7.8

1×104

5

-7.8

5×10

5

5

-9.6

3×103

6

-8.7

4

6

P-L 0 mol% PE-PEG 3 mol% PE-PEG

2× 10

ACKNOWLEDGMENT We acknowledge the financial support from FAPESP, CAPES, CNPq and INCT-FCx.


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(9) Rausch, J. M.; Marks, J. R.; Rathinakumar, R.; Wimley, W. C. Beta-sheet pore-forming peptides selected from a rational combinatorial library: mechanism of pore formation in lipid vesicles and activity in biological membranes. Biochemistry 2007, 46, 12124-12139. (10) Stromstedt, A. A.; Ringstad, L.; Schmidtchen, A.; Malmsten, M. Interaction between amphiphilic peptides and phospholipid membranes. Curr. Opin. Colloid Interface Sci. 2010, 15, 467-478. (11) Matsuzaki, K.; Murase, O.; Fujii, N.; Miyajima, K. An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation. Biochemistry 1996, 35, 11361–11368. (12) Matsuzaki, K. Magainins as paradigm for the mode of action of pore forming polypeptides. Biochim. Biophys. Acta 1998, 1376, 391-400. (13) Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1999, 1462, 55–70. (14) Ladokhin, A. S.; White, S. H. 'Detergent-like' permeabilization of anionic lipid vesicles by melittin. Biochim. Biophys. Acta 2001, 1514, 253–260. (15) Silva, Jr., P. I.; Daffre, S.; Bulet, P. Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family. J. Biol. Chem. 2000, 275, 33464-33470.


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(16) Miranda, A.; Miranda, M. T. M.; Jouvensal, L.; Vovelle, F.; Bulet, P.; Daffre, S. Gomesin. A Powerful antimicrobial peptide isolated from the Brazilian tarantula spider Acanthoscurria gomesiana. In: Animal Toxins: State of the Art. Perspectives in Health and Biotechnology. Ed. M. E. Lima, Belo Horizonte, Editora UFMG. 2009, 323-343. (17) Moreira, C. K.; Rodrigues, F. G.; Ghosh, A.; Varotti, F. P.; Miranda, A.; Daffre, S.; JacobsLorena, M.; Moreira, L. A. Effect of the antimicrobial peptide gomesin against different life stages of Plasmodium spp. Exp. Parasitol. 2007, 116, 346-53. (18) Barbosa, F. M.; Daffre, S.; Maldonado, R. A.; Miranda, A.; Nimrichter, L.; Rodrigues, M. L. Gomesin, a peptide produced by the spider Acanthoscurria gomesiana, is a potent anticryptococcal agent that acts in synergism with fluconazole. FEMS Microbiol. Lett. 2007, 274, 279-86. (19) Sacramento, R. S.; Martins, R. M.; Miranda, A.; Dobroff, A. S.; Daffre, S.; Foronda, A. S.; De Freitas, D.; Schenkman, S. Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii. Parasitology 2009, 136, 813-821. (20) Rodrigues, E. G.; Dobroff, A. S.; Cavarsan, C. F.; Paschoalin, T.; Nimrichter, L.; Mortara, R. A.; Santos, E. L.; Fazio, M. A.; Miranda, A.; Daffre, S.; Travassos, L. R. Effective topical treatment of subcutaneous murine B16F10-Nex2 melanoma by the antimicrobial peptide gomesin. Neoplasia 2008, 10, 61-68. (21) Mandard, N.; Bulet, P.; Caille, A.; Daffre, S.; Vovelle, F. The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider. Eur. J. Biochem. 2002, 269, 1190-1198.


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(22) Fázio, M. A.; Jouvensal, L.; Vovelle, F.; Bulet, P.; Miranda, M. T.; Daffre, S.; Miranda, A. Biological and structural characterization of new linear gomesin analogues with improved therapeutic indices. Biopolymers 2007, 88, 386-400. (23) Domingues, T. M.; Riske, K. A.; Miranda, A. Revealing the Lytic Mechanism of the Antimicrobial Peptide Gomesin by Observing Giant Unilamellar Vesicles. Langmuir 2010, 26, 11077–11084. (24) Seelig, J. Titration calorimetry of lipid-peptide interactions. Biochim. Biophys. Acta 1997, 1331, 103–116. (25) Seelig, J. Thermodynamics of lipid-peptide interactions. Biochim. Biophys. Acta 2004, 1666, 40– 50. (26) Blume, G.; Cevc, G. Liposomes for the sustained drug release in vivo. Biochim. Biophys. Acta 1990, 1029, 91-97. (27) Immordino, M. L.; Dosio, F.; Cattel, L. Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int. J. Nanomedicine 2006, 1, 297– 315. (28) Kenworthy, A. K.; Hristova, K.; Needham, D.; McIntosh., T. J. Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 1995, 68, 1921-1936. (29) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Hydration of Polyethylene GlycolGrafted Liposomes. Biophys. J. 1998, 74, 1371–1379.


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(30) Weinstein, J. N.; Yoshikami, S.; Henkart, P.; Blumenthal, R.; Hagins, W. A. Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker. Science 1977, 195, 489–492. (31) Fázio, M. A.; Oliveira Jr., V. X.; Bulet, P.; Miranda, M. T.; Daffre, S.; Miranda, A. Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability. Biopolymers 2006, 84, 205–218. (32) Rouser, G.; Fkeischer, S.; Yamamoto, A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970, 5, 494-496. (33) Verly, R. M.; Rodrigues, M. A.; Daghastanli, K. R. P.; Denadai, A. M. L.; Cuccovia, I. M.; Bloch Jr., C.; Frézarde, F.; Santorod, M. M.; Piló-Veloso, D.; Bemquerer, M. P. Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes. Peptides 2008, 29, 15-24. (34) Wenk, M. R.; Seelig, J. Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation. Biochemistry 1998, 37, 3909-3916. (35) Wieprecht, T.; Beyermann, M.; Seelig, J. Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure. Biochemistry 1999, 38, 1037710387. (36) McLaughlin, S. The electrostatic properties of membranes. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113-136.


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Langmuir

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

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TABLE OF CONTENTS


Langmuir

P e p tid e in to L ip id

L ip id in to P e p tid e T im e ( m in ) 0

5 0

1 0 0

1 5 0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

k c a l/m o l in je c ta n t

0

5 0

1 0 0

1 5 0

0

e b

a

0 .0

T im e ( m in )

0

5 0

1 0 0

1 5 0

2 0 0

f

0 .0

-0 .2

µc a l / s

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

Page 38 of 43

-0 .2 -0 .4

-0 .6

-0 .4

0 m o l%

0 .0

3 m o l%

P E -P E G

P E -P E G

0 m o l%

d c

0

3 m o l%

P E -P E G

g

P E -P E G

h

-2

-0 .2

-4 -0 .4 -6 -0 .6 -8 -0 .8 -1 0 -1 .0

0

-1 2 5

1 0

1 5

2 0

L ip id /P e p tid e

0

5

m o la r r a tio

1 0

1 5

2 0

2 5

3 0

3 5

0 .0 0

ACS Paragon Plus Environment

0 .0 5

0 .1 0

0 .1 5

P e p tid e /L ip id

0 .0 0

0 .0 5

m o la r r a tio

0 .1 0

0 .1 5

0 .2 0

Page 39 of 43

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

Langmuir

164x93mm (150 x 150 DPI)


Langmuir

L ip id in to P e p tid e

P e p tid e in to L ip id

L /P 0

5

1 0

1 5

2 0

2 5

P /L 0

5

a 4

1 0

1 5

2 0

0 .0 0

2 5

b

0 .0 5

0 .1 0

0 .1 5

d

c 4

R e la tiv e L ig h t S c a tte r in g

5

L ig h t S c a tte r in g ( a .u .)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Page 40 of 43

3 b u ffe r b u ffe r 2

1

3 4

2 1 3

2

1 0 0

0 m o l% 5 0

1 0 0

1 5 0

3 m o l%

P E -P E G 0

T im e ( m in )

5 0

1 0 0

1 5 0

0 m o l%

P E -P E G 2 0 0

0


5 0

1 0 0

3 m o l%

P E -P E G 1 5 0

0

T im e ( m in )

5 0

1 0 0

1 5 0

P E -P E G 2 0 0

Page 41 of 43

1 0 0

a ) 0 .0 2 P /L

8 0

6 0

g o m e s in

4 0

%

le a k a g e

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

Langmuir

T X -1 0 0

2 0

0 m o l% 3 m o l% 0 0

1 0 0

1 0

2 0

P E G P E G

3 0

4 0

5 0

6 0

b ) 0 .2 0 P /L

8 0

4 0

le a k a g e ( 2 0 m in )

le a k a g e

6 0

%

1 0 0

%

2 0

8 0 6 0 4 0 2 0 0 0 .0 0

0 .0 5

0 .1 0

0

0 .1 5

0 .2 0

0

P /L 5

1 0

1 5

T im e ( m in u te s )


2 0

2 5

Langmuir

c u m u l a n t h e a t ( µc a l )

c u m u l a n t h e a t ( µc a l )

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

Page 42 of 43

0

a 0 m o l% 3 m o l%

-5 0

P E G P E G

-1 0 0

-1 5 0

L -P -2 0 0 0

5

1 0

1 5

2 0

2 5

3 0

lip id /p e p tid e 0

b

-2 0 -4 0 -6 0 -8 0 -1 0 0

P -L -1 2 0 0 .0 0

0 .0 5

0 .1 0

p e p tid e /lip id ACS Paragon Plus Environment

0 .1 5

Page 43 of 43

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

Langmuir

181x90mm (150 x 150 DPI)


Interaction of the Antimicrobial Peptide Gomesin ... - ACS Publications

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