Amphiphilicity Is a Key Determinant in the Membrane Interactions of

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Amphiphilicity is a key determinant in the membrane interactions of synthetic 14-mer cationic peptide analogs Matthieu Fillion, Maxime Goudreault, Normand Voyer, Burkhard Bechinger, and Michèle Auger Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00961 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on November 26, 2016

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Biochemistry

Amphiphilicity is a key determinant in the membrane interactions of synthetic 14-mer cationic peptide analogs

Matthieu Filliona, Maxime Goudreaulta, Normand Voyerb, Burkhard Bechingerc and Michèle Augera*

a

Department of Chemistry, Regroupement québécois de recherche sur la fonction,

l’ingénierie et les applications des protéines, Centre de recherche sur les matériaux avancés (CERMA), Centre québécois sur les matériaux fonctionnels (CQMF), Université Laval, Québec, QC, Canada, G1V 0A6 b

Department of Chemistry, PROTEO, Université Laval, Québec, QC, Canada, G1V 0A6

c

Université de Strasbourg, CNRS, UMR7177, Institut de Chimie, 4, Rue Blaise Pascal, 67070 Strasbourg, France

*Address correspondence to: Michèle Auger, Department of Chemistry, PROTEO, CERMA, CQMF, Université Laval, Québec, QC, Canada, G1V 0A6, Tel.: 418-656-3393, Fax: 418-656-7916, E-mail: [email protected]

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FUNDING This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Fonds de Recherche du Québec Nature et Technologies (FRQNT), the Regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines (PROTEO), the Centre de recherche sur les matériaux avancés (CERMA) et the Centre québécois sur les matériaux fonctionnels (CQMF). The financial contributions of the Agence Nationale de la Recherche (projects MemPepSyn 14-CE340001-01, LabEx Chemistry of Complex Systems 10-LABX-0026_CSC), and the RTRA International Center of Frontier Research in Chemistry are gratefully acknowledged. M.F. would like to acknowledge graduate scholarships from NSERC and FRQ-NT and an intern scholarship from PROTEO.

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ABBREVIATIONS AMP, antimicrobial peptide; ATR, attenuated total reflectance; CE, crown ether; DMPC, 1,2-dimyristoylphosphatidylcholine;

DMPG,

1,2-dimyristoylphosphatidylglycerol;

EDTA, ethylenediamine-tetraacetic acid disodium; FRPA, fluoroquinolone-resistant Pseudomonas aeruginosa; Fmoc, fluorenylmethyloxycarbonyl; FTIC, fluorescein isothiocyanate;

HCl,

hydrochloric

acid;

HEPES,

4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; MAS, magic angle spinning; MLV, multilamellar vesicle; MRSA, methicillin-resistant Staphylococcus aureus; NMR, nuclear magnetic resonance; POPC, 1-palmitoyl-2-oleyol-sn-glycero-3phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; SUV, small unilamellar vesicle; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; TPPM, twopulse phase modulation; VRE, vancomycin-resistant Enterococcus.


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ABSTRACT Cationic antimicrobial peptides are a component of the innate immune system of several organisms and represent an interesting alternative to fight multiresistant bacteria. In this context, we have elaborated a synthetic peptide scaffold allowing the study of the impact of different molecular determinants on the membrane interactions. The aim of the present study was to elucidate the mechanism of action of two cationic peptides that derive from a neutral 14-mer template peptide and where the hydrophilic portion is composed of a crown ether. The R5R10 peptide is active in the presence of both negatively charged and zwitterionic membranes (non-selective) and adopts an α-helical conformation, whereas the R4R11 peptide is more active in the presence of negatively charged membranes (selective) and forms intermolecular β-sheet structures. Both the membrane topology and the location of the peptides have been assessed using solid-state NMR and ATR-FTIR spectroscopy. In addition, fluorescence experiments have been performed on different membrane mixtures to evaluate the ability of the peptides to induce a positive curvature to the membrane. Overall, for both the R5R10 and R4R11 peptides, the results are consistent with a mechanism of action similar to the sinking-raft model in which the peptides are mainly lying flat on the membrane surface and impose a bending stress to the membrane, thus leading to the formation of pores. Furthermore, the difference of membrane selectivity between R5R10 and R4R11 peptides is due to their differing amphipathic properties which modulate the membrane activity on zwitterionic model membranes.


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Over the years, overuse and misuse of antibiotics have led bacteria to acquire resistance mechanisms. Antibiotic resistance is considered a major worldwide problem as the number of nosocomial infections due to resistant or multidrug-resistant bacteria is consistently increasing.1 Currently, the arsenal of antibiotics is becoming less effective to treat infections involving resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and fluoroquinoloneresistant Pseudomonas aeruginosa (FRPA).2 Furthermore, the new antibiotics release on the market are often analogs of existing antibiotics which in turn lead to rapid acquisition of resistance by bacteria.3 In fact, since the early 80’s, lipopeptides and oxazolidinones are the only novel classes of antibiotics that have reached the market.4 In this context, it is of great importance to concentrate our efforts on searching and developing alternatives. Among the promising ones, there are the naturally occurring cationic antimicrobial peptides (AMP). These molecules are a key component in the innate immune system from lower to higher organisms such as bacteria and mammals.5 Despite their structural diversity, they share common characteristics such as a short length, a global positive charge, and an amphiphilic character.6 Antimicrobial peptides cause a rapid destruction of the pathogenic agents and are characterized by a large spectrum of activity. Previous studies indicate that AMPs specifically target the membrane of pathogens where they induce the formation of defects that will eventually cause collapse of the transmembrane electrochemical gradient, thus leading to cell death.7-9 In comparison with conventional antibiotics whose mechanisms of action generally rely on altering a specific target, development of resistance to AMPs is less likely to occur since they act on generalized targets.10 However, the mechanisms of action of cationic antimicrobial


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peptides are still unclear even though several of them have been proposed in the literature.11, 12 An important feature of the mechanisms of action is the peptide membrane topology, i.e. their location in the membrane. For example, in the case of the sinking-raft mechanism, the peptide lies on the membrane surface in close contact with phospholipid polar head groups, whereas the peptide has a transmembrane topology in the case of the barrel-stave and toroidal pore mechanisms. In addition, accumulation of peptides on the membrane surface may impose curvature strain to the membrane as observed for the toroidal pore and sinking-raft mechanisms.13 Indeed, the peptides impose a positive curvature strain which triggers the fusion of the inner and outer leaflets of the membrane. Therefore, to synthesize and design more potent antimicrobial peptides, a better understanding of their modes of action requires determining both their membrane topology and their propensity to alter the membrane intrinsic curvature. Initially, we have designed and synthesized a 21-mer peptide that acts as an ion channel.14 This synthetic peptide is composed of 15 leucine residues and 6 phenylalanine residues that are modified with a 21-C-7 crown ether (CE). The choice of leucine residues pertains to their high propensity to induce an α-helical conformation and the rationale behind the choice of using modified phenylalanine residues is their hydrophilic property and their ability to chelate cations, allowing for channel activity. By studying the shorter 7- and 14-mer analogs, it has been observed that only the 14-mer peptide is able to induce leakage of calcein confined within vesicles made of zwitterionic and anionic phospholipids which mimic eukaryotic and prokaryotic cellular membranes, respectively.15 To gain selectivity against negatively charged vesicles, we have systematically substituted leucine residues with basic residues such as lysine, arginine,


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and histidine. The 14-mer template peptide, as well as the mono- and bisubstituted cationic analogs, have been extensively studied in previous investigations. 16-18 As a brief summary, the model 14-mer peptide (Figure 1) has an α-helical conformation both in solution and in interaction with lipid bilayers. Its membrane topology has been determined by the combination of various solid-state NMR experiments. The overall conclusion is that the peptide lies on the membrane surface and acts by a mechanism of action similar to the sinking-raft model. For the cationic analogs, the conformation depends on the position(s) of substitution of the basic residues in the helical representation. When the charged residues are located near the CE, the peptides have an α-helical conformation whereas when the charged residues are located far from the CE in a helical representation, the peptides self-assemble to form intermolecular β-sheet structures. In addition, by performing calcein leakage assays, it has been shown that the selectivity of the cationic analogs is directly linked to the peptide conformation since only the β-sheet aggregated analogs are selective towards vesicles made of anionic phospholipids. The analogs chosen for the current study are illustrated in Figure 1 and each peptide contains a labeled L-leucine residue (1-13C; 15N) at position 7 in the primary sequence. We have focused our current investigation on arginine-containing derivatives of the model 14-mer peptide because they are generally more potent in terms of antimicrobial activity in comparison with lysine- and histidine-containing derivatives (unpublished work). The aim of this contribution is to determine the detailed mode of action of both selective and non-selective bicationic derivatives using various spectroscopic methods and membrane-mimicking systems. Moreover, since few biophysical studies have been


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carried out on linear β-sheet peptides, our peptide design provides an innovative system to establish comparative studies between α-helical and β-sheet peptides. More specifically,

31

P and 15N solid-state NMR experiments have been performed on oriented

samples to obtain information on both the membrane disruptive activity and the topology of the α-helical peptide. In order to determine the degree of insertion of the peptides in the interfacial region of the membrane, a rotational-echo double-resonance technique (REDOR) has been applied to measure

15

N-31P internuclear distances. In addition, we

have used ATR-FTIR with linearly polarized light to determine both the conformation and the membrane topology of the cationic analogs in oriented films of DMPC and DMPG. Furthermore, the orientation of the lipid chains has been evaluated by measuring the linear dichroism of the CH2 symmetric stretching vibration. The ability of the peptides to impose a bending stress on the membrane was determined by fluorescence spectroscopy.


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MATERIALS AND METHODS Materials 1,2-dimyristoylphosphatidylcholine

(DMPC),

1,2-dimyristoylphosphatidylglycerol

(DMPG), 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without purification. CHCl3, CH3OH, D-(+)-trehalose dehydrate, EDTA, HEPES, HFIP, NaH2PO4, and Na2HPO4 were purchased from Sigma-Aldrich (St-Louis, MO). Deuterium oxide was obtained from CDN isotopes (Pointe-Claire, QC). Water used for buffer preparation was distilled and deionized using a Barnstead NANOpurII system (resistivity of 18.2 MΩ/cm; Boston, MA) with four purification columns. All solvents were of reagent grade or HPLC grade quality, purchased commercially and used without any further purification. N-Fmoc-protected amino acids were purchased from Matrix Innovation (Québec, QC). The labeled L-leucine residue (1-13C, 99%;

15

N, 98%+) was

purchased from Cambridge Isotope Laboratories (Andover, MA). All other chemicals were of reagent grade. Glass plates with dimensions of 8×22 mm were purchased from Marienfeld (Lauda-Königshofen, Germany).

Peptide synthesis All the analogs were synthesized using a solid-phase synthesis approach on a Wang resin by sequentially adding N-Fmoc-protected amino acids as previously described.19 Peptide crude purity was checked by reverse-phase HPLC using an Agilent 1050 chromatograph (Agilent Technologies, Santa Clara, CA) with a gradient of solvents A [90% H2O/5% 9 ACS Paragon Plus Environment

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CH3CN/5% 2-propanol/0.1% trifluoroacetic acid (TFA)] and B (50% CH3CN/50% 2propanol/0.1 % TFA) over 45 min. Final characterization was conducted using a LC/MSTOF Agilent 6210 mass spectrometer (Agilent technologies, Santa Clara, CA) equipped with electrospray ionization. We have estimated that the crude purity of the R5R10 and R4R11 peptides is superior to 85%. For the labeled peptides, an isotopically labeled Lleucine residue (1-13C;

15

N) has been protected with a Fmoc group and incorporated at

position 7 in the peptide sequence.

Sample preparation Oriented samples – glass plates Samples of phospholipids mechanically oriented between glass plates were prepared from 20 mg of POPC and the adequate mass of peptide to obtain phospholipid/peptide molar ratios of 60/1 or 100/1. The powder mixture was dissolved in 100 µL of HFIP and deposited onto 20 ultrathin cover glasses (8×22 mm, thickness 00).20 The lipid suspension was allowed to dry in air and the glass plates were placed in a high vacuum chamber to remove all traces of organic solvent. Then, the glass plates were stacked on top of each other. Hydration of sample was done at 37°C (96% relative humidity using a saturated K2SO4 solution) by placing the sample for a duration of 24 hours in an incubator. After completion of the hydration process, the stacked glass plates were wrapped with teflon tape and sealed in plastic wrapping prior to data acquisition.

Lyophilized samples – REDOR


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Biochemistry

These samples were prepared by weighing 40 mg of DMPC and the adequate mass of peptide to obtain a phospholipid/peptide molar ratio of 20/1. The powder mixtures were dissolved in 180 µL of CHCl3/MeOH (1/1 v/v). The residual solvent was removed under a gentle stream of nitrogen gas, followed by an overnight lyophilization. The dried samples were hydrated with 180 µL of 100 mM HEPES, 5 mM EDTA (pH 7.4) buffer, giving a total proportion of 20% (w/w) lipids in buffer. Trehalose, a carbohydrate lyoprotectant, was also added to an equivalent of 20% of the dry weight of phospholipids in order to preserve important hydrogen bonds.21 Preparation of multilamellar vesicles (MLVs) was ensured by repeating 5 cycles of vigorous vortexing, freezing (liquid N2), thawing (37°C), and vortexing. The sample was rapidly frozen in liquid nitrogen (N2), lyophilized overnight, and then packed into a 4 mm NMR tube prior to data acquisition.

Multilamellar vesicles – ATR-FTIR Prior to sample preparation, peptides were solubilized in 10 mM HCl and lyophilized overnight. This process was repeated 5 times in order to remove all traces of TFA. MLV samples were prepared by mixing the necessary amount of phospholipids (7 mg of DMPC or DMPG) dissolved in organic solvent and peptides dissolved in CHCl3/MeOH (1/1 v/v) to obtain a phospholipid/peptide molar ratio of 60/1. The residual solvent was removed under a gentle stream of nitrogen gas and lyophilized overnight. The dried samples were hydrated with 40 µL of 10 mM phosphate buffer (pH 7.4) made with D2O, giving a total proportion of 11% (w/w) lipids in buffer. To obtain a homogenous dispersion of MLVs, 5 cycles of vigorous vortexing, freezing (liquid N2), thawing (37°C), and vortexing were performed. Oriented films were spontaneously formed by depositing


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20 µL of the lipid suspension on each side of the germanium crystal and spread with a Teflon bar.

Unilamellar vesicles – Dye-release assay MLVs were prepared according to the same procedure described for ATR-FTIR samples. However, the total mass of phospholipids was 15 mg and lyophilized samples were hydrated with 650 µL of 100 mM HEPES, 5 mM EDTA, and 80 mM calcein (pH 7.4) buffer, giving a 20 mM liposomal suspension. After performing five vortex/freeze/thaw cycles, the liposomal suspension was tip sonicated to ensure formation of small unilamellar vesicles (SUVs). Unencapsulated calcein was removed by size-exclusion chromatography using a column filled with Sephadex G-50 gel swollen in the external buffer. To make sure that the phospholipid-to-peptide molar ratio is adequate, the lipid concentration was quantified by the Bartlett phosphate technique.22

Solid-state NMR experiment 31

P NMR experiments – glass plates

Proton-decoupled 31P solid-state NMR spectra were acquired with a Bruker Avance wide bore 300 MHz spectrometer. The glass plate samples were inserted into a flat-coil solidstate NMR probehead with the bilayer normal oriented parallel relative to the magnetic field direction.23 The spectra were obtained at 121.5 MHz with a Hahn-echo sequence and TPPM proton decoupling.24, 25 Using 512 data points, the spectra were acquired with a pulse length of 5 µs for a 90° pulse (50 kHz B1 field), a recycle delay of 3 s, and an interpulse delay of 50 µs. The spectral width was 36.5 kHz and a line broadening of 50


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Biochemistry

Hz was applied to all spectra. A total of 500 scans were acquired for the glass plate samples. The chemical shifts were referenced relative to external 85% H3PO4 (0 ppm).

15

N NMR experiments – glass plates

The proton-decoupled

15

N cross polarization (CP) spectra were acquired on a Bruker

Advance 750 MHz wide bore spectrometer. The 15N NMR spectra were obtained at 76.5 MHz using a cross-polarization (CP) pulse sequence with continuous wave proton decoupling. Using 780 data points, typically 50 000 scans were acquired with a 1H-15N CP contact time of 1 ms and a recycle delay of 4 s. The spectral width was 100 kHz, and a line broadening of 300 Hz was applied to all oriented 15N solid-state NMR spectra. The chemical shift was referenced relative to external

15

NH4Cl (39.3 ppm) corresponding

approximately to 0 ppm for liquid NH3.26

REDOR experiments The spectra were acquired with a Bruker Advance 400 MHz spectrometer. The sample was packed in a 4 mm NMR tube and inserted in a magic angle spinning (MAS) probe. REDOR experiments required the acquisition of two spectra, one with phase-inversion pulses on the 31P channel to produce the 15N NMR spectrum S and one without pulses to produce the S0 spectrum27. The ratio of the difference between the two spectra (∆S = S0 S) and S0 as a function of the dipolar evolution time generates a dephasing curve allowing the determination of the magnitude of the dipolar coupling, DIS. The latter parameter was determined using the Bessel function method.28 The dipolar coupling is


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readily converted into the internuclear distance between two heteronuclear nuclei rIS with the following equation:

DIS =

γ I γ S hµ 0 16π 3 rIS3

(1)

where µ0 is the vacuum permeability constant, γI and γS are the gyromagnetic ratio of the I and S spins, respectively, h is the Plank constant, and the dipolar coupling constant DIS is in Hz. The spinning speed was kept constant at 5000 Hz. Typically, 20000 scans were acquired for each increment and the 1H-15N CP contact time was 1 ms with a match spinlocked CP of 56.8 kHz. Spectra were obtained with a recycle delay of 4 s and 1H decoupling field strength of 124.1 kHz. Spectra were processed with a 100 Hz line broadening before Fourier transformation. In order to correct for errors due to the flip angle, resonance offset effects, and variation in the B1 field, the XY8 phase cycling was employed.29 The B1 field value was 97.6 kHz for

31

P 180° pulses and 62.9 kHz for

15

N

180° pulses. All the REDOR experiments were conducted at -10°C in order to reduce motional averaging. The chemical shift was referenced relative to external 15NH4Cl (39.3 ppm) corresponding approximately to 0 ppm for liquid NH3.

Attenuated total reflectance FTIR spectroscopy The attenuated total reflectance spectra were recorded with a Nicolet Magna 760 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) equipped with an ATR unit (model TMP-220; Harrick Scientific Co., Ossining, NY) and a nitrogen cooled MCT (mercury-cadmium-telluride) A detector. The sample was deposited on a parallelogram germanium ATR crystal (50×20×2 mm, with 45° faces; Wilmad Glass Co., Inc., Buena, NJ). Prior to sample deposition, the germanium crystal was successively cleaned with 14 ACS Paragon Plus Environment

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Biochemistry

CHCl3 and MeOH. A total of 256 scans were averaged for each polarization (parallel (p) and perpendicular (s) to the plane of incidence) at room temperature with a resolution of 2 cm-1. The polarized light was produced with a ZnSe wire-grid polarizer (Specac, Orpington, UK). The spectra were corrected by subtracting reference spectra acquired for each polarization with a clean crystal. All data manipulations were performed with the Grams/AI 8.0 software (Galactic Industries, Salem, MA). The 3100-2750 cm-1 and 18501550 cm-1 spectral regions were baseline-corrected using a linear function. The dichroic ratios were determined by analyzing the ratio of the peak heights of the parallel polarized spectrum over the perpendicular polarized spectrum. For a system having both axial symmetry and uniaxial orientation as for the acyl chains and α-helical peptides and assuming and uniaxial orientation with respect to the normal to the ATR crystal plane, an order parameter relating the orientation of the principal axis of the system and the normal of the ATR crystal can be calculated with the following equation: 〈 P2 (cos θ )〉 =

R ATR − 2.00 2 ⋅ ATR R + 1.45 ( 3cos 2 γ − 1)

(2)

where RATR is the dichroic ratio and γ is the angle between the transition moment of a given vibration and the principal axis of the molecule.30, 31 This equation is obtained by considering the thick film approximation and a germanium crystal with an incident angle of 45°. For phospholipids having acyl chains in an all-trans conformation, the orientation of the CH2 symmetric stretching mode transition moment is well-defined with an angle γ of 90°.32 On the other hand, for proteins or peptides having an α-helical secondary structure, values ranging from 24° to 40° are found in the literature for the orientation of the


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transition moment of the amide I mode. An angle γ of 35° has been selected.33 Assuming an infinitely narrow distribution of orientations, the mean angle θ, which defines the orientation between the principal axis of the phospholipids or the helix and the bilayer normal, can be calculated from the order parameter with the following equation. 〈 P2 (cos θ )〉 =

3cos 2 θ − 1 2

(3)

Fluorescence measurements The membrane leakage activity of the peptides was evaluated by measuring the dequenching of calcein released in the medium. Fluorescence intensity was recorded using an excitation wavelength of 490 nm and an emission wavelength of 515 nm on a Horiba Jobin-Yvon spectrofluorimeter (Longjumeau, France). 3.4 mL of buffer and 50 µL of vesicle suspension were added in a 1 cm quartz cuvette. Stirring and acquisition were then started. After 50 seconds, a suitable amount of peptide solubilized in TFE was injected. Then, at 400 seconds, 10 µL of 10% Triton X-100 were added to disrupt the membrane integrity, thus allowing the measurement of the maximum fluorescence intensity.

%leakage =

(I

pept

− I0 )

( I max − I 0 )

(4)

where Ipept is the fluorescence intensity at time t after peptide addition, I0 the fluorescence intensity before addition of peptide, and Imax the fluorescence intensity after addition of Triton X-100 at time 400 seconds.


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RESULTS 31

P and 15N solid-state NMR

Solid-state NMR is a well suited technique for studying membrane proteins and membrane-active peptides in fluid bilayers, i.e. under conditions similar to their native environment.34 15N solid-state NMR has been extensively used to investigate the topology of membrane-active peptides.35, 36 For peptides adopting an α-helical conformation,

15

N

chemical shift values measured with oriented samples can be directly correlated with membrane topology because of the orientation dependence of the chemical shift interaction.37,

38

Consequently, in samples of phospholipids mechanically oriented

between glass plates with the membrane normal parallel relative to the magnetic field direction, the

15

N chemical shift value is below 100 ppm for a peptide that lies on the

membrane surface and around 200 ppm for a peptide having a transmembrane topology 39

. In this study, the membrane topology of the α-helical peptide R5R10 has been

investigated in samples of POPC mechanically oriented between glass plates. Before determining the membrane topology, 31P solid-state NMR experiments have been performed to evaluate the macroscopic alignment of the phospholipids. At both phospholipid-to-peptide molar ratios (60/1 and 100/1), a dominant NMR signal is observed at approximately 30 ppm (Figure 2A and 2B) that is assigned to phosphatidylcholines in the fluid state, oriented with their long axe parallel relative to the magnetic field. There is a smaller NMR signal at approximately 16 ppm which may be due to a small population of phospholipids experiencing membrane thinning.40,

41

In

addition, another small population of phospholipids is not oriented properly as seen by the residual powder intensities in the highfield region. However, the presence of R5R10


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does not perturb significantly the membrane integrity, thus allowing 15N solid-state NMR measurements to be done. On the

15

N NMR spectrum of POPC/R5R10 (Figure 2 C), a

dominant signal is observed at approximately 70 ppm and this chemical shift value is associated with a peptide lying flat on the membrane surface.

In addition, this is

consistent with the results obtained for the 14-mer template peptide which adopts a surface orientation regardless of the bilayer hydrophobic thickness. However, the

15

N

NMR spectrum of the POPC/R5R10 sample (Figure 2C) is quite broad and a small residual powder pattern is observed, thus suggesting that the peptide, globally or only at the labeled site, does not adopt a well-defined in-plane membrane topology. This could be due to conformational heterogeneity at the site of the

15

N and/or to a tendency to

aggregate. Both a larger heterogeneity in the membrane orientation of the peptide and the presence of membrane misalignments and distortions have been proposed to increase the 15

N resonance bandwidth.42 In addition, since the peptide incorporates an isotopically

labeled L-leucine residue (1-13C;

15

N), the dipolar interaction between the

15

N and

13

C

nuclei may be a source of line broadening. In order to minimize peptide aggregation, experiments have been performed at a higher phospholipid-to-peptide molar ratio of 100 to 1 (Figure 2D). The signal-to-noise ratio is lower under these conditions and the spectral contribution at approximately 70 ppm is still relatively broad as observed for the POPC/R5R10 60/1 system. Overall, the results suggest that R5R10 peptide aggregates are localized on the membrane surface and that some peptides involved in pore formation may experience a different membrane topology.

REDOR measurements


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Biochemistry

The location of both R5R10 and R4R11 peptides in DMPC membranes has been investigated using the REDOR technique allowing the determination of internuclear distance constraints between a pair of heteronuclear nuclei.43 More specifically, REDOR relies on determining the dipolar coupling between heteronuclear nuclei and the distance can be extracted by fitting the experimental data points with the universal REDOR dephasing curve generated with the Bessel function expansion. In this study, estimation of the relative distance between the peptides and the phospholipids was achieved by measuring the dipolar coupling interaction between the 15N nucleus of the labeled leucine residue at position 7 in the primary sequence (15N-Leu7-R5R10 and R4R11) and the 31P nucleus of the phosphate moiety of lipids. In order to minimize the effect of motional averaging on the dipolar coupling measured, experiments were performed at low temperature (-10°C) and on lyophilized powder of DMPC MLVs. Results of the 15N{31P} REDOR experiments for both R5R10 and R4R11 analogs are depicted in Figure 3. The dephasing was measured up to 40 ms as the signal-to-noise ratio becomes lower for longer dipolar evolution times, which is mainly due to spin-spin relaxation phenomena. For the R5R10 analog, the mean magnitude of the dipolar coupling measured is 11.2 Hz corresponding to a mean relative distance of 7.6 ± 0.7 Å. It is consistent with the distance measured for the 14-mer template peptide reconstituted in zwitterionic DMPC MLVs.16 Similar results were expected since the 14-mer and R5R10 peptides have the same conformation and the only difference arises from the substitution of two leucine residues by arginine residues, thus giving an overall charge of +2 for R5R10. The distance measured suggests a close proximity between the peptide helical backbone and the phosphate groups, thus indicating that the amphiphilic peptide is located in the interfacial


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region of the membrane as seen for other antimicrobial peptides such as magainin 2 and PGLa.21, 44 For the R4R11 analog, it was impossible to determine a distance by fitting the experimental data points. As seen on the graph (Figure 3 B), the maximum value for the 15

N{31P} dephasing is lower than 0.05 and dephasing values close to 0 were obtained at

longer dipolar evolution times. Consequently, the absence of coherent REDOR dephasing for the peptide forming intermolecular β-sheet structures in the presence of DMPC (cf. below) strongly suggests that the nuclei are > 8 Å apart and the peptide label is not in close proximity with the phosphate moieties.45 Likewise, a similar dephasing behavior has been observed for R4R11 peptide labeled at position 3 in the primary sequence (15NLeu3-R4R11, Figure S1).

ATR-FTIR studies Acyl chain orientation Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) is a well-established technique for studying biological samples especially in the context of peptides or proteins interacting with lipid bilayers.46, 47 Moreover, polarized ATR-FTIR can be used for determining the membrane topology of membrane-active proteins and peptides.48 The location of R5R10 and R4R11 peptides in the membrane has been determined in interaction with oriented films of DMPC and DMPG multilayers formed on germanium crystal at ambient temperature. Before investigating the membrane topology, the oriented phospholipid films have been characterized in the presence and absence of peptides. The orientation of the lipid acyl chains relative to the crystal normal


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Biochemistry

(angle θ) has been determined using the dichroic ratio (R) values obtained from the CH2 symmetric stretching vibrations (2850 cm-1), assuming an angle of 90° between the transition moment and the principal axis of the phospholipids, and an infinitely narrow distribution of orientations.31,

49

The calculated order parameter values for

pure DMPC and DMPG are 0.762 and 0.663, corresponding to tilt angles θ of 23.5° and 28.3°, respectively (Table 1). These values are in good agreement with other values reported previously.33, 50-53 The R5R10 and R4R11 peptides do not significantly alter the order and/or the orientation of DMPC acyl chains as seen by values of 0.76 (θ=24°) and 0.73 (θ=25°) for the DMPC/R5R10 and DMPC/R4R11 systems, respectively (Table 1). However, R5R10 and R4R11 peptides, although to a lesser extent, trigger a change in the order and/or in the orientation of the DMPG acyl chains. Indeed, the calculated order parameter values for DMPG acyl chains are 0.78 and 0.735 in the presence of R5R10 and R4R11 peptides, respectively, corresponding to tilt angles of 22.5 and 24.8° (Table 1).

Peptide orientation The conformation of analogs of the 14-mer peptide bearing two lysines has been previously determined by infrared spectroscopy in the transmission mode and is dependent on the position(s) of the cationic residues relative to the CE bearing residues in the helical wheel representation.18 The conformation of arginine-containing analogs has also been investigated in solution and membrane environments using infrared spectroscopy and circular dichroism (unpublished work). Results of these investigations demonstrate that the conformation of arginine-containing peptides follows the same trend


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as their bis-lysine counterparts. Nevertheless, the secondary structure of R5R10 and R4R11 peptides has been assessed by ATR spectroscopy to investigate the potential alteration of the peptide conformation that could result from their interactions with the germanium crystal. The amide I’ band is characteristic of the carbonyl stretching vibration of the peptidic bond and is sensitive to the hydrogen bonding network, which is unique for each secondary structure.54 For the R5R10 peptide in the presence or absence of phospholipids (Figure 4A), a band centered at approximately 1655 cm-1 is observed, characteristic of peptides adopting an α-helical conformation. For the R4R11 peptide, the spectral pattern is different and two characteristic bands centered at approximately 1625 and 1685 cm-1 are observed in the presence of both DMPC and DMPG multilayers, as well as in the absence of lipids (Figure 4B). These bands are assigned to antiparallel βsheet structures and most likely result from the self-assembly of linear β-strand peptides leading to the formation of intermolecular β-sheet structures. Even though R5R10 and R4R11 peptides adopt predominantly their native secondary structure when interacting with the germanium crystal, the distribution of peptide conformations is greater than for the peptide in solution (results not shown), suggesting that the germanium crystal slightly alters the peptide secondary structure. To determine the membrane topology of the α-helical peptide R5R10, linear dichroism of the amide I’ band has been used to calculate the angle θ that defines the relative orientation between the principal axis of the helix and the normal of the germanium crystal. As seen in Table 1, the dichroic ratio values are near 2 when the peptide is in interaction with phospholipid multilayers whereas the dichroic ratio value is lower when the peptide is only interacting with the germanium crystal. More specifically, order


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Biochemistry

parameter values of 0.07 and 0.03 have been obtained with DMPC and DMPG multilayers, respectively, corresponding to tilt angles of 52.1 and 53.6° (Table 1). Generally, a dichroic ratio near 2 is obtained for randomly oriented peptides, thus suggesting that R5R10 peptide does not adopt a specific orientation in interaction with lipids and/or form aggregates. When the R5R10 peptide is directly deposited on the germanium crystal, the dichroic ratio is 1.6, which corresponds to an order parameter value of -0.3 (θ = 69°). Thereby, the peptide adopts a more specific orientation in interaction with the germanium crystal as the dichroic ratio value diverges from 2. For the β-aggregated R4R11 peptide, the determination of its orientation is more complicated since a β-sheet must be considered as having a biaxial symmetry. Consequently, it requires two dichroic ratio values (amide I and II bands) to define the orientation as previously described by Marsh.55 However, in the case of R4R11 peptide, the determination of a precise orientation angle is not relevant since this peptide selfassembles to form intermolecular β-sheet amorphous aggregates, which have been observed by transmission electron microscopy (TEM) (unpublished work). Nevertheless, dichroic ratio values of 1.50 with DMPC and 1.63 with DMPG have been calculated for the R4R11 peptide from the amide I’ band. On the other hand, amide I’ band of R4R11 peptide in interaction with the germanium crystal gives rise to a dichroic ratio value of 1.19, which is significantly lower than the ones obtained in lipid multilayers.

Dye-release assay Fluorescence measurements based on the dequenching of the calcein fluorophore have been widely used to study the membrane permeabilization by cationic antimicrobial


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peptides.56,

57

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We have determined the membrane leakage activity of both R5R10 and

R4R11 peptides in interaction with POPC or POPG unilamellar vesicles, which mimic eukaryotic and prokaryotic cell membranes, respectively. As seen in Figure 5A, the R5R10 peptide is highly active in the presence of POPC or POPG since the percentage of leakage is approximately 90%. High activity of the R5R10 peptide in the presence of POPG is expected because of the attractive electrostatic interactions between the cationic peptides and the anionic phospholipids insuring a high peptide concentration in the membrane. On the other hand, its high activity in interaction with the zwitterionic lipid POPC could be due to the amphiphilic nature of its α-helical conformation that confers a hydrophobic moment.58 However, the R4R11 peptide is moderately active in the presence of POPG vesicles, even though attractive electrostatic interactions are present (Figure 5B). Its lower activity in comparison to the R5R10 peptide could be explained by the fact that the R4R11 peptide is not amphiphilic in its most favorable conformation. The weak activity of the R4R11 peptide in the presence of POPC vesicles is not surprising taking into account the absence of electrostatic interactions and its non-amphiphilic conformation. Overall, these results demonstrate the importance of both electrostatic interactions and amphiphilic character as molecular determinants for the peptide-induced leakage in POPC and POPG vesicles. There are many examples in the literature emphasizing the ability of natural antimicrobial peptides to impose curvature strain on the membrane bilayers as an important part of the mechanism of action.13, 59 For example, it has been shown that the neutral 14-mer peptide activity is increased in the presence of the inverse-cone shaped lysophosphocholine (LPC) and decreased in the presence of POPE.15 The amphipathic α-helical R5R10


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Biochemistry

peptide is a class A peptide since it has a large hydrophilic section containing the bulky phenylalanine residues modified with CE and a narrower hydrophobic section.60 Consequently, insertion of the R5R10 peptide in membranes could potentially induce a positive curvature strain to the membrane by increasing the lateral pressure in the interfacial region. To investigate the effects of membrane curvature on pore formation of both R5R10 and R4R11 peptides, a certain amount of phosphatidylethanolamine (POPE), a lipid having a negative intrinsic curvature, was added to the system. Results presented in Figure 5A indicate that the addition of 30% of POPE in POPC membranes triggers a significant decrease of the calcein leakage at all phospholipid-to-peptide molar ratios investigated for the α-helical R5R10 peptide. Despite the non-amphipathic conformation of the R4R11 peptide, the incorporation of 30% of POPE triggers a decreases by 16 and 25% of calcein release at phospholipid-to-peptide molar ratios of 60/1 and 120/1, respectively (Figure 5B). These results indicate the propensity of both R5R10 and R4R11 peptides to induce a positive curvature in POPC model membranes. The same experiments have been performed in the presence of POPG model membranes even though substituting a fraction of POPG by POPE alters the surface charge. For the R5R10 peptide, a substitution of 30% POPG by POPE leads to minor changes of the membrane activity at all phospholipid-to-peptide molar ratios tested (Figure 5A). Experiments have also been performed on a 3/7 lipid mixture of POPG/POPE, which is often used as mimic of the membrane composition of Escherichia coli.61 Results indicate a significant decrease of the leakage activity for both R5R10 and R4R11 peptides at each phospholipid/peptide molar ratio tested, which indicates that the peptides induce a positive curvature to the membrane at a higher proportion of POPE relative to POPG.


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Therefore, the peptide conformation does not seem to be an essential prerequisite for the induction of a positive curvature strain since R5R10 and R4R11 peptides behave similarly towards systems that contain PE phospholipids.


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Biochemistry

DISCUSSION The goal of the present work was to shed light on the mode of action of non-selective and selective cationic analogs bearing 21-C-7 crown ether phenylalanine residues. The substitution pattern of the cationic residues in the primary sequence dictates the peptide conformation, thus allowing to do a comparative study between very similar peptides in composition, but one adopting an α-helical conformation (R5R10) and the other a β-sheet conformation (R4R11). The peptide conformation has been confirmed by ATR-FTIR spectroscopy. It was possible to rule out a detergent-like mechanism as the R5R10 and R4R11 peptides do not compromise the membrane integrity and/or induce formation of isotropic phases. Indeed, we have observed no significant contribution at the 31P isotropic chemical shift in solid-state NMR and the linear dichroism of the CH2 symmetric stretching band of the lipids measured by ATR indicates well oriented lipids. However, the R5R10 and R4R11 peptides interacting with the anionic phospholipid DMPG trigger an ordering and/or reorientation of DMPG acyl chains. This may be due to the attractive electrostatic interactions between the anionic phospholipids and the cationic peptides. In fact, we have previously shown by infrared spectroscopy that the lysine-containing peptides increased the phase transition temperature of DMPG by approximately 9°C, thus stabilizing the gel phase.18 Therefore, the phospholipid acyl chains have an increased conformational order and this is related to higher order parameters and lower θ angle values, as observed for both DMPG/R5R10 and DMPG/R411 systems (Table 1).33, 53 15

N solid-state NMR experiments have been performed on POPC oriented samples in

order to assess the membrane topology of the α-helical peptide. Analysis of the spectra suggests that the R5R10 peptide is mainly located on the membrane surface. However,


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the spectral pattern is quite broad and this might be due to peptide aggregation and/or peptides exhibiting different membrane topologies. In fact, the

31

P spectral pattern

strongly suggested that R5R10 induces membrane defects with high local curvature. For example, a toroidal pore geometry in POPC bilayers could lead to a broadening of the resonance in the

15

N spectra and the appearance of a powder spectral pattern.62 The

results presented in Figure 2C suggest that there is a small residual powder pattern on the 15

N spectrum. However, it is difficult to distinguish since the signal-to-noise ratio is

relatively low. The membrane topology of the R5R10 peptide has also been investigated by polarized ATR-FTIR spectroscopy and the conclusions were consistent with the ones drawn from

15

N solid-state NMR. Dichroic ratio values of approximately 2 have been

measured for the α-helical peptide reconstituted in DMPC and DMPG multilayers, thus indicating that the peptide does not adopt a well-defined orientation. Measurements of dichroic ratio values of approximately 2 could also be indicative of peptide aggregation. Similar conclusions were drawn by Fa-Xiang Ding et al. for the study of short fragments of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae in DMPC multilayers.63 It is important to note that we cannot distinguish whether the θ angle value calculated is an average of peptide populations having different membrane topology or that the R5R10 peptide is precisely oriented near the magic angle (54.7°). However, by considering the hydrophobic mismatch between the length of the hydrophobic portion of the peptide and the hydrophobic bilayer thickness, it is unlikely that the R5R10 peptide is embedded into the membrane bilayer.64,

65

Since lower dichroic ratios have been measured for the

intermolecular β-sheet R4R11 peptide in interaction with both DMPC and DMPG multilayers, it strongly suggests that the peptide aggregates are most likely tilted on the


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Biochemistry

membrane surface. Indeed, Castano et al. have performed simulations for the fusion peptide FP23 that relate the dichroic ratio value of the amide I band (1625 cm-1) with an angle that defines the rotation around the chain axis of a β-sheet initially oriented parallel relative to the lipid bilayer plane.66 By analyzing the simulations performed with experimental conditions closer to our experiments (i.e. with a bilayer deposited on a germanium ATR crystal), an angle between 35 and 40° can be approximated for the R4R11 peptide in interaction with DMPC and DMPG multilayers, respectively. It should be noted that we cannot rule out a scenario in which the dichroic ratio value measured for the R4R11 peptide is an average between a smaller population of peptides in close contact with the germanium crystal and a larger population of peptides interacting with the phospholipids, thus justifying the dichroic ratio values diverging away from 2. When the R5R10 and R4R11 peptides are deposited on the germanium crystal, the dichroic ratio values are significantly lower than the ones measured in the presence of phospholipids. In the case of the R5R10 peptide, this suggests that the peptide adopts a more specific orientation whereas for the R4R11 peptide, this suggests that the peptide aggregates are almost lying flat on the surface of the germanium crystal. Indeed, for peptides or proteins having an antiparallel β-sheet structure and lying completely flat on the membrane surface, a dichroic ratio value of 1 is expected.66 Therefore, we hypothesize that the germanium crystal may act as a supporting material making interactions with peptides and thus, altering the conformational profile. REDOR experiments have been conducted to measure heteronuclear distances between the

15

N labeled peptides and the

31

P nucleus of DMPC lipids. A

15

N-31P distance of

7.6 ± 0.7 Å has been measured for the α-helical R5R10 peptide, whereas the dephasing


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behavior of the β-sheet R4R11 peptide cannot be fitted with the universal REDOR dephasing curve. A close proximity between the R5R10 peptide and the phosphate moiety is not surprising because of its amphipathic nature. Furthermore, it could explain the higher permeabilizing activity of the R5R10 peptide interacting with zwitterionic vesicles in comparison with the R4R11 peptide. Indeed, the farther location of the R4R11 peptide from the interfacial region in POPC vesicles could limit its ability to induce formation of defects. Furthermore, the short

15

N-31P distance measured for the R5R10

peptide reconstituted in DMPC MLVs enables to eliminate the barrel-stave mechanism. In fact, if R5R10 peptides were completely embedded in a characteristic transmembrane fashion, the

15

N labeled leucine residue at position 7 would have been located near the

center of the bilayer and, consequently, the15N-31P distance would have been significantly longer. On the other hand, a farther location of the R4R11 peptide from the hydrophobic core of the membrane is not surprising since it is not amphiphilic under its more preferred conformation, as opposed to the R5R10 peptide. In fact, Hong et al. have studied the location of the β-sheet cationic antimicrobial peptides protegrin-1 in POPC/cholesterol 1.2/1 model membranes using the proton spin diffusion technique. They have measured a peptide-lipid distance of 20 ± 2 Å and a peptide-water distance of 2 ± 2 Å which led to the conclusion that protegrin-1 lies on the surface of bilayers mimicking the mammalian membrane.67 However, a distance of approximately 20 Å would have been impossible to measure for the heteronuclear pair 15N-31P using the REDOR technique. Fluorescence dye release measurements have been performed to evaluate the membranepermeabilizing activity and the ability of the peptides to induce a positive curvature to the membrane. Taken together, the results show that the membrane activity is dependent on


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Biochemistry

both the electrostatic interactions and the amphiphilic character. The highly amphipathic α-helical R5R10 peptide induces a high release of calcein in both types of model membranes, whereas the non-amphipathic β-sheet R4R11 peptide is more efficient in inducing the release of calcein confined within vesicles of anionic phospholipids. There is a precedent in the literature reporting that β-sheet peptides may display a higher degree of selectivity towards bacterial cells and bacterial mimicking membranes than their αhelical peptide counterparts.68 It is noteworthy that the activity of the R5R10 peptide is higher in interaction with POPC vesicles than POPG vesicles at phospholipid-to-peptide molar ratios of 120/1 and 240/1. This is indicative of the stronger ability of the R5R10 peptide to permeabilize POPC membranes than POPG membranes even though the higher affinity of the R5R10 peptide for POPG is expected because of the attractive electrostatic interactions. Studies conducted by Wieprecht et al. on analogs of the natural AMP maganin 2 and Vogt and al. on the histidine-containing peptide LAH4 support this conclusion.69, 70 Furthermore, addition of cone-shaped phospholipids, such as POPE, in lipid vesicles of POPC led to a significant reduction of the membrane activity of both the R5R10 and R4R11 peptides, suggesting that they induce a positive curvature strain to the membrane bilayer. Induction of a positive curvature strain to the membrane triggers the fusion of the inner and outer leaflets, which in turn constrains the phospholipids to tilt. Therefore, these tilted lipids located in the pore lumen could be responsible for the spectral powder pattern observed in the

31

P solid-state NMR spectra of oriented POPC samples in the

presence of the R5R10 peptide. In interaction with POPG vesicles, a higher proportion of POPE is required to observe the decrease of the leakage activity of both peptides. Indeed,


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for both the R5R10 and R4R11 peptides, a substitution of 30% POPG by POPE leads to minor changes in the membrane activity at each phospholipid-to-peptide molar ratios investigated (Figure 5). Considering a toroidal pore, the pore-lining phospholipids would exhibit a negative curvature in a dimension parallel to the membrane surface and a positive curvature in a dimension perpendicular to the plane of the membrane.71 Consequently, under certain conditions, addition of lipids having a negative curvature such as PE might stimulate pore formation. For the POPG/POPE 3/7 system, the membrane activity of both peptides is significantly decreased, thus indicating that the peptides induce a positive curvature strain on the membrane. The bending stress imposed on the membrane by the R5R10 peptide is higher than the one imposed by the R4R11 peptide since incorporation of PE has a greater impact on the membrane activity of the former peptide. However, we cannot exclude the possibility that the decrease of activity for the R4R11 peptide may partially be due to the decrease of the membrane charge density, as the activity of this peptide is more dependent on electrostatic interactions. To better characterize the molecular mechanisms involved in the membrane interactions of the R5R10 and R4R11 peptides, it would be of interest to perform dye-leakage assays in a lipid competitive environment rather than in a single lipid environment.72 In the case of the α-helical R5R10 peptide, we propose a mode of action that is similar to the sinking-raft mechanism involving the formation of disordered toroidal pores.73, 74 On an experimental basis, the disordered toroidal mechanism has also been proposed for pleurocidin, an amphipathic α-helical cationic antimicrobial peptide, and Tp10, an amphipathic α-helical cell-penetrating peptide derivative.75, 76 The R5R10 peptide has an amphipathic α-helical conformation and would form aggregates in solution that are in


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Biochemistry

equilibrium with monomers. The first step would involve the binding of the monomers and/or peptide aggregates to the membrane surface. After reaching a certain threshold concentration, accumulation of the peptides on the membrane surface would destabilize the membrane intrinsic curvature and induce a positive curvature strain, which would lead to the formation of transient disordered toroidal pores. Contrary to the toroidal pore mechanism in which the α-helical peptides adopt a transmembrane topology, the peptides do not adopt a well-defined orientation in the disordered toroidal pore model. Indeed, a fraction of peptides is located near the pore center, while the other peptides are lying on the membrane surface close to the poor edge. Then, depending on the pore stability, the peptides may translocate across the membrane in order to relieve the stress between the inner and outer leaflets, thus leading to the pore closure. Consequently, in the eventuality that these membrane active peptides bear some antimicrobial activities, the most likely mechanism responsible for killing bacteria would be the release of the cellular content through transient defects. However, we cannot exclude the possibility that translocation of the peptides represents only a step before killing bacteria by altering an intracellular target or by other mechanisms. Indeed, in addition to their membrane permeabilizing activity, studies have demonstrated that cationic antimicrobial peptides can exert their antibacterial activity in multiple ways.77 For the β-sheet R4R11 peptide, proposing a mechanism of action is less straightforward since the spatial arrangement of the peptide aggregates is not known and the peptide is not amphipathic in its active conformation. In the literature, Frantz et al. have elaborated a model for the interaction of the linear β-sheet peptide cateslytin with bacterial-like membrane domains that does not involve formation of toroidal pores. In that model, the


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peptide monomers form aggregates on the membrane surface which lead to the formation of rigid (thicker) domains enriched with anionic phospholipids and softer (thinner) domains enriched with zwitterionic phospholipids.78 Consequently, the permeabilizing activity of cateslytin is predicted to occur through the phase boundary defects between thick and thin membrane domains. However, this mechanism of action implies that the defects are relatively small which is unlikely for the ones induced by the R4R11 peptide. Indeed, this peptide is active in inducing the release of both small molecules such as calcein and the 3000 kDa FITC-dextrans entrapped in POPC/POPG 1/1 liposomes (Fig. S6). Dextrans are best modeled as prolate ellipsoids having a short axis of approximately 20 Å, thus suggesting that the R4R11 peptide most likely forms transient pores having an inner diameter of 20 Å or more.79 Therefore, we propose a mechanism of action that is similar to the one described for the R5R10 peptide with the difference that the R4R11 peptide is less deeply inserted into the interfacial region of the membrane since it is not amphipathic. However, it is not possible to claim undoubtedly that the R4R11 peptide induces formation of pores having a toroidal geometry in POPC model membranes. Indeed, spectral simulations were performed to analyze the

31

P spectra acquired for

lysine-containing derivatives reconstituted in macroscopically oriented DMPC samples. It is interesting to note that a greater perturbation of the lipids was observed with selective peptides compared to that observed with non-selective peptides.40 A mechanistic model based on the sinking-raft mechanism has also been proposed by the group of research of Wimley for linear peptides having a β-sheet conformation and potent membrane-destabilizing activities.80 More work is therefore necessary to decipher the molecular details of the R4R11 peptide mode of action.


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Biochemistry

CONCLUSION The mode of action of R5R10 and R4R11 peptides, cationic analogs of the 14-mer template peptide, was investigated by various experiments performed on model membranes. The results suggest that the membrane activity of both α-helical and linear βsheet analogs is via a mechanism of action similar to the sinking-raft model and involves the formation of transient disordered toroidal pores. More specifically, this implies that the peptides induce a positive curvature to the membrane and that the majority of the peptides lie along the membrane surface with a smaller fraction of peptides interacting with the phospholipids in the pore lumen. Our results also demonstrate that the depth of insertion into the interfacial region is dependent on the amphiphilic character of the peptide and that it is an important factor for the membrane activity towards mammalianmimicking membrane. Overall, these results show that the mechanism of action of the cationic derivatives is independent on the membrane conformation and that the amphipathic character is an important molecular determinant to considerer in order to design more potent and selective antimicrobial peptides.


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ACKNOWLEDGEMENTS The authors would like to thank Pierre Audet, Christopher Aisenbrey and Jean-François Rioux-Dubé for their technical assistance in the solid-state NMR and polarized ATR measurements.


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SUPPORTING INFORMATION 15

N{31P} REDOR dephasing curve for

15

N-Leu3-R4R11 peptide reconstituted into

lyophilized DMPC multilamellar vesicles (Figure S1), amide I’ spectral region of ATR spectra acquired with parallel (P) and perpendicular (S) polarized lights for the R5R10 peptide with DMPC multilayers, DMPG multilayers, and in the absence of phospholipids (Figure S2), amide I’ spectral region of ATR spectra acquired with parallel (P) and perpendicular (S) polarized lights for the R4R11 peptide with DMPC multilayers, DMPG multilayers, and in the absence of phospholipids (Figure S3), kinetic measurements of calcein leakage induced by the presence of the R5R10 peptide in POPC, POPG, POPC/POPE (7/3), POPG/POPE (7/3), and POPG/POPE (3/7) SUVs (Figure S4), kinetic measurements of calcein leakage induced by the presence of the R4R11 peptide in POPC, POPG, POPC/POPE (7/3), POPG/POPE (7/3), and POPG/POPE (3/7) SUVs (Figure S5), kinetic measurements of the 3 kDa FITC-dextran leakage induced by the R4R11 peptide in POPC/POPG 1/1 SUVs (Figure S6).


Biochemistry

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Biochemistry

Table 1: ATR studies of R5R10 and R4R11 peptides in interaction with the germanium crystal, DMPC multilayers, and DMPG multilayers at a phospholipid/peptide molar ratio of 60/1. Spectra are reported in the supplementary information (Figures S2 and S3).

CH2 symmetric stretching vibration

Amide I’ vibration

Wavenumber (cm-1)

RATR

Angle θ (deg)

Wavenumver (cm-1)

RATR

Angle θ (deg)

DMPC

2850

1.049 ±0.002

0.762 ±0.002

23.5 ±0.1

—

—

—

—

+R5R10

2850

1.08 ±0.02

0.76 ±0.02

24 ±1

1656

2.13 ±0.03

0.07 ±0.01

52.1 ±0.4

+R4R11

2850

1.05 ±0.03

0.73 ±0.03

25 ±2

1624

1.5 ±0.1

—

—

DMPG

2850

1.141 ±0.007

0.663 ±0.007

28.3 ±0.3

—

—

—

—

+R5R10

2850

1.03 ±0.01

0.78 ±0.01

22.5 ±0.7

1658

2.05 ±0

0.03 ±0

53.6 ±0

+R4R11

2850

1.073 ±0.007

0.735 ±0.008

24.9 ±0.4

1627

1.63 ±0.01

—

—

R5R10

—

—

—

—

1653

1.5 ±0.1

-0.3 ±0.1

69 ±5

R4R11

—

—

—

—

1625

1.19 ±0.02

—

—


Biochemistry

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Page 44 of 51

FIGURE CAPTIONS Figure 1: A) Structure of the 14-mer template peptide. B) Primary sequences of the arginine-containing analogs investigated in the present study. 31

Figure 2:

P solid-state NMR spectra of POPC mechanically oriented between glass

plates in the presence of R5R10 peptide at phospholipid/peptide molar ratios of A) 60/1 and B) 100/1. 15N solid-state NMR spectra of POPC mechanically oriented between glass plates in the presence of R5R10 peptide at phospholipid/peptide molar ratios of C) 60/1 and D) 100/1. All spectra were acquired at 37 °C. Figure 3: peptide

15

N{31P} REDOR dephasing curves for A) R5R10 peptide and B) R4R11

reconstituted

into

lyophilized

DMPC

multilamellar

vesicles

at

a

phospholipid/peptide molar ratio of 20/1. The spinning speed was kept constant at 5000 Hz and experiments were carried out at -10 °C. The red solid line in A) is the REDOR universal curve calculated using the Bessel function method. It represents the best-fit of the experimental values of ∆S/S0 and corresponds to a mean 15N-31P internuclear distance of 7.6 ± 0.7 Å and a dipolar coupling constant of 11.2 Hz. The dotted lines illustrate the distribution of distances. Figure 4: ATR spectra of the amide I’ spectral region of A) R5R10 peptide and B) R4R11 peptide in the absence of lipids and in the presence of DMPC and DMPG multilayers. The spectra displayed have been acquired with parallel polarized light (P) at a phospholipid/peptide molar ratio of 60/1. All measurements were done at ambient temperature. Figure 5: Percentage of calcein leakage of A) R5R10 peptide and B) R4R11 peptide in the presence of POPC, POPC/POPE (7/3), POPG, POPG/POPE (7/3), and POPG/POPE


Page 45 of 51

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Biochemistry

(3/7) SUVs at a phospholipid/peptide molar ratio of 60/1,120/1, and 240/1 in A) and 60/1 and 120/1 in B). All measurements were done at 37 °C. Kinetics measurements are reported in the supplementary information (Figures S4 and S5).


Biochemistry

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Page 46 of 51

FIGURES

Figure 1


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Biochemistry

Figure 2


Biochemistry

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Figure 3


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Biochemistry

Figure 4


Biochemistry

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Figure 5


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Biochemistry

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Amphiphilicity Is a Key Determinant in the Membrane Interactions of

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