Effect of Phosphatidic Acid on Biomembrane: Experimental and

Article pubs.acs.org/JPCB

Effect of Phosphatidic Acid on Biomembrane: Experimental and Molecular Dynamics Simulations Study Urszula Kwolek,† Waldemar Kulig,‡ Paweł Wydro,† Maria Nowakowska,† Tomasz Róg,*,‡ and Mariusz Kepczynski*,† †

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland Department of Physics, Tampere University of Technology, P.O. Box 692, FI-33101 Tampere, Finland

‡

S Supporting Information *

ABSTRACT: We consider the impact of phosphatidic acid (namely, 1,2-dioleoyl-sn-glycero-3-phosphate, DOPA) on the properties of a zwitterionic (1,2-dipalmitoyl-sn-glycero-3phosphocholine, DPPC) bilayer used as a model system for protein-free cell membranes. For this purpose, experimental measurements were performed using differential scanning calorimetry and the Langmuir monolayer technique at physiological pH. Moreover, atomistic-scale molecular dynamics (MD) simulations were performed to gain information on the mixed bilayer’s molecular organization. The results of the monolayer studies clearly showed that the DPPC/DOPA mixtures are nonideal and the interactions between lipid species change from attractive, at low contents of DOPA, to repulsive, at higher contents of that component. In accordance with these results, the MD simulations demonstrated that both monoanionic and dianionic forms of DOPA have an ordering and condensing effect on the mixed bilayer at low concentrations. For the DOPA monoanions, this is the result of both (i) strong electrostatic interactions between the negatively charged oxygen of DOPA and the positively charged choline groups of DPPC and (ii) conformational changes of the lipid acyl chains, leading to their tight packing according to the so-called “umbrella model”, in which large headgroups of DPPC shield the hydrophobic part of DOPA (the conical shape lipid) from contact with water. In the case of the DOPA dianions, cation-mediated clustering was observed. Our results provide a detailed molecular-level description of the lipid organization inside the mixed zwitterionic/PA membranes, which is fully supported by the experimental data.

1. INTRODUCTION

alters the state of phosphate headgroup protonation, making the PA lipid a pH biosensor. The biological significance of PA was the motivation for researchers to carry out experimental and computing studies aimed at explaining the impact of PA on the properties of zwitterionic membranes. The miscibility of PA/phosphocholine (PC) binary mixtures containing saturated lipids of various chain lengths was studied using differential scanning calorimetry (DSC),8−10 and the mixtures comprising unsaturated lipids were examined directly by fluorescence microscopy.11,12 Blume et al. showed that the miscibility of a PA/PC system (chain length n of 14 or 16 C atoms) strongly depends on the degree of ionization of the PA headgroups and thus on pH of the surrounding environment.8,9 The authors suggested that at pH 7, at which PA is negatively charged, electrostatic repulsion between the PA molecules and hydrogen bonds between the PA and PC headgroups (complex formation) favor the mixing of the two components.8,9 Lowering pH to 4 induced large changes in the mixing behavior due to the lower ionization degree of the PA headgroups. The reduction in electrostatic

Phosphatidic acid (PA, 1,2-diacyl-sn-glycero-3-phosphate) is the simplest diacyl-glycerophospholipid; it consists of glycerol, to which two fatty acids are esterified at the sn-1 and sn-2 carbons, and the phosphate group is attached at the sn-3 position. Therefore, PA is a negatively charged (anionic) molecule at physiological pH. Although PA is present only in small amounts (often less than a few mol %) in biological membranes, it is crucial for cell survival.1 Its role in the vital processes occurring in cells has been previously discussed in several reviews.2−5 Most significantly, PA is at the center of membrane phospholipid biosynthesis, and as a consequence, the level of PA is carefully controlled to maintain lipid homeostasis.5 PA plays important role in regulating cell proliferation; therefore, elevated expression and/or activity of enzymes that generate PA is commonly detected in human cancer.2 Cancer cells are known to produce PA to avoid apoptosis.6 PA has been also identified as an important signaling molecule in both plants and animals.3 In plants, its formation is triggered in response to various biotic and abiotic stress factors, including low temperature, drought, salinity, or wounding. Recently, it was shown that PA signaling can be dynamically regulated by changes in pH.7 The change in pH © 2015 American Chemical Society

Received: April 15, 2015 Revised: July 9, 2015 Published: July 13, 2015 10042

DOI: 10.1021/acs.jpcb.5b03604 J. Phys. Chem. B 2015, 119, 10042−10051

Article

The Journal of Physical Chemistry B

strong interactions between both lipids. We used Langmuir monolayer measurements to study the intermolecular interactions in the PC/PA mixture. This method allowed us to determine thermodynamic parameters of lipid mixing. MD simulations were performed to gain information on the mixed bilayer’s molecular organization. We consider the properties of the DPPC membranes containing low amounts of monoanionic and dianionic DOPA, because both these forms exist in biomembranes at physiological pH. First, the impact of DOPA on the area per lipid and bilayer thickness was determined. Next, we consider the arrangement of DPPC hydrocarbon chains inside the membrane. Finally, we focus on the possibility of hydrogen bonds and charge pairs forming between lipid molecules, between lipids and water, and between lipids and sodium cations.

repulsion and the concomitant increase in attractive hydrogen bonding interactions between the PA headgroups may result in immiscibility of the saturated lipids. The experiments performed at pH 12, at which the PA component is double charged, indicated the possible phase separation of both lipids. This was explained by stating that “other strongly attractive interactions overcompensate the electrostatic effects.”9 The miscibility in the PC/PA membranes can be also controlled by the monovalent ion concentration. It was shown that 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) are uniformly mixed in the pH range 5−8, at room temperature and high concentration of KCl.11 However, the irreversible separation of lipids into two domains was observed upon lowering the K+ concentration at pH 5. In addition to the experimental methods, molecular dynamics (MD) simulations have been applied to study the PA/PC bilayers.13−15 The 50 ns simulations were performed for DOPC bilayers containing 10 mol % of either monoanionic or dianionic DOPA in 0.2 M NaCl salt.13 Interestingly, the results indicated that both forms of DOPA phosphate groups were located at the same depth as the phosphates of DOPC in the membrane; however, interactions between the lipid molecules were not addressed in this study. Cheng et al. performed the 50 ns all-atom MD simulations of a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer containing 20 mol % of 1-palmitoyl-2-oleoylphosphatidic acid (POPA) in the monomeric form and 20 mol % of cholesterol at 30 °C.14 These simulations revealed that the lipid organization in the membrane is regulated by various interactions between different types of molecular pairs formed in the system; however, POPA molecules had no strong tendency to interact with each other. Broemstrup and Reuter simulated an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) with 1,2-dimyristoyl-sn-glycero-3phosphate (DMPA) at 37 °C.15 It was shown that the DMPC/DMPA bilayer has a lower area per lipid and higher order parameter than the pure DMPC membrane, indicating tighter chain packing in the mixed bilayer than in DMPC. The effect of monovalent ions (Na+) on the formation of salt bridges was analyzed, and preferential clustering of DMPA around Na+ was demonstrated. On the basis of the analysis of the literature, one can conclude that the intermolecular interactions in the PC/PA membrane, thus its molecular organization, are not fully recognized, especially in the case of the PA dianion. In this article, we present the results of complementary experimental and atomic-scale MD simulation studies for the effects of PA on the properties of a PC bilayer, used as a model biomembrane. Binary mixtures of saturated phosphatidylcholine (DPPC) and unsaturated phosphatidic acid (DOPA) were examined. DPPC was chosen for the study, because it is well studied both experimentally and computationally.16 In addition, its melting temperature is above 0 °C, which enables examination of the thermotropic behavior of a mixed DPPC/DOPA bilayer by a microDSC method. DOPA has in its structure two cis-double bonds bending the hydrocarbon chain and phosphate group bearing a negative charge due to dissociation under our experimental conditions. Both of these functions would affect the organization of DPPC molecules in its bilayer. The bent hydrocarbon chains of DOPA should introduce disordering in the dense packing of the DPPC chains, whereas the negative charge at the headgroup should result in the appearance of the

2. MATERIALS AND METHODS 2.1. Materials. Phospholipids, 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), are synthetic products of high purity (≥99%) that were purchased from Sigma-Aldrich. Chloroform and methanol were purchased from Sigma-Aldrich (HPLC grade, ≥99.9%). The ultrapure Milli-Q water used in the experiments had a surface tension of 72.6 mN/m (at 20 °C) and resistivity of 18 MΩ cm. 2.2. Monolayer Experiments. The experiments were carried out with a KSV 2000 Langmuir trough (KSV Instruments Ltd., Helsinki, Finland) (total area of 870 cm2) equipped with two movable barriers. The surface pressure was measured with an accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. Brewster angle microscopy (BAM) experiments were performed using an ultraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at 658 nm, a 10× objective, and a CCD camera. The spatial resolution of the BAM images was 2 μm. The microscope and the film trough were placed on a table (Standa Ltd., Vilnius, Lithuania) with an active vibration isolation system (VarioBasic_40, Halcyonics, Gö ttingen, Germany). The lipids were dissolved in a chloroform/methanol (9:1 v/ v) mixture to form stock solutions. Spreading solutions were prepared from the stock solutions and were deposited onto the subphase with a Hamilton microsyringe that is precise to 1.0 μL. The volume of the spreading solutions varied between 150 and 200 μL. After spreading, the monolayers were left for 20 min and then the compression was initiated with the barrier speed of 5 cm2/min (0.025 nm2 molecule−1 min−1). The temperature was controlled by a circulating water system. All of the experiments were repeated at least twice to ensure consistent results. The isotherms were reproducible within an error of ±0.02 nm2 molecules−1. 2.3. Differential Scanning Calorimetry (DSC). DSC experiments were performed using a NanoDSC TA Instruments calorimeter with 0.3 mL capillary cells. The measurements of lipid suspensions (2 mg/mL total lipid concentration and varying XDOPA) were performed from 3 to 65 °C at a scan rate of 0.5 °C/min. Thermograms for the buffer were acquired under identical conditions and were subtracted from excess heat capacity curves. Tm was defined as the temperature of the peak maximum. The enthalpy was obtained by integrating the area under the transition peak. 10043


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The Journal of Physical Chemistry B 2.4. MD Simulations. We performed atomistic-scale MD simulations of three model systems containing 116 molecules of DPPC and 12 of DOPA (∼10 mol %), ∼3800 water molecules, and an appropriate amount of Na+ counterions. We considered two protonation states of DOPA: monoanionic and dianionic (referred to as DOPA− and DOPA2−). As a reference system, we used pure DPPC in the liquid-crystalline phase, as described in our previous studies.17 An initial structure of the mixed bilayer was constructed from an equilibrated DPPC membrane17 by replacing 12 randomly selected molecules with DOPA. Prior to the MD simulations, the system was optimized with 500 steps of the steepest-descend algorithm. MD simulations (200 or 400 ns long) were conducted using the GROMACS 4.5 software package.18 To parametrize all of the lipid molecules, the all-atom OPLS force field was employed,19 with a recent extension for lipids.17 Partial charges for the DOPA phosphate group at two protonation states were derived according to the procedure described previously.17 Briefly, the charges were derived for a small molecular fragment (methyl phosphate in this case, Me−PO4H− and Me−PO42−). The structure of the compound was first optimized using secondorder Møller−Plesset perturbation theory (MP2), with the 631G** basis set. Next, the electrostatic potential was calculated. A polarizable continuum model (PCM) was used to model the effect of a polar environment.20 These calculations were performed using the Gaussian09 package.21 Finally, the partial atomic charges were calculated using the RESP program.22 The derived charges are given in Table S2 (Supporting Information). A TIP3P model that is compatible with the OPLS-AA force field was used for the water.23 The time step was set to 2 fs, and the simulations were carried out at 1 bar and two temperatures: 37 °C, which is below the Tm of DPPC (the system with DOPA−, 400 ns), and 50 °C, which is above the Tm (for the systems with DOPA− and DOPA2−, 200 ns). The v-rescale method was used to couple the temperature with separate heat baths for the membrane and the rest of the system, with time constants of 0.1 ps.24 The reference pressure was set to 1 bar; it was coupled using the semi-isotropic Parrinello−Rahman barostat.25 For the longrange electrostatic interactions, the particle-mesh Ewald (PME) method was used.26 The linear constraint solver (LINCS) algorithm was used to preserve the covalent bond lengths.27

mole fractions (XDOPA) in phosphate buffer (pH 7.4) were prepared and analyzed by DSC. The interactions between DPPC and DOPA were studied using monolayer experiments. 3.1. DSC Studies. The melting temperatures of DOPA and DPPC differ considerably. DOPA is a fluid above −8 °C,28 whereas DPPC is in the gel phase up to 41.6 °C. Therefore, we first experimentally determined the phase diagram for the DPPC/DOPA system to obtain information about the mixed bilayer’s physical state at different temperatures. DSC thermograms of the DPPC/DOPA samples of various compositions are shown in Figure S1 (Supporting Information). The thermogram of neat DPPC exhibited two endothermic peaks, corresponding to a pretransition (Lβ′ phase to ripple phase, Pβ′) and the main chain-melting transition (Pβ′ phase to a lamellar fluid phase, Lα). The values of chain-melting temperature (Tm) and the enthalpy of transition (Hm) are in agreement with literature data (Table S1, Supporting Information). The incorporation of DOPA into the DPPC membrane resulted in significant changes in the gel-to-liquid-crystalline phase transition (Pβ′ phase to Lα phase). This is associated with an enrichment of the system in the unsaturated compound. As the DOPA content in the mixture increased, the phase transition became considerably broader and moved toward lower temperatures. As expected, no peak was observed for the neat DOPA in the studied temperature range (3−65 °C); the membrane of this lipid is in the fluid state above 0 °C. On the basis of the DSC measurements, a pseudobinary phase diagram can be constructed by plotting transition temperatures against the sample composition. The heat capacity curves were analyzed using the tangent method (see, e.g., ref 29) to determine the onset and completion temperatures (i.e., estimated temperatures of the transition from a single-phase state to two-phase coexistence). As a result, the phase boundaries among the gel phase, liquid-crystalline phase, and their coexistence region for the mixed system were determined (Figure 2). 3.2. Monolayer Experiments. The selected surface pressure−area (π−A) isotherms recorded during the compression of the mixed DPPC/DOPA films at 25 and 37 °C on the buffer subphase are shown in Figure 3 (also Figure S3,

3. RESULTS The properties of the DPPC/DOPA binary system were studied; the chemical structures of the lipids are presented in Figure 1. A series of DPPC/DOPA samples varying by DOPA

Figure 1. Chemical structures of 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA).

Figure 2. Phase diagram of the DPPC/DOPA system. The melting temperature of pure DOPA was taken from ref 28. 10044


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Figure 4. (A) Dependence of the mean area per lipid molecule (A12) on the composition for the DPPC/DOPA mixed monolayers at a surface pressure of 30 mN/m and at 37 °C (blue squares) and 25 °C (red circles). (B) Values of excess free energy of mixing (ΔGExc) versus the composition of the DPPC/DOPA monolayers at π = 30 mN/m. The dotted lines are added to guide the eye and have no physical meaning.

Figure 3. Selected surface pressure (π)−area (A) isotherms for DPPC/DOPA mixed monolayers measured at 25 °C (A) and 37 °C (B).

Supporting Information). The isotherms for the pure DPPC and DOPA monolayers (also Figure S2, Supporting Information) are in good agreement with those reported in the literature.30 The isotherms for pure DPPC show pseudoplateau regions, indicating that the monolayer undergoes a phase transition during compression. At high surface pressures, the DPPC monolayer was in the liquid-condensed (LC) state. However, the monolayer became more fluid as the temperature increased from 25 to 37 °C, as seen in the BAM images (Figures S4 and S5, Supporting Information). In regard to DOPA, the shape of the π−A curves indicates the liquid state of the film at both temperatures, as confirmed by the BAM images. For the DPPC/DOPA isotherms, the phase transition characteristic of the DPPC monolayer shifted toward higher surface-pressure values as the DOPA mole fraction increased and completely vanished at XDOPA ≥ 0.3 for 37 °C and XDOPA ≥ 0.7 for 25 °C. As expected, the monolayers containing DOPA, an unsaturated lipid, were less organized due to the steric hindrance created by the presence of the cis-double bond in the acyl chains. The properties of lipid monolayers correlate with those of bilayers at surface pressures in the range 30−35 mN/m.31 Therefore, further analysis was performed for π = 30 mN/m. To analyze the miscibility of the monolayer components, the average molecular area (A12) was plotted as a function of the monolayer composition (Figure 4A). The dashed straight lines correspond to ideal mixing of the film components. The A12 values showed weak deviations from the ideal mixing (the additivity rule). They changed irregularly with the film’s composition; for both temperatures, the monolayers with XDOPA ≤ 0.3 displayed a small contraction of the area per

molecule, whereas the further increase in DOPA content caused the mixed film to expand. The values of excess free energy of mixing (ΔGExc) were calculated and plotted as a function of the film composition (Figure 4B) to evaluate the magnitude of the interaction between the molecules in the mixed systems. As can be seen, the ΔG Exc versus X DOPA showed irregularity. For the monolayers with XDOPA ≤ 0.3 negative deviations from the ideal behavior were observed, which means that the interactions between the molecules were more attractive than those in pure component films. The location (XDOPA ≈ 0.2) and depth of the minimum that appeared in the curves are the same for the monolayers studied at both temperatures, indicating that the strongest attractive interactions between the lipid molecules occurred in the monolayer, when DPPC and DOPA molecules were mixed at the ratio of 4:1. The negative value of ΔGExc shows that mixing of the monolayer components was thermodynamically favorable. The situation is quite different for the mixed films with XDOPA > 0.3. In this case the values of ΔGExc were positive, which suggests that the intermolecular interactions were less favorable compared to those in the respective one-component monolayers. The strongest repulsion between lipid molecules was observed in the DPPC/DOPA monolayers with the compositions of 1:1 and 1:2 at 37 and 25 °C, respectively, according to the maximum position in the ΔGExc versus XDOPA dependency. A similar shape of the ΔGExc versus composition of membrane was recently observed for the system containing cardiolipin (CL) and DPPC.32 CL can be considered as a dimer 10045


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Figure 5. Snapshots of the configurations at the end of the simulation for DPPC/DOPA− at 50 °C (A), DPPC/DOPA2− at 50 °C (B), and DPPC/ DOPA− at 37 °C (C). The DOPA molecules are shown as tan spheres. The DPPC lipids are shown as gray sticks. The phosphate groups and Na+ cations are presented as red and blue spheres, respectively. The water molecules were removed for clarity.

is very close to the experimental data (ca. 0.62934 or 0.6435 nm2) determined at the same temperature. As can be seen, the addition of DOPA led to the membrane’s condensation. At 50 °C, this effect was greatest for the DPPC/DOPA2− system. The A value calculated for DPPC/DOPA− at 37 °C was similar to that obtained for DPPC/DOPA2− at 50 °C. It clearly indicates that the bilayer at the lower temperature was in the liquid phase, as the area was much larger than that observed for the saturated bilayer in the gel phase (e.g., 0.46 nm2 for DSPC at 25 °C).36 The membrane thickness was calculated as the distance between the points in the opposite leaflets at which the densities of water and lipids are equal (Table 1). As expected, membrane thickness was inversely correlated with A. The average tilt angle of the palmitoyl chains was calculated as the angle between the bilayer normal and the average vector linking the C atom at the end of the acyl chain with the carbonyl C atom of the same chain. The tilt values averaged over both sn-1 and sn-2 tails are given in Table 1. At 50 °C, the tilt angle correlated well with the area per lipids. The effect of DOPA on the lipid packing in the bilayer can be deduced from changes in the deuterium order parameter, SCD, which is defined as follows:37

of two PA molecules linked via additional glycerol. The excess free energy of mixing measured for the DPPC/CL system showed a minimum at XCL = 0.1. This value is 2 times lower than that observed for DOPA. 3.3. MD Simulations. MD simulations of systems comprising 10 mol % of DOPA and differing in their degree of protonation (DPPC/DOPA− and DPPC/DOPA2− systems) were performed at 37 or 50 °C. Figure 5 shows selected snapshots illustrating the organization of the mixed bilayers at the end of the simulations. Several features of the systems became apparent at the end of the simulation. The DOPA molecules were almost evenly distributed in all three membranes. The DPPC/DOPA− bilayer at 50 °C (Figure 5A) was characterized by a noticeable chain disorder that is peculiar to the liquid phase of a membrane. In the case of DPPC/DOPA2− at 50 °C and DPPC/DOPA− at 37 °C, respectively (Figure 5B,C), the presence of two regions with very different molecular ordering patterns was observed. In the first region, the lipid molecules were stretched out and tightly packed in a manner characteristic of the gel phase. The upper and lower monolayers were separated by a distance of approximately double the chain length; as a result, the membrane was significantly thicker. In the second region, the hydrocarbon chains were disordered and the monolayers were partially interdigitated, which reduced the bilayer’s thickness. Such a membrane structure resembles the ripple phase (Pβ′) and was previously observed in the MD simulations but is not typical for biological membranes.33 Four standard parameters describing the organization of the lipid bilayers were calculated: the area per lipid molecule (A), bilayer thickness, tilt angle, and order parameter (SCD) of the DPPC acyl tails. A was calculated as the mean “x−y” area of the simulation box in the membrane plane divided by the number of molecules in a single leaflet (Table 1). The value for DPPC

SCD =

A (nm2) Thickness (nm) Tilt angle (deg)

DPPC/ DOPA− at 50 °C

DPPC/ DOPA2− at 50 °C


0.63 3.92

0.600 4.28

0.563 4.60

0.561 4.69

17.6

16.1

14.44

17.0

(1)

where θi is the angle between a C−D bond (C−H in simulations) of the ith carbon atom and the bilayer normal. The angular brackets denote averaging over time for molecules in the bilayer. The deuterium order parameter provides information on the probability of occurrence of each of the C−C bond rotamers and, consequently, the number of gauche defects per chain, as well as their projected average length onto the bilayer normal.38 The rotation around the C−C bonds is assumed to be subject to a trans−gauche torsional potential. This parameter is commonly used in its negative form (−SCD), as was done in this paper. In all-trans conformation, the most ordered configuration of the saturated hydrocarbon chain, −SCD, reaches the value of 0.5. The appearance of gauche rotamers (the gauche defects in the chain) leads to a gradual decrease in the order parameter. The SCD parameter’s profiles along two DPPC acyl chains are shown in Figure 6. The results are in agreement with the trends observed for A and membrane thickness. The presence of DOPA increased the ordering of the DPPC hydrocarbon tails; the effect was much stronger for DOPA2− at 50 °C.

Table 1. Area per Lipid Molecule (A), Membrane Thickness, and Tilt Angle Obtained from the MD Simulationsa DPPC at 50 °C

3 1 (cos2 θi) − 2 2

a

The estimated errors for A, thickness and tilt angle are 0.001, 0.02, and 0.2, respectively. 10046


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Figure 6. Comparison of the order parameters (shown as negative values of SCD) for the sn-1 (a) and sn-2 (b) alkyl chains in DPPC, calculated for pure DPPC membrane and the membrane containing 10 mol % of DOPA.

The mass density profiles across the membrane of selected lipid atoms and water were calculated (Figure 7) to elucidate upon the structure of the mixed bilayers. The profiles for pure DPPC are shown in Figure S6 (Supporting Information). Figure 7A shows that, for the DPPC/DOPA− membrane in the liquid state, the distributions of phosphates from both lipids along the bilayer normal were characterized by a single symmetrical peak. However, the DOPA phosphates were located slightly deeper in the bilayer (ca. 0.26 nm), compared to those groups of DPPC. For the system with DOPA2−, we observed that the distributions of the phosphate, choline, and carbonyl groups of DPPC and the DOPA phosphates had two maxima. Although the data were scattered due to the more complex distribution, the location of the DOPA phosphates was also slightly deeper (0.2−0.3 nm) in this case than that of DPPC. In the case of the simulation performed at 37 °C, when the membrane was more condensed and ordered, the DOPA phosphates were also located deeper (0.3−0.4 nm) compared to DPPC. The Na+ counterions located at the water− membrane interface and collocated with the DOPA phosphates. The water penetrated deeper in the system containing DOPA2− than in the system containing DOPA− (Figure S7, Supporting Information). Next, we calculated the numbers of intermolecular interactions between the lipids, water, and ions. We considered hydrogen bonds (H-bonds), charge pairs, and ion binding. To evaluate the existence of these interactions, we used geometrical definitions derived from our previous studies.39 H-bonds were assumed to be formed when the distance between the hydrogen

Figure 7. Density profiles of selected lipid atoms and water along the bilayer normal for DPPC/DOPA− at 50 °C (a), DPPC/DOPA2− at 50 °C (b), and DPPC/DOPA− at 37 °C (c).

acceptor and donor was ≤0.325 nm and the angle between the OH bond and the donor−acceptor vector was ≤35°. The charge pairs between positively charged quaternized ammonium groups of choline (N+(CH3)3) and negatively charged O atoms of lipids were evaluated on the basis of a distance cutoff of ≤0.4 nm;39 contacts of Na+ with the lipid O atoms were evaluated on the basis of a distance cutoff of ≤0.35 nm.40 In the case of the DOPA−−DPPC pair, H-bonds could form between the P−OH groups of DOPA and oxygen in the DPPC phosphate group. For lipid−water pairs, H-bonds could form between O atoms of lipid phosphates and an OH group of a water molecule (lipid−O···H−OH) or between P−OH groups of DOPA− and an O atom of a water molecule (DOPA−OH··· OH2). The calculated numbers of intermolecular interactions are given in Table 2. DOPA− formed a large number of charge pairs with the DPPC choline groups and a small number of direct H-bonds. At lower temperatures, the number of charge pairs slightly increased as the number of direct H-bonds decreased. The number of contacts with Na+ was rather small for DOPA−, particularly at 37 °C. For DOPA2−, a substantial increase in hydration and contacts with Na+ was observed, but at the expense of direct interactions with the choline groups of DPPC (charge pairs). 10047


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mixtures with DOPA content of 30 mol % or higher were in the liquid phase. 4.2. Miscibility of DPPC/DOPA Membranes. Phospholipid miscibility in the hydrated bilayer is a subject of fundamental importance for membrane biophysics.10 Lipid miscibility is mostly governed by the difference in the pair interaction energies among the like-pairs and mixed-pairs formed in the mixture. Lateral phase separation and domain formation can occur when the like-pair interactions are significantly stronger than that of the mixed-pairs. We investigated the intermolecular interactions between phospholipid species using the Langmuir monolayer technique, which allowed us to determine the excess free energy of mixing (Figure 4B). The results clearly show that the DPPC/DOPA mixtures were nonideal, due to intermolecular interactions, but their nature is strongly dependent on the DOPA content in the monolayer. The negative value of ΔGExc for the system with low mole fractions of DOPA reflects the tendency for the two lipids to mix in the monolayer. The monolayer condensation indicates that the mixed-pair interactions were stronger than the interactions between like-pair molecules in the mixed monolayer. The attractive forces between the DOPA and DPPC molecules prevailed over the fluidizing effect of the cis double bonds in the DOPA molecules. On the contrary, the positive ΔGExc values for the films containing larger amounts of DOPA indicate that the lipid miscibility was not thermodynamically favorable. However, there were no signs of phase separation as indicated by the BAM images. This means that the repulsive interactions between the mixed pairs were rather weak (as indicated by the ΔGExc value at the maximum) to cause the immiscibility of the DPPC/DOPA membrane at pH 7.4. 4.3. Molecular View of the Interactions inside the DPPC/DOPA Membranes. We performed MD simulations to gain insight into the molecular organization of the DPPC/ DOPA bilayer at low anionic lipid content. The results indicate that the addition of 10 mol % of DOPA to the DPPC bilayer increased the order of the hydrocarbon tails and decreased the area per lipid. Thus, DOPA has ordering and condensing properties under these conditions. This phenomenon can be explained by considering two factors: the charge nature of DOPA and its conical shape. A DOPA headgroup is relatively small, whereas two oleoyl tails constitute a large hydrophobic part of the molecule. Thus, the DOPA molecule has a conical shape with small hydrophilic and large hydrophobic parts.41,42 Due to the negative charge, DOPA− and DOPA2− can interact electrostatically with N+(CH3)3 groups of DPPC or Na+ counterions. However, we observed a different number of interactions between these forms of the anionic lipid and the cationic species. In the case of DOPA−, the predominant interaction with DPPC was the formation of charge pairs between the negative oxygen of DOPA and the positively charged choline group of DPPC. The choline moiety is a large, bulky group bearing a positive charge spread over three methyl groups and having partially hydrophobic properties, but it is able to form charge pairs with nonwater oxygens.43 A number of charge pairs formed between DOPA− and Na+ cations was ca. 3 times smaller. In addition to the Coulomb interaction, also the conical shape of DOPA can contribute to the condensing effect of this lipid on the zwitterionic membrane. It was shown that neutral lipids with the conical shapes, such as diacylglycerol, ceramide, or cholesterol possess condensing properties in PC

Table 2. Average Number of Intermolecular Interactions between DOPA, DPPC, Water, and Na+ a

no. no. no. no. no.

of of of of of

H-bonds for DOPA−DPPC H-bonds for DOPA−water H-bonds for DPPC−water charge pairs for DOPA−DPPC charge pairs for DOPA−Na+


DPPC/ DOPA2− at 50 °C


0.42 7.01 7.63* 2.71 0.91

10.78 7.26* 0.88 5.33

0.33 7.33 7.51* 2.95 0.88

a The numbers are given per one DOPA (*DPPC) molecule. The errors are smaller than 0.01.

4. DISCUSSION The main objective of this study was to explore the effect of phosphatidic acid on the properties of a zwitterionic membrane, used as a model system for protein-free cell membranes. For this purpose, the behavior of binary mixtures of DPPC and DOPA at physiological pH was studied as a function of composition and temperature. DPPC is a zwitterionic lipid with two saturated acyl chains (Figure 1), and DOPA has two cisdouble bonds in its structure, which causes the 30° bend of oleoyl chains at the C9 position. Moreover, DOPA, as a phosphomonoester, has two pKa’s. For 10 mol % of DOPA in the DOPC membrane, pKa1 and pKa2 were determined to be 3.2 ± 0.3 and 7.92 ± 0.03 at 20 °C,41 which implies that at pH 7.4, ca. 80% of DOPA molecules were in the monoanionic form and the rest was in the dianionic form. 4.1. Comparison of DSC and Monolayer Experiments. At higher surface pressures, the molecular packing and physical state of the mixed monolayers were found to be strongly dependent on the film composition and temperature. At 25 °C, the mixed monolayer with XDOPA = 0.1 was in the LC state, which was confirmed by the BAM images (Figure S5, Supporting Information) as well as the Cs−1 vs π plot (Figure S3D, Supporting Information). At 30 mol % content of DOPA, the LC and liquid-expanded (LE) phases coexisted in the monolayer; however, most of the film material was in the LC phase (Figure S5, Supporting Information). For the monolayer containing 50 mol % DOPA, the film was mainly in the liquid state, but one could see that the monolayer was not fully homogeneous (very small domains can be seen in the BAM image, Figure S5, Supporting Information). These observations may explain the presence of the shallow minimum in the Cs−1 vs π curve for this monolayer (Figure S3, Supporting Information). At higher content of DOPA, the monolayers were completely homogeneous and, similarly to pure DOPA film, were in the liquid state. These observations are in very good agreement with the DSC results obtained for the DPPC/ DOPA mixed bilayers. The phase diagram (Figure 2) indicates that at 25 °C, the bilayer with XDOPA ≤ 0.1 was in the gel phase and the mixed bilayers containing XDOPA ≥ 0.66 were in the liquid-crystalline phase, whereas the coexistence of liquid crystalline−gel phase was observed within these limits. A similar correlation between the monolayer and bilayer’s properties could also be observed at 37 °C. The DSC measurements indicated that the mixed bilayer with XDOPA between 0.027 and 0.27 coexisted with the liquid crystalline− gel phase; for XDOPA ≥ 0.27, the bilayer was in the liquidcrystalline phase. In the case of the mixed monolayer with XDOPA = 0.1, the shallow minimum in the Cs−1 vs π curve indicated the coexistence of the LC−LE phases, whereas the 10048


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The Journal of Physical Chemistry B membranes.44−46 The proposed mechanism of this condensation is known as an “umbrella model”.47 In this model, lipids with larger headgroups (like PCs) shield the hydrophobic part of the conical lipids from contact with water; to make this possible, these lipid tails need to increase their order and reduce the occupied surface area, creating additional space for the hydrophobic part of the other lipid. This picture agrees well with our results for DOPA−: (i) DOPA molecules are located ca. 0.2−0.4 nm deeper into the bilayer, and (ii) the incorporation of DOPA into the membrane causes an increase in the DPPC acyl chain order parameter, which creates additional space for the hydrophobic part of DOPA. Figure 8A

effectiveness of the membrane condensation was larger compared to the DPPC/DOPA− bilayer, as indicated by the A value. The formation of ion bridge was observed to exist in the case of most negatively charged lipids (like phosphatidylserine, PS), whose headgroups are comparable in size with the PC headgroup.48 The strong adsorption of Na+ cations on the surface of DMPC/DMPA membrane and Na+-mediated clustering of DMPA lipids together was shown in MD simulations.15 It was found that Na+ is mostly bound to clusters of two lipids. 4.4. Comparison of MD Simulations and Experimental Results. A comparison of the MD and experimental data should be done with necessary care. We constructed two systems with fixed protonation states in the MD simulations, whereas in the real system, both forms coexist in the membrane, although the monoprotonated state is more frequent in the physiological medium. Also, the comparison of the experimental and MD simulation results was limited to a temperature of 37 °C. Due to experimental limitations, we were unable to carry out measurements at temperatures >40 °C, whereas the simulation of membranes in the gel phase (25 °C) requires a much longer time scale. However, we can compare the data for the physiologically relevant system (pH 7.4 and 37 °C). Both the monolayer experiments and computer simulations indicated that the introduction of low content of DOPA to a zwitterionic membrane resulted in its contraction. Moreover, the value of A calculated by the MD simulations for the DPPC/DOPA− system at 37 °C (Table 1) was very close to the value of A12 determined for the monolayer with XDOPA = 0.1 at π = 30 mN/m and at the same temperature (Figure 4A). Additionally, our simulations indicated that the introduction of DOPA reduced the tilt angle of the DPPC chains in the membrane. The same effect of DPPA on DPPC in the monolayer was previously shown experimentally.49 The increasing amount of the anionic lipid resulted in a drastic reduction of the tilt angle in the DPPC-rich domains.

Figure 8. Snapshots showing: (A) a charge pair forming in the DPPC/ DOPA− membrane at 50 °C and arranged according to the “umbrella model”; (B) the complex involving an ion bridge in the simulated DPPC/DOPA2− membrane at 50 °C. Na+ (blue sphere) is coordinated with two DPPC lipids and one DOPA lipid. The DOPA and DPPC acyl chains are shown as orange and magenta sticks, respectively.

5. CONCLUSIONS The Langmuir monolayer and DSC experiments supported by the MD simulations provided interesting insights into the properties of a zwitterionic (DPPC) bilayer, used as a model of cellular membranes, after incorporating various amounts of phosphatidic acid (DOPA). Our results showed that DOPA molecules affect the level of molecular ordering in membranes and their thermotropic properties. These effects are strongly dependent on the DOPA content in the system and, to some extent, on the experimental conditions. Under physiological conditions (pH 7.4) and at low DOPA content (XDOPA ≤ 0.3), the mixing of DPPC and DOPA lipids in monolayers is thermodynamically favorable due to the strong attractive electrostatic interactions between their headgroups, resulting in the monolayer’s condensation. The situation changes significantly at higher DOPA content in the system (XDOPA > 0.3). There, we observed the expansion of DPPC/DOPA mixed films, compared to the ideal mixing of both lipids. This means that mixing at higher DOPA content is thermodynamically unfavorable, although there is no visible phase separation. In agreement with the experiments, our MD simulations revealed the reduced area per lipid after introducing 10 mol % of either monoanionic or dianionic DOPA to the DPPC bilayer. However, the MD simulations showed important differences in the behavior of the bilayer containing mono- and dianionic forms of DOPA. We see evidence that DOPA2− has a far

shows the charge pair of DOPA and DPPC adopting the arrangement in accordance with the umbrella model. Although the umbrella model does not explain all aspects of the DOPA condensing effect, it certainly contributes to the mechanisms of the membrane condensation observed in our studies. The appearance of two negative charges at the DOPA phosphate groups had a very pronounced effect on the intermolecular interactions in the mixed membrane. Strong binding of Na+ cations to the DPPC/DOPA2− membrane was observed, whereas the DPPC cholines did not appear to play an important role in interactions with DOPA. The adsorbed sodium cations formed ion bridges between different lipids. Figure 8B illustrates an example of a cluster made of one DOPA2− and two DPPC molecules around Na+. However, we observed that cation-mediated clustering may include different numbers of DPPC and DOPA2− molecules and the complexes lasted for several nanoseconds during the simulation. Additionally, one DOPA dianion can coordinate up to two Na+ ions. This led to the formation of larger structures. Ultimately, the 10049


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(5) Athenstaedt, K.; Daum, G. Phosphatidic Acid, a Key Intermediate in Lipid Metabolism. Eur. J. Biochem. 1999, 266, 1−16. (6) Steed, P. M.; Chow, A. H. M. Intracellular Signaling by Phospholipase D as a Therapeutic Target. Curr. Pharm. Biotechnol. 2001, 2, 241−256. (7) Young, B. P.; Shin, J. J. H.; Orij, R.; Chao, J. T.; Chen, S. C.; Guan, X. L.; Khong, A.; Jan, E.; Wenk, M. R.; Prinz, W. A.; et al. Phosphatidic Acid is a pH Biosensor That Links Membrane Biogenesis to Metabolism. Science 2010, 329, 1085−1088. (8) Johann, C.; Garidel, P.; Mennicke, L.; Blume, A. New Approaches to the Simulation of Heat-Capacity Curves and Phase Diagrams of Pseudobinary Phospholipid Mixtures. Biophys. J. 1996, 71, 3215−3228. (9) Garidel, P.; Johann, C.; Blume, A. Nonideal Mixing and Phase Separation in Phosphatidylcholine-Phosphatidic Acid Mixtures as a Function of Acyl Chain Length and pH. Biophys. J. 1997, 72, 2196− 2210. (10) Inoue, T.; Nibu, Y. Phase Behavior of Hydrated Lipid Bilayer Composed of Binary Mixture of Phospholipids with Different Head Groups. Chem. Phys. Lipids 1999, 100, 139−150. (11) Cambrea, L. R.; Haque, F.; Schieler, J. L.; Rochet, J.-C.; Hovis, J. S. Effect of Ions on the Organization of Phosphatidylcholine/ Phosphatidic Acid Bilayers. Biophys. J. 2007, 93, 1630−1638. (12) Lamberson, E. R.; Cambrea, L. R.; Rochet, J.-C.; Hovis, J. S. Path Dependence of Three-Phase or Two-Phase End Points in Fluid Binary Lipid Mixtures. J. Phys. Chem. B 2009, 113, 3431−3436. (13) Kooijman, E. E.; Tieleman, D. P.; Testerink, C.; Munnik, T.; Rijkers, D. T. S.; Burger, K. N. J.; de Kruijff, B. An Electrostatic/ Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins. J. Biol. Chem. 2007, 282, 11356− 11364. (14) Cheng, M. H.; Liu, L. T.; Saladino, A. C.; Xu, Y.; Tang, P. Molecular Dynamics Simulations of Ternary Membrane Mixture: Phosphatidylcholine, Phosphatidic Acid, and Cholesterol. J. Phys. Chem. B 2007, 111, 14186−14192. (15) Broemstrup, T.; Reuter, N. Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers. Biophys. J. 2010, 99, 825−833. (16) Marrink, S. J.; Lindahl, E.; Edholm, O.; Mark, A. E. Simulation of the Spontaneous Aggregation of Phospholipids into Bilayers. J. Am. Chem. Soc. 2001, 123, 8638−8639. (17) Maciejewski, A.; Pasenkiewicz-Gierula, M.; Cramariuc, O.; Vattulainen, I.; Róg, T. Refined OPLS-AA Force Field for Saturated Phosphatidylcholine Bilayers at Full Hydration. J. Phys. Chem. B 2014, 118, 4571−4581. (18) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 435−447. (19) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 1996, 118, 11225−11236. (20) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. New Developments in the Polarizable Continuum Model for Quantum Mechanical and Classical Calculations on Molecules in Solution. J. Chem. Phys. 2002, 117, 43−54. (21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; et al. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004. (22) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A WellBehaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model. J. Phys. Chem. 1993, 97, 10269−10280. (23) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935. (24) Bussi, G.; Donadio, D.; Parrinello, M. Canonical Sampling Through Velocity Rescaling. J. Chem. Phys. 2007, 126, 1−7.

greater influence on the ordering of the DPPC hydrocarbon tails, compared to the monoanionic form at the same temperature. This is likely due to the difference in interactions between two forms of DOPA and DPPC. The DOPA monoanions interact with zwitterionic lipids mainly via the formation of charge pairs between the negatively charged oxygen of DOPA and the positively charged choline groups of DPPC. An additional reason for the membrane condensation is undoubtedly related to the “umbrella model,” in which bulky headgroups of DPPC shield the hydrophobic part of DOPA (the conical shape lipid) from contact with water; as a result, the acyl chains of both lipids become tightly packed and the membrane becomes thicker. In the case of DPPC/DOPA2−, sodium cations readily adsorb at the membrane surface and form ion bridges linking strongly the lipid molecules, which results in the smaller surface area of the membrane. These observations may be important for understanding the significance of PA as a pH biosensor in cellular membranes.

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ASSOCIATED CONTENT

S Supporting Information *

DSC data. DSC heating thermograms for the DPPC/DOPA aqueous dispersions. Description of analysis of the isotherms. Surface pressure (π)−area (A) isotherms. BAM images for the DPPC/DOPA monolayers. Partial charges derived for methyl phosphate. Density profiles of selected lipid atoms along the bilayer normal DPPC bilayer 50 °C. Density profile of water along bilayer normal. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b03604.

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AUTHOR INFORMATION

Corresponding Authors

*M. Kepczynski. Tel: +48 12 6632020. Fax: +48 12 6340515. E-mail: [email protected]. *T. Róg. Tel: +358 40 198 1010. Fax: +358 3 3115 3015. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was financed by the National Science Centre Poland on the basis of decision number DEC-2012/07/B/ ST5/00913. The European Research Council (Advanced Grant project CROWDED-PRO-LIPIDS) and the Academy of Finland Center of Excellence program are thanked for financial support (W.K., T.R.). CSC − Finnish IT Centre for Scientific Computing (Espoo, Finland) is acknowledged for its computer resources.

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