A Study of the Interaction between a Family of Gemini Amphiphilic

Article pubs.acs.org/JPCB

A Study of the Interaction between a Family of Gemini Amphiphilic Pseudopeptides and Model Monomolecular Film Membranes Formed with a Cardiolipin Marcelina Gorczyca,† Beata Korchowiec,*,‡ Jacek Korchowiec,† Sonia Trojan,† Jenifer Rubio-Magnieto,§ Santiago V. Luis,§ and Ewa Rogalska*,∥ †

Department of Theoretical Chemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, ul. R. Ingardena 3, 30-060 Krakow, Poland § Departamento de Química Inorgánica y Orgánica, Universitat Jaume I, Avda. Sos Baynat, s/n, 12071 Castellón, Spain ∥ Structure et Réactivité des Systèmes Moléculaires Complexes, BP 239, CNRS/Université de Lorraine, 54506 Vandoeuvre-lès-Nancy cedex, France ‡

S Supporting Information *

ABSTRACT: The interaction between five gemini amphiphilic pseudopeptides (GAPs) differing by the length of the central spacer and a model membrane lipid, 1,3-bis[1,2-dimyristoyl-sn-glycero-3phospho]-sn-glycerol (cardiolipin) were studied with the aim to evaluate their possible antimicrobial properties. To this end, monomolecular films were formed at the air/water interface with pure cardiolipin or cardiolipin/GAPs mixtures; film properties were determined using surface pressure and surface potential measurements, as well as polarization-modulation infrared reflection− absorption spectroscopy. Moreover, to better understand the GAPs-phospholipid interaction at the molecular level, molecular dynamics simulations were performed. The results obtained indicate that the length of the central spacer has an effect on the interaction of GAPs with cardiolipin and on the properties of the lipid film. The GAPs with the longer linkers can be expected to be useful for biological membrane modification and for possible antimicrobial applications.

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INTRODUCTION

the rather low amount of cardiolipin present in bacterial membranes, it plays an important biological role. Indeed, CL is involved in protein translocation 6 and stabilization of membrane proteins.7 Furthermore, CL is necessary for the prokaryotic energy-transducing system.8 It can be observed that the involvement of mitochondria in Barth syndrome seems plausible because tafazzin, the mutated enzyme, is localized in mitochondria and so is cardiolipin, its primary metabolic target; few atomistic or CG simulations showed specific binding sites of CL at cytochromes.9 Gemini amphiphilic pseudopeptides (GAPs) are molecules having in its structure two polar (amino acid-derived units) and two apolar moieties, which are typically lipophilic chains, and they are linked together with a spacer.10−12 Because of the presence of two amino groups, the polar head of GAPs can be positively charged depending on the pH of the environment.13

Biological membranes are complex systems composed of a large variety of lipids and proteins. The lipids are organized into a bilayer, which not only support proteins, but also interact, modify and regulate their function.1 The type of lipids present in these membranes differs significantly depending on the kind of cell. The Escherichia coli membrane consists of only three main lipids, namely phosphatidylethanolamine (PE), a zwitterionic lipid which represents the 70% of the total molar phospholipid content, phosphatidylglycerol (PG) (20−25%) and cardiolipin (CL) (5−10%).2 Cardiolipin, unlike most of the other phospholipids, carries four acyl chains of varying length and saturation. In its dimeric-like structure, the two phosphatidyl moieties are linked by a central glycerol group.3 Moreover, cardiolipin bears two negative charges due to the presence of two phosphate groups. Consequently, the structure of CL is characterized by a large hydrophobic region and a relatively small, negatively charged head.4 Cardiolipin is found in Gram-negative and Gram-positive bacteria, as well as in mitochondrial membranes.5 Despite of © 2015 American Chemical Society

Received: March 17, 2015 Revised: May 8, 2015 Published: May 9, 2015 6668

DOI: 10.1021/acs.jpcb.5b02575 J. Phys. Chem. B 2015, 119, 6668−6679

Article

The Journal of Physical Chemistry B

film technique, as well as molecular dynamics simulations. The miscibility of cardiolipin and GAPs was studied using surface pressure and surface potential measurements. The polarizationmodulation infrared reflection−absorption spectroscopy (PMIRRAS) was used to gain more information on the intra- and intermolecular interactions in the mixed GAP/phospholipid films. The experimental observations were compared to the theoretical results obtained from computational calculations. Molecular dynamics yielded information concerning the degree of hydration of the polar heads present in the film forming molecules. Moreover, the ordering of pure component and mixed monolayers was analyzed in terms of order parameter, rotational order parameter and tilt angle of hydrocarbon chains.

The high surface activity and the biocompatibility of these GAPs makes them particularly interesting for different applications, such as contaminated soil or water remediation, enhanced petroleum recovery, health and personal care products, crop protection, preparation of high-porosity materials, drug entrapment and release, etc.14−19 Another potential application for the gemini amphiphilic compounds is to improve the efficiency of gene delivery, as they have many features of good nonviral carriers.20 Furthermore, gemini surfactants have been proposed as active antibacterial agents.21 The C2-symmetry GAPs considered in this work (Figure 1) have interesting self-assembling properties. Indeed,

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EXPERIMENTAL SECTION Materials and Reagents. 1,3-Bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt) (cardiolipin-CL;) was purchased from Avanti Polar Lipids (99% pure; CAS No. 63988-21-6). The gemini amphiphilic pseudopeptides (GAPs) used here were as follows GAP II: (2S)-2-({[4-(decyloxy)phenyl]methyl}amino)-N{2[(2S)-2-({[4-(decyloxy) phenyl]methyl}amino)3methylbutanamido]ethyl}-3-methylbutanamide. GAP III: (2S)-2-({[4-(decyloxy)phenyl]methyl}amino)-N{3[(2S)-2-({[4-(decyloxy) phenyl]methyl}amino)3methylbutanamido]propyl}-3-methylbutanamide. GAP IV: (2S)-2-({[4-(decyloxy)phenyl]methyl}amino)-N{4[(2S)-2-({[4-(decyloxy) phenyl]methyl}amino)3methylbutanamido]butyl}-3-methylbutanamide. GAP V: (2S)-2-({[4-(decyloxy)phenyl]methyl}amino)-N{5[(2S)-2-({[4-(decyloxy) phenyl]methyl}amino)3methylbutanamido]pentyl}-3-methylbutanamide. GAP VI: (2S)-2-({[4-(decyloxy)phenyl]methyl}amino)-N{6[(2S)-2-({[4-(decyloxy) phenyl]methyl}amino)3methylbutanamido]hexyl}-3-methylbutanamide. The chemical structures of the compounds employed in this study are shown in Figure 1. These molecules were synthesized and purified as previously described.30,31 All GAPs are at least 99.8% pure. It should be noted that at pH 5.6 the average charge in GAPs is close to 2+ due to the coexistence of predominant diprotonated and minor monoprotonated species, as indicated by distribution diagrams.13 Spectrophotometric grade chloroform and methanol (both from Sigma-Aldrich) were used for preparing phospholipid and GAPs solutions. Compression Isotherms. The surface pressure and electrical surface potential measurements were carried out with a KSV 2000 Langmuir balance (KSV Instruments, Helsinki). A Teflon trough (7.5 cm × 3.6 cm × 0.5 cm) with two hydrophilic Delrin barriers (symmetric compression) was used in compression isotherm experiments. The surface pressure was measured by the Wilhelmy method using platinum plate. Surface potential was monitored by means of KSV Spot 1 with a vibrating electrode and a stainless steel as a counter electrode immersed below the water surface. Before each use, the trough and the barriers were cleaned using wipes soaked in chloroform, gently brushed with ethanol and then rinsed with Milli-Q water. All solvents used for cleaning were of analytical grade. Aqueous subphases for monolayer experiments were prepared with Milli-Q water, which had a surface tension of 72.8 mN m−1 at 20 °C, pH 5.6. The subphase temperature (20 °C) was maintained by thermostat Lauda RE 104. The apparatus was closed in a Plexiglas box. In order to eliminate the influence of contaminant, all impurities were removed from

Figure 1. Chemical structures of the GAPs studied (A) with n = 2, 3, 4, 5, 6, abbreviated as GAP II, GAP III, GAP IV, GAP V, and GAP VI, respectively, and CL (B).

these tailor-made molecules with adjusted properties may be responsive to environmental stimuli like pH.22−24 Recently, we reported on the behavior of a family of four different GAPs in a membrane environment by using a mixed monolayer formed with chosen phospholipids.25 A cardiolipin is particularly interesting as a model lipid for studying the interaction with GAPs, because it forms stable, condensed films due to the presence of four hydrocarbon chains; moreover, the negatively charged polar head of this lipid may be sensitive to the interaction with the positively charged polar head present in GAPs. Langmuir films are extensively used as model systems to mimic natural membranes and to provide information on lipidpeptide or lipid−protein interactions.26−29 In this model, parameters such as temperature and surface pressure, as well as lipid and subphase composition, can be easily adjusted to mimic biological conditions. Our previous study using Langmuir films showed that properties of GAPs/phospholipids mixed films were controlled by an intrinsic conformational flexibility of a four-carbon linker present in the GAPs polar groups.25 Here, to get a deeper understanding of the behavior of GAPs, the effect of the length of this central linker is studied. To this end, five different GAPs bearing an ethyl, propyl, butyl, pentyl or hexyl linker were used to form mixed films with 1,3-bis[1,2dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (cardiolipin, CL); properties of these films were studied using the Langmuir 6669


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frequency of 74 kHz. This allows simultaneously measurement of spectra for the two polarizations, the difference providing surface specific information and the sum providing the reference spectrum. As the spectra are measured simultaneously, the effect of water vapor is largely removed. The PMIRRAS spectra of the film-covered surface, S(f), as well as that of the pure water, S(w), were measured and the normalized difference ΔS/S = [S(f) − S(w)]/S(w) is reported. 6000 interferogram scans (10 scans per second) have been acquired for each spectrum. In the mid-IR region, the wavenumber at which the half-wave retardation takes place can be freely selected. Here, the maximum of PEM efficiency was set to either 1500 or 2900 cm−1 for analyzing the different regions of the spectra. The spectral range of the device is 800−4000 cm−1 and the resolution is 8 cm−1. Computational Details. The simulation setup included water slab bounded by two vacuum slabs. Two symmetric monolayers, each composed of 100 molecules, were placed at the vacuum/water interfaces. Pure CL, GAP II and GAP VI monolayers, as well as mixed GAP II/CL and GAP VI/CL monolayers of 0.1 and 0.5 molar fraction of the given GAPs, were investigated. The GAP and CL molecules were randomly distributed in a two-dimensional surface layer. While the CL headgroup is negatively charged, the value of the charge is not unequivocal. Indeed, both single and double charged CLs were proposed.4,37−41 The 1− charge observed at physiological pH is due to the pKa values of the two phosphate groups in the CL polar head (2.8 and 7.5−9.5). However, depending on the local pH and CL concentration, the deprotonation of both phosphate groups cannot be excluded. In line with simulations of CL bilayers published in the literature,4,37 a doubly deprotonated CL headgroup was here studied. The CL headgroup negative charge (2−)42 was neutralized by sodium ions. Protonated amino groups of GAPs were used in the simulations because pseudopeptides carry two positive charges under the experimental conditions.13 Chloride ions were added to neutralize the positive charge (2+) in GAPs. The water slabs comprised from 30000 to 100000 water molecules, depending on the system. The NAMD243 package was used for simulations performed with the CHARMM27 force field.44 The water molecules were described by the TIP3P model.45 The simulations were performed under a constant number of particles (N), constant temperature (T), constant normal pressure (pN), and constant surface tension (γ): NpNγT ensemble.46 Three dimensional boundary conditions were used. Depending on the system, the length of the simulation box normal to the monolayer (z axis) was changed from 970 to 3200 Å. A particle−mesh Ewald algorithm was used to calculate the electrostatic energies and forces with 1 Å grid spacing for three dimensions.47 The van der Waals interactions were cutoff at 12 Å. A smoothing function with a 10 Å switching distance was applied. The initial configuration off all systems was obtained after 20 ps NVT simulation. The NpNγT simulations with 1 fs time steps were carried out for 50 ns. After this a final production run of 3 ns was performed for each model. The normal pressure, temperature, and surface tension were set to 1 atm, 20 °C, and 44.8 mN m−1, respectively. Equilibration was checked by means of convergence of pressure normal to monolayer and temperature, as well as the average surface per molecule.

the subphase surface by sweeping and suction. When the surface pressure fluctuation was found to be lower than 0.2 mN m−1 during a compression stage, monolayers were spread from calibrated solutions (concentrations around 0.5 mg mL−1) using a microsyringe (Hamilton Co., USA). The CL stock solution was prepared in a chloroform/methanol mixture (4:1 v/v), GAPs were dissolved in chloroform. Stock solutions of all the investigated compounds were used to prepare 0.1, 0.3, 0.5, 0.7, and 0.9 GAPs/CL molar fraction mixtures, respectively. The stability of the surface potential signal was checked before each experiment, after cleaning the water subphase. When the ΔV signal had reached the constant value, it was zeroed and the film was spread on the subphase. Each film was allowed to equilibrate and the solvent to evaporate for 15 min and then it was compressed at the rate of 5.0 mm min−1 barrier −1. Each compression isotherm was repeated at least three times. A PC computer and KSV software were used to control the experiments. The standard deviation was ±0.5 Å2 with mean molecular area, ± 0.2 mN m−1 with surface pressure and ±0.01 V with surface potential measurements. The collapse surface pressure (Πcoll), area per molecule at collapse pressure (Acoll) and collapse surface potential (ΔVcoll) values of the monolayers, as well as the compressibility modulus (Cs−1)32 defined as

CS−1 = −A(∂Π/∂A)T

(1)

were determined directly from the surface pressure−area (Π− A) and surface potential-area (ΔV−A) isotherms. The excess free enthalpy of mixing, ΔGmix, was also determined from Π−A isotherms using the following formula: ΔGmix =

∫0

Π

[A12 − (x1A1 + x 2A 2 )] dΠ

(2)

where A12 is the mean molecular area in the mixed monolayer at a given surface pressure, A1 and A2 are the mean molecular areas of the pure components 1 and 2 at the same surface pressure, and x1 and x2 are the mole fractions of the two lipid components in the mixed film.33,34 We want to mention that thermodynamic terms used here were defined in our previous papers.35,36 Namely, in accordance with the definition of Gibbs energy of mixing, ΔGmix = G − (x1G1 + x2G2), eq 2 gives ΔGmix instead of ΔGexc given by Goodrich at al.33 and Bacon et al.34 Polarization-Modulation Infrared Reflection−Absorption Spectroscopy (PM-IRRAS). The PM-IRRAS spectra of pure phospholipid, pure GAPs and mixed GAPs/phospholipid monolayers of 0.1 and 0.5 molar fraction of the given GAPs spread on pure water subphase were registered at 20 °C. The Teflon trough dimensions were 36.5 cm × 7.5 cm × 0.5 cm; other experimental conditions were as described in the preceding paragraph. The PM-IRRAS measurements were performed using a KSV PMI 550 instrument (KSV Instruments Ltd., Helsinki, Finland). The PMI 550 instrument contains a compact Fourier Transform IR-spectrometer equipped with a polarization-modulation (PM) unit on one arm of a goniometer, and a MCT detector on the other arm. The incident angle of the light beam can be freely chosen between 40 and 90°; here, the incident angle was 79°. The spectrometer and the PM unit operate at different frequencies, allowing separation of the two signals at the detector. The PM unit consists of a photoelastic modulator (PEM), which is an IRtransparent ZnSe piezoelectric lens. The incoming light is continuously modulated between s- and p-polarization at a 6670


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RESULTS AND DISCUSSION

Pure GAPs Monolayers. The compression isotherms of pure GAPs monolayers on water subphase are presented in Figure 2.

Figure 2. Compression isotherms of pure GAPs. Solid lines: Π−A isotherms. Dashed lines: ΔV−A isotherms. Subphase: pure water. Temperature: 20 °C. GAP II (black), GAP III (red), GAP IV (blue), GAP V (magenta), and GAP VI (cyan).

The GAPs studied form stable monolayers at the air−water interface. It can be observed that the length of the central spacer affects the profiles of the isotherms. The molecular area at the collapse observed in the Π-A isotherms slightly increases with increasing the length of the spacer (from 63.0 Å2 in the case of GAP II to 69.6 Å2 for GAP VI), while the values of the collapse pressure are similar (31.1−30.3 mN m−1). The maximum values of Cs−1 are 90.1, 87.8, 79.2, 69.3, and 65.5 mN m−1 with GAP II, GAP III, GAP IV, GAP V, and GAP VI, respectively. These values indicate that the character of the films formed with all GAPs is situated between the liquid expanded (LE) and the liquid condensed phase (LC).32 This is in accordance with our previous study, in which GAPs containing different amino acids and a butylenic spacer were characterized.25 It should be also pointed out that the monolayers become more compressible with an increase of the length of the central linker as indicated by the decreasing CS−1 values. Accordingly, the films formed with GAP II and GAP III are more rigid compared to those formed with GAP V and GAP VI. The pseudopeptide with a butylenic spacer (GAP IV) can be considered as an intermediary between the two precedent groups. The ΔV values at the collapse for all films are close and equal to 0.64, 0.63, 0.64, 0.65, and 0.67 V for GAP II, GAP III, GAP IV, GAP V, and GAP VI, respectively. These values show that the orientation of the aliphatic chains in all five derivatives is comparable at the maximal compression of the films. Interestingly, the profiles of the ΔV−A isotherms show that gas−liquid expanded phase transition occurs at higher molecular areas (around 225 Å2) for GAP IV, GAP V, and GAP VI compared to GAP II and GAP III (around 200 Å2). This result indicates that molecules belonging to the two subfamilies n = 2, 3 or n = 4, 5, 6, undergo different rearrangements upon compression. Obviously, above the limiting length n = 4 of the linker molecules acquire a higher conformational mobility. GAPs/CL Binary Mixtures. The Π−A and ΔV−A isotherms of GAP/CL monolayers spread on pure water at 20 °C are presented in Figure 3. Mixtures with 0.1, 0.3, 0.5, 0.7,

Figure 3. Π−A (solid lines) and ΔV−A (dashed lines) isotherms of binary mixtures of GAP II/CL (A) and GAP VI/CL (B) spread on water at 20 °C: blue, xGAP = 0; green, xGAP = 0.1; purple, xGAP = 0.3; red, xGAP = 0.5; cyan, xGAP = 0.7; magenta, xGAP = 0.9; black, xGAP = 1.0.

and 0.9 mol fraction of a given GAP were used. Here, only the isotherms of GAP II/CL and GAP VI/CL mixtures are presented, while the isotherms obtained with other mixtures are placed in the Supporting Information (Figure S1). The collapse parameters of the isotherms for GAP II/CL and GAP VI/CL are listed in Table 1. It can be seen that the profiles of the isotherms corresponding to mixed films depend on the composition. The surface pressure isotherm of pure CL shows a steeper slope compared to the mixtures. With an increase in the GAPs molar fraction, the isotherms become similar to those obtained with pure GAPs. Moreover, the presence of GAPs in the films results in a decrease of the molecular area at the collapse (Table 1). The Acoll values for the equimolar GAPs/CL mixtures are lower compared to those of pure GAPs. In all cases studied, it is also observed that mixed films with xGAP = 0.1, 0.3, 0.5 collapse at higher surface pressures than those formed with pure compounds. The value of Cs−1 for CL reveals that the monolayer is in a liquid condensed phase. For all the investigated binary GAPs/CL systems at xGAP = 0.1, 0.3, 0.5 the condensed state is also observed at high compression. The mixed monolayers with a higher content of GAPs (xGAP ≥ 0.7) can be considered as situated between a liquid expanded and a liquid condensed phase. This indicates that the presence of GAPs induces a more liquid-like character of the mixed films. The isotherms corresponding to xGAP = 0.5 display a plateau between the LE and LC phase, with the transition pressure depending on the length of the spacer; the latter is well seen in the plot Cs−1 vs Π (Figure S2 in Supporting Information). Indeed, with an increased length of the spacer in GAPs, a minimum corresponding to the phase transition appears in the plot at higher surface pressures. 6671


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The Journal of Physical Chemistry B Table 1. Characteristic Parameters of GAPs/CL Isotherms at 20 °C GAP II/CL

GAP VI/CL

xGAP

Acoll (Å2)

Πcoll (mN m−1)

ΔVcoll (V)

Cs−1 (mN m−1)

0.0 0.1 0.3 0.5 0.7 0.9 1.0 0.0 0.1 0.3 0.5 0.7 0.9 1.0

76.6 71.6 64.4 53.9 66.3 66.2 63.0 76.6 73.6 67.2 55.1 77.4 75.4 69.6

51.5 55.7 61.6 65.8 37.0 32.6 31.1 51.5 56.3 55.9 65.0 35.0 31.0 30.3

0.26 0.32 0.46 0.64 0.64 0.64 0.64 0.26 0.32 0.44 0.65 0.65 0.65 0.67

647.4 580.2 545.3 429.8 97.3 98.5 90.1 647.4 338.4 313.8 230.7 82.9 70.4 65.5

Figure 4. Mean molecular area (MMA) and Gibbs energy of mixing (ΔGmix) as a function of xGAP, calculated at Π = 20 (●), 24 (▲), and 28 (■) mN m−1 for mixed films of GAP II/CL (A, B) and GAP VI/CL (C, D). The straight lines represent the additive mixing.

It can be seen in Figure 3 that for the xGAP = 0.9, 0.7, or 0.5 mixtures the ΔV values at the collapse point are close to pure GAPs, while for the films with xGAP = 0.3 or 0.1 these values decrease. This effect shows the importance of the negatively charged polar head of CL in the ΔV measurements. The effect of single components on film properties can be observed in the mixed films based on the ΔV isotherms. Indeed, with xGAP = 0.3 or 0.5, a higher ordering of the film forming molecules upon compression is observed in the region 70−130 Å2, compared to pure GAP. In the case of xGAP = 0.7 or 0.9, the ordering effect can be observed in the region 145−240 Å2. Miscibility Analysis and Thermodynamic Properties. The variation of the values of the mean molecular area (MMA) and Gibbs energy of mixing (ΔGmix) as a function of xGAP for mixed GAPs/CL films are presented in Figure 4 (for GAP II and GAP VI) and in Figure S3 in the Supporting Information (for GAPs III−V). Both positive and negative deviations from linearity in the MMA values are observed. However, the sign of the deviations depends on the molar composition, on surface pressure and on the length of the central spacer of GAP. For GAP II/CL, the MMA values are lower compared to the ideal system at xGAP =

0.1, 0.3, and also at 0.5 for the higher surface pressures. It can be seen that the negative deviations disappear for longer linkers. In the case of GAP VI/CL, the positive deviations are observed in the whole range of the monolayers composition. For the GAPs III−V, the maximal positive deviations in the MMA appear in the molar fraction range of 0.5−0.7, which suggests repulsive intermolecular interactions between molecules in the mixed films. On the other hand, negative ΔGmix values were obtained at xGAP = 0.1, except GAP VI. For xGAP = 0.3 at n ≤ 5, and also for the equimolar GAP II/CL mixture at the highest surface pressures, ΔGmix remains negative. This suggests that in this range of the monolayer composition the interactions between a given GAP and cardiolipin are more attractive compared to molecules in pure films. Interestingly, the ΔGmix values become positive with the increasing xGAP or the length of the central spacer, with a maximum at xGAP = 0.7 (for GAP II and GAP III) or 0.5 (for GAP IV−VI). This indicates a stronger repulsion between molecules in the mixed films compared to those in their respective pure monolayers. In general, it can be concluded that mixed GAP/CL films formed with pseudopep6672


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experiments showing a more fluid-like character of the films formed with GAPs containing a longer spacer group. In the PM-IRRAS spectra of pure CL monolayers (Figure 6, green line) characteristic lipid vibrations can be observed.56−58 The vas(CH2) and vs(CH2) bands at 2919 and 2844 cm−1, respectively, indicate high conformational ordering of the alkyl chains. These results are in accordance with the compressibility modulus value which is characteristic for a highly packed and well-ordered monolayer. The CL v(CO) band observed at 1749 cm−1 suggests a low hydration of the carbonyl groups. Indeed, this band is usually situated at approximately 1730 cm−1 in hydrated lipids.53,59,60 The PM-IRRAS spectra of mixed GAPs/CL films with xGAP = 0.1 and 0.5 are shown in Figure 6. In the mixed systems with a lower pseudopeptide content, the vas(CH2) and vs(CH2) signals appear at wavenumbers close to that of the pure cardiolipin (Table 3). In the case of the equimolar mixtures the differences are more important and the disorganizing effect of GAP molecules is visible both by the vas(CH2) blue shift and, in particular, the v(CO) red shift compared to pure CL. The latter indicates a higher hydrogen bonding of the carbonyl groups in the mixed systems, which may be due to higher penetration of water molecules in the polar headgroup region in the disorganized mixed films. It can be noted that differentiation between GAPs with different spacer length was not evidenced with PM-IRRAS. Molecular Dynamics Simulation. To better understand the GAP-phospholipid interaction at the molecular level, molecular dynamics simulations were carried out. GAP II and GAP VI, were taken into account, to elucidate the effect of the linker length. In the first step MD simulations of monolayers formed with pure GAP II and GAP VI were performed. The snapshots of GAP II and GAP VI films at the end of the simulation are showed in Figure 7. The observed lack of ordering can be attributed to strong electrostatic repulsive interactions between the polar headgroups; the latter penetrate to a different degree into the water phase. To monitor conformations adopted by the alkyl chains, a trans-order parameter, S, was calculated from the simulation trajectories using

tides with shorter central spacers are thermodynamically more stable. Polarization-Modulation Infrared Reflection−Absorption Spectroscopy. PM-IRRAS spectra48,49 of pure compounds and selected mixtures were performed and the regions corresponding to CH2 and CO stretching vibrations have been analyzed. This powerful tool provides information about the molecular structure and orientation of the films constituents. The frequencies of vas(CH2) and vs(CH2) are sensitive to the conformation of alkyl chains. Indeed, values lower than 2920 and 2850 cm−1 indicate chain ordering (alltrans conformation), while higher values suggest chain disordering (presence of gauche conformers).50−52 The CO stretching of the phospholipid ester group (around 1730 cm−1) can provide information concerning hydration of the headgroup, and its shift to lower wavenumbers indicates significant hydrogen bonding.53−55 The PM-IRRAS spectra of pure GAPs films in the region of methylene stretching bands at Π = 28 mN m−1 are collected in Figure 5 and the characteristic wavenumbers are given in Table 2.

Figure 5. PM-IRRAS spectra in the region of methylene stretching vibrations νas(CH2) and νs(CH2) of pure GAPs monolayers recorded at Π = 28 mN m−1. Subphase: pure water. Temperature: 20 °C. GAP II (black), GAP III (red), GAP IV (blue), GAP V (magenta), and GAP VI (cyan). Dashed lines: original spectra; solid lines: fitted peaks.

S = (3 cos2 θ − 1)/2

Table 2. Asymmetric and Symmetric Stretching Vibrations of Methylene Groups in Pure GAPs Films at Π = 28 mN m−1 GAP GAP GAP GAP GAP

II III IV V VI

vas(CH2) (cm−1)

vs(CH2) (cm−1)

2927 2925 2928 2929 2932

2851 2856 2861 2858 2858

(3)

where θ refers to successive dihedral angles in the carbon skeleton. S = 1 indicates that all carbon atoms in the chain are in all-trans conformation, while S = 0 corresponds to a random orientation and total disorder of the alkyl chains.61 The transorder parameter, S, obtained for GAP II and GAP VI decyl chains is presented in Figure 8A, while the average values of S over the entire chain length are listed in Table 4. It can be seen that the trans-order parameter of GAP chains decreases as the length of the central spacer increases. This indicates that GAP II chains are more ordered compared to GAP VI. This result is consistent with the PM-IRRAS spectra showing a higher number of gauche conformers in the case of long linker GAPs. Moreover, the order parameter profiles reveal a lower orientational order for the methylene groups located close to the polar heads. A further insight into the orientation of molecules in the monolayers was provided by the tilt angle (ϑ) of the decyl chains present in GAPs relative to the normal to the water surface. To estimate the tilt angle, a vector connecting the first and the last carbon atom in both acyl chains of GAPs was used.

The vas(CH2) and vs(CH2) bands of pure GAP monolayers appear at wavenumbers higher than 2920 and 2850 cm−1, respectively, which indicates disordering of the two hydrocarbon chains and the CH2 groups present in the linker. It was also noticed that the peak positions of the methylene stretching vibrations showed a dependence on the spacer length of the gemini amphiphilic pseudopeptides. In the case of GAPs with longer spacers the blue shift is more pronounced, indicating a higher number of gauche conformers of the chains and linker. These results are consistent with the compression isotherm 6673


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Figure 6. PM-IRRAS spectra, collected at Π = 28 mN m−1, in the spectral regions of methylene (left column), and carbonyl (right column) stretching vibrations. Temperature: 20 °C. Results obtained with pure CL and mixed GAP/CL monolayers at xGAP = 0.1 (A, B) and at xGAP = 0.5 (C, D). Pure CL film (green), GAP II/CL (black), GAP III/CL (red), GAP IV/CL (blue), GAP V/CL (magenta) and GAP VI/CL (cyan). Dashed lines: original spectra. Solid lines: fitted peaks.

Table 3. Characteristic Vibrational Wavenumbers in Pure CL and in GAP/CL Mixed Films Obtained from PM-IRRAS Spectra

xGAP = 0.1

xGAP = 0.5

CL GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP

II/CL III/CL IV/CL V/CL VI/CL II/CL III/CL IV/CL V/CL VI/CL

vas(CH2) (cm−1)

vs(CH2) (cm−1)

v(CO) (cm−1)

2919 2918 2919 2918 2920 2919 2921 2922 2922 2923 2922

2844 2849 2845 2849 2846 2846 2843 2851 2852 2849 2844

1749 1744 1743 1745 1745 1745 1743 1739 1740 1740 1744

Figure 7. Snapshots (xz cut) from the simulation of GAP II (A) and GAP VI (B) films. The number of water in y-direction is limited to 10 Å width. 6674


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Figure 8. Average trans-order parameter (A) and tilt angle (B) of GAPs hydrocarbon chains: GAP II (black) and GAP VI (cyan). Figure 9. Density profiles for water and different groups of GAP II (A) and GAP VI (B). Water (black), amino groups (red), amide groups (purple), CH(CH3)2 (magenta), CH3 (yellow), CO (green), O (blue), C6H4 (cyan). Inset: zoom of the density profiles.

Table 4. Average S, ϑ, and G Values for Mixed GAPs/CL Systems Obtained from Molecular Dynamics Simulations GAP II/CL

GAP VI/CL

xGAP

S

ϑ (deg)

G

0 0.1 0.5 1.0 0.1 0.5 1.0

0.88 0.87 0.84 0.71 0.88 0.84 0.68

30.0 27.6 31.4 42.2 24.2 34.5 47.6

0.50 0.18 0.04 0.07 0.06 0.15 0.01

width of the water interface indicates that GAP films are rather unstable compared to phospholipid monolayers, with water molecules penetrating between the amino, amide, carbonyl and isopropyl groups, as well as phenyl rings and oxygen atoms of GAPs; the density profiles for the terminal methyl groups are not clearly separated from water profiles. These results are in accordance with the large distributions of the tilt angle of GAP alkyl chains. The snapshots of pure CL, GAP II/CL, and GAP VI/CL films obtained at the end of the simulation run are shown in Figure 10. For the two component monolayers, MD simulations were performed at xGAP = 0.1 or 0.5. It can be observed that CL (Figure 10 A) contributes to GAP ordering in the xGAP = 0.1 mixed films (Figure 10 B, C); it is not the case with xGAP = 0.5 (Figure 10 D, E). In a pure CL monolayer, an average value of S for lipid chains is 0.88 (Table 4) indicating that hydrocarbon chains are mostly in all-trans conformation. The variability of the average S value (Figure S4 A,B in Supporting Information) is typical for phospholipid monolayers showing higher orientational order for the central methylene groups in the hydrocarbon chains compared to the last ones.63 It can be seen from the density profiles (Figure 11) that water penetrates into the polar headgroup region, while the methylene groups at the end of the alkyl chains are dehydrated. The water profile for CL monolayer is sharp and typical for phospholipids. The conformational properties of the mixed GAP/CL monolayers were analyzed by calculating the order and rotational order parameters and the tilt angle of GAPs and CL alkyl chains in the binary systems. In the case of CL, the tilt vector was defined by the carbon atom adjacent to the carbonyl group and the last carbon atom in the each chain. The results obtained are displayed in Figures S4 and S5 in Supporting Information and average values of all parameters are given in

The tilt angle probability distribution of GAP II and GAP VI alkyl chains is shown in Figure 8B; the average values of the tilt angles are reported in Table 4. The broad tilt angle probability distribution observed in both systems indicates different orientations of the alkyl chains. However, a slightly higher tilt angle is observed for GAP VI compared to GAP II. This result is in accordance with ΔΠ measurements indicating that films formed with GAPs bearing longer linkers are more liquid-like compared to GAPs with shorter linkers. One more parameter characterizing the alkyl chains is the rotational order parameter, G, defined as G = 2cos2(φ) − 1

(4)

where φ is the azimuthal angle of the tilt vector; G = 0 or ±1 corresponds to a complete orientational disordering or perfect ordering, respectively.62 An average value of this rotational order parameter for GAP II and GAP VI is close to 0 (Table 4), indicating a high disorder in the monolayer xy plane. The interfacial behavior of GAPs was evaluated through the density profiles along z-axis (Figure 9). The overall density profiles are very similar for both compounds. Distribution of the functional groups in GAPs is broad and asymmetric, indicating their penetration into the water phase; this behavior can be attributed to electrostatic repulsion between the protonated amino groups. It can be observed as well that the water profile is less sharp compared to phospholipids.36 The 6675


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Figure 10. Snapshots (xz cut) from the simulation of the CL (A) monolayer and mixed GAP II/CL (B, D) and GAP VI/CL (C, E) monolayers at xGAP = 0.1 (B, C) and at xGAP = 0.5 (D, E). The number of water in y-direction is limited to 10 Å. Color code: CL, cyan; GAP II, orange; GAP VI, pink; water, blue.

chains compared to the pure CL film. This effect is more pronounced with GAP VI compared to GAP II. Moreover, the order parameter S of the CL acyl chains decreases with the increasing GAPs contents. Radial distribution function (RDF) is a useful quantity to describe the structure of the monolayer and hydration of the headgroup atoms.61 Two- and three-dimensional RDFs are defined as follows:64 g 2D(r ) =

N(r ) 2πrρδr

(5)

N(r ) 4πr 2ρδr

(6)

and

Figure 11. Density profiles for water and different groups of CL. Water (black), PO4− (orange), OH (purple), CO (cyan), and CH3 (yellow).

g 3D(r ) =

where N(r) is number of water oxygen atoms in a spherical shell at a distance r and thickness δr from a reference atom, and ρ is the number density calculated as the ratio of the number of atoms to the area of circle (2D) or volume of sphere (3D).

Table 4. It can be seen that a small amount of GAPs decreases the ϑ angle of the CL hydrocarbon chains. On the other hand, for xGAP = 0.5, the width of ϑ value distribution increases and a higher value of the average ϑ is observed for the hydrocarbon 6676


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Figure 12. Two-dimensional radial pair distribution function in binary mixtures GAP II/CL (A) and GAP VI/CL (B): blue, xGAP = 0; green, xGAP = 0.1; red, xGAP = 0.5; black, xGAP = 1.0.

The structure of monolayer can be easily monitored by the 2D RDF. This function was calculated between C4 atoms of alkyl chains. The C4 atoms in the two GAP lateral chains are equivalent while the two symmetry related subgroups (sn-1 and sn-2) were averaged in the case of CL (Figure 12). As observed with compression isotherms (Figure 3), the pure GAP films below 150 Å2 exist in a LE phase. This state of the monolayer is observed as well with RDF. Indeed, the low first peak and a decreasing number of peaks with distance observed in the black curves confirm the liquid-like character of the monolayer. In the other systems, the long-range ordering can be observed. The highest ordering is observed with pure CL and with mixed xGAP = 0.1 films. The RDF peaks cover the whole range of r. In the case of the xGAP = 0.5 systems, coexistence of the LE and LC phases is observed (see Figure 3). The intensity of the first few peaks is lower and the peaks disappear at lower r values compared to the pure CL or xGAP = 0.1 monolayers. Molecular dynamics simulations provide information about the degree of hydration of the polar heads. The 3D RDF can be used to monitor the number of water oxygen atoms around reference atoms X, namely carbonyl oxygen, hydroxyl oxygen, or phosphorus atoms in CL, [gX−(OH2)(r)] (Figure S6 in Supporting Information). While the data for pure CL and GAPs/CL xGAP = 0.1 are similar, in the case of xGAP = 0.5 the first hydration peaks are more intensive. The same observations hold true for all reference atoms X (panels A−C and D−F) and are in agreement with the v(CO) stretching vibration results. The perturbation observed between the first and the second hydration sphere in the 3D RDF plots indicates counterion penetration to the monolayer (Figure S6; the area between the first two peaks should be compared). This effect may play a role in hydration of the polar heads in the case of the negatively charged pure CL and GAPs/CL xGAP = 0.1 and, to a much lesser degree in the case of the xGAP = 0.5 mixture bearing an overall neutral charge. Figure S6 displays the hydration number obtained by integrating 3D RDF over the first solvation shell (Table 5). In each case, the first solvation shell was defined as water molecules occupying the distance from atom X to the first minimum in the corresponding radial distribution function. The hydration of the PO and P−O−C oxygen atoms present in the PO4− groups, as well as the C−O−C oxygens was determined. It can be observed that hydration numbers of all CL oxygen atoms in GAPs/CL monolayers are slightly higher compared to the pure CL monolayers. Indeed, these oxygen atoms are more hydrated in mixed films compared to the pure CL films. Such observation is in agreement with the v(CO)

Table 5. Hydration Number in the First Solvation Shell of Different Oxygen Atoms of CL Molecule in Pure CL and Mixed GAPs/CL Monolayers GAP II/CL

GAP VI/CL

xGAP

PO

P−O−C

CO

C−O−C

O−H

0 0.1 0.5 0.1 0.5

2.30 2.35 2.34 2.35 2.38

0.73 0.74 0.73 0.74 0.74

0.90 0.92 0.93 0.91 0.92

0.41 0.44 0.46 0.43 0.50

1.79 1.80 1.81 1.83 1.86

stretching vibration results. The stronger hydration of CL headgroups in the equimolar mixtures was demonstrated in the PM-IRRAS spectra, which show a redshift of the v(CO) frequency in the mixed GAPs/CL films compared to pure CL (see Table 3). It can be observed that in both GAPs the linker is increasingly in gauche conformation with an increasing content of CL in the mixed films; this effect is more pronounced in the case of GAP VI compared to GAP II (Figures S7 in Supporting Information). The fraction of trans to gauche conformers in GAP II linker is equal to 63.5, 49.6, and 30.0% for xGAP = 1.0, 0.5, and 0.1, respectively. The corresponding data for GAP VI are 25.4, 11.3, and 9.8%. This result is not unexpected, because the conformational liberty is higher in GAP VI compared to GAP II, as shown with a wider first band. It can be supposed that polar head disordering observed in the films and, consequently, polar head hydration depend on the conformational mobility of the GAP linker. This conjecture is in accordance with a lower thermodynamic stability of the mixed films formed with long linker GAPs and with a higher hydration demonstrated with PM-IRRAS compared to GAPs with the short linkers.

■

CONCLUSIONS Cardiolipin is an important component of different biological membranes. This negatively charged diphosphatidylglycerol lipid bearing four hydrocarbon chains forms highly organized, condensed films. The affinity of GAPs for cardiolipin is due to the hydrophobic lateral chains, and to the positively charged polar head present in GAPs. It can be expected that a possible antimicrobial activity of GAPs would be related to their capacity to disorganize lipid membranes. From this point of view, the effect of the variation of the length of the central linker in GAPs is of a particular interest. Both compression isotherms and thermodynamic results indicate a more liquid-like character and a stronger repulsion between molecules forming mixed films compared to those 6677


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(5) Hoch, F. L. Cardiolipins and Biomembrane Function. Biochim. Biophys. Acta, Rev. Biomembr. 1992, 1113, 71−133. (6) du Plessis, D. J. F.; Nouwen, N.; Driessen, A. J. M. The Sec Translocase. Biochim. Biophys. Acta, Biomembr. 2011, 1808, 851−865. (7) Gold, V. A. M.; Robson, A.; Bao, H.; Romantsov, T.; Duong, F.; Collinson, I. The Action of Cardiolipin on the Bacterial Translocon. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10044−10049. (8) Gomez, B., Jr.; Robinson, N. C. Quantitative Determination of Cardiolipin in Mitochondrial Electron Transferring Complexes by Silicic Acid High-Performance Liquid Chromatography. Anal. Biochem. 1999, 267, 212−216. (9) Schlame, M.; Ren, M. Barth Syndrome, a Human Disorder of Cardiolipin Metabolism. FEBS Lett. 2006, 580, 5450−5455. (10) Infante, M. R.; Pérez, L.; Pinazo, A.; Clapés, P.; Morán, M. C.; Angelet, M.; García, M. T.; Vinardell, M. P. Amino Acid-Based Surfactants. C. R. Chim. 2004, 7, 583−592. (11) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.; Soderman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Garcia Rodriguez, C. L.; Guedat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; van Eijk, M. C. P. Gemini Surfactants: New Synthetic Vectors for Gene Transfection. Angew. Chem., Int. Ed 2003, 42, 1448− 1457. (12) Menger, F. M.; Littau, C. A. Gemini Surfactants: a New Class of Self-Assembling Molecules. J. Am. Chem. Soc. 1993, 115, 10083− 10090. (13) Marti, I.; Ferrer, A.; Escorihuela, J.; Burguete, M. I.; Luis, S. V. Copper(II) Complexes of Bis(Amino Amide) Ligands: Effect of Changes in the Amino Acid Residue. Dalton Trans. 2012, 41, 6764− 6776. (14) Cavalli, S.; Albericio, F.; Kros, A. Amphiphilic Peptides and Their Cross-Disciplinary Role as Building Blocks for Nanoscience. Chem. Soc. Rev. 2010, 39, 241−263. (15) Kokkoli, E.; Mardilovich, A.; Wedekind, A.; Rexeisen, E. L.; Garg, A.; Craig, J. A. Self-Assembly and Applications of Biomimetic and Bioactive Peptide-Amphiphiles. Soft Matter 2006, 2, 1015−1024. (16) Lyu, Y.-Y.; Yi, S. H.; Shon, J. K.; Chang, S.; Pu, L. S.; Lee, S.-Y.; Yie, J. E.; Char, K.; Stucky, G. D.; Kim, J. M. Highly Stable Mesoporous Metal Oxides Using Nano-Propping Hybrid Gemini Surfactants. J. Am. Chem. Soc. 2004, 126, 2310−2311. (17) Paria, S. Surfactant-Enhanced Remediation of Organic Contaminated Soil and Water. Adv. Colloid Interface Sci. 2008, 138, 24−58. (18) Zhao, X.-B.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.-H.; Hauser, C. A. E.; Zhang, S.-G.; Lu, J.-R. Molecular Self-Assembly and Applications of Designer Peptide Amphiphiles. Chem. Soc. Rev. 2010, 39, 3480−3498. (19) Schramm, L. L.; Stasiuk, E. N.; Marangoni, D. G. Surfactants and Their Applications. Ann. Rep. Prog. Chem., Sect. C 2003, 99, 3−48. (20) Kumar, M.; Jinturkar, K.; Yadav, M. R.; Misra, A. Gemini Amphiphiles: A Novel Class of Nonviral Gene Delivery Vectors. Crit. Rev. Ther. Drug Carrier Syst. 2010, 27, 237−278. (21) Almeida, J. A. S.; Marques, E. F.; Jurado, A. S.; Pais, A. A. C. C. The Effect of Cationic Gemini Surfactants upon Lipid Membranes: An Experimental and Molecular Dynamics Simulation Study. Phys. Chem. Chem. Phys. 2010, 12, 14462−14476. (22) Luis, S. V.; Alfonso, I. Bioinspired Chemistry Based on Minimalistic Pseudopeptides. Acc. Chem. Res. 2014, 47, 112−124. (23) Rubio, J.; Alfonso, I.; Burguete, M. I.; Luis, S. V. Interplay between Hydrophilic and Hydrophobic Interactions in the SelfAssembly of a Gemini Amphiphilic Pseudopeptide: from NanoSpheres to Hydrogels. Chem. Commun. 2012, 48, 2210−2212. (24) Rubio, J.; Marti-Centelles, V.; Burguete, M. I.; Luis, S. V. Synthesis and Organogelating Ability of Bis-Urea Pseudopeptidic Compounds. Tetrahedron 2013, 69, 2302−2308. (25) Rubio-Magnieto, J.; Luis, S. V.; Orlof, M.; Korchowiec, B.; Sautrey, G.; Rogalska, E. Effects of Gemini Amphiphilic Pseudopeptides on Model Lipid Membranes: A Langmuir Monolayer Study. Colloids Surf., B 2013, 102, 659−666.

present in the pure monolayers. The disorganization of the mixed films shows as well in a higher number of gauche conformers and a higher polar head hydration evidenced with PM-IRRAS. The results obtained with molecular modeling support the disorganization of the mixed films and penetration of water molecules into the polar headgroup region. Differentiation between GAPs was possible based on compression isotherms and thermodynamic. The latter studies showed that derivatives with longer central spacers decrease film ordering and stability to a higher degree compared to those with shorter linkers. Molecular modeling showed that longer linkers adopt more disorganized, gauche conformations compared to short linkers. We propose that the intrinsic conformational flexibility of the long linkers is decisive for rupturing the interaction between the CL polar heads and for their hydration. Hydration of the polar heads would be responsible in turn for film disordering. Accordingly, a further engineering of GAP structures will be based on the molecules containing the hexyl linker.

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ASSOCIATED CONTENT

S Supporting Information *

Figures showing compression isotherms of mixed GAPs/CL monolayers, compressibility and miscibility analysis, order parameter, and tilt angle probability distribution functions of CL hydrocarbon chains in mixed GAP II/CL and GAP VI/CL monolayers. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jpcb.5b02575.

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AUTHOR INFORMATION

Corresponding Authors

*(B.K.) E-mail: [email protected]. Telephone: +48 (12) 663 22 51. Fax: +48 (12) 634 05 15. *(E.R.) E-mail: [email protected]. Telephone: +33 3 83 68 43 45. Fax: +33 3 83 68 43 22. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.G. acknowledges the financial support from the project Interdisciplinary Ph.D. Studies “Molecular sciences for medicine” cofinanced by the European Social Fund within the Human Capital Operational Programme. This research was supported in part by PL-Grid Infrastructure. Financial support by the MINECO (CTQ2012-38543-C03-01) and UJI (P11B2013-38) is acknowledged.

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REFERENCES

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A Study of the Interaction between a Family of Gemini Amphiphilic

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