Article pubs.acs.org/JPCB
Structural Degradation and Swelling of Lipid Bilayer under the Action of Benzene Alexey Odinokov*,† and Denis Ostroumov†,‡ †
Photochemistry Center of the Russian Academy of Sciences, 7a Novatorov ul., Moscow, 119421, Russia Moscow Institute of Physics and Technology, 9 Institutskiy per., Dolgoprudny, Moscow Region, 141700, Russia
‡
S Supporting Information *
ABSTRACT: Benzene and other nonpolar organic solvents can accumulate in the lipid bilayer of cellular membranes. Their effect on the membrane structure and fluidity determines their toxic properties and antibiotic action of the organic solvents on the bacteria. We performed molecular dynamics simulations of the interaction of benzene with the dimyristoylphosphatidylcholine (DMPC) bilayer. An increase in the membrane surface area and fluidity was clearly detected. Changes in the acyl chain ordering, tilt angle, and overall bilayer thickness were, however, much less marked. The dependence of all computed quantities on the benzene content showed two regimes separated by the solubility limit of benzene in water. When the amount of benzene exceeded this point, a layer of almost pure benzene started to grow between the membrane leaflets. This process corresponds to the nucleation of a new phase and provides a molecular mechanism for the mechanical rupture of the bilayer under the action of nonpolar compounds.
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INTRODUCTION Benzene and other volatile organic compounds (VOC) are common constituents in petroleum products and polymeric materials, which are ubiquitous in the present-day environment. A better understanding of their biochemical activity is of great importance in medicine and environmental control. Inside the organism, VOCs accumulate in the lipid membranes, in common with other hydrophobic compounds. Structural changes in membranes caused by the penetration of hydrophobic molecules contribute to their mode of action.1,2 It has been shown that alterations in membrane properties lead to the inhibition of the activity of embedded proteins,3 which is the case for anesthetic drugs.4−6 This mechanism provides an explanation for the effect of VOCs on the central nervous system, which includes tiredness, depression, dizziness, and so forth.7 Another practically relevant case of interaction between VOCs and biological membranes concerns the survival of bacteria in the heterogeneous medium with the mixture of water and organic solvent. Such conditions often occur in industry and in remediation of soil or groundwater polluted with organic solvents.8,9 Success in this area depends on the ability of the given bacterial strain to live and proliferate in a highly contaminated environment.10−12 With the presence of condensed organic phase in the immediate vicinity, molecules of VOC can accumulate in the cellular membrane in very high concentration, thus deteriorating its function and slowing down the growth of bacteria.13 There are many experimental © 2015 American Chemical Society
indications of the severe effect of organic molecules on both bacterial proliferation and physicochemical properties of the lipid membranes.3,14 This typically includes swelling of the membrane and increase in its fluidity and permeability, which accelerate passive transfer of hydrophilic molecules and protons. Another consequence is a dysfunction of proton pumps and other membrane-embedded proteins. As a result, intracellular homeostasis becomes impaired, which leads to inhibition of bacterial growth and cellular death. Molecular dynamics (MD) simulations regarding finite concentrations of different small molecules inside the lipid bilayer can be found in the literature. Simulations of this type have been reported for polar molecules noted for their biological activity, such as dimethyl sulfoxide,15−20 ethanol,21−23 other alcohols,19,20 and acetone.24 These studies directly illustrated the changes in lipid packing and acyl chain order appearing in lipid membrane under the influence of polar molecules and offered a molecular basis for their mode of action. Special mention must be made of the studies on chloroform,25,26 which is a prototypical anesthetic drug. Despite being hydrophobic, the chloroform molecule possesses large permanent dipole moment, which causes dramatic differences when compared to purely nonpolar compounds. This is why it cannot serve as an example of nonpolar organic solvent. Such Received: September 27, 2015 Revised: October 27, 2015 Published: November 10, 2015 15006
DOI: 10.1021/acs.jpcb.5b09420 J. Phys. Chem. B 2015, 119, 15006−15013
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DL = 8.0 ± 1.4 × 10−8 cm2 s−1, which are reasonably close to the experimental values (AL = 59.9,38 60.639 Å2 and DL = 5.95,40 9.041 × 10−8 cm2s−1). The dependence of the deuterium order parameter SCD on the position in acyl chain also agrees with experiment42 (see Figure 1).
an example can be found in ref 25, where carbon tetrachloride (CCl4) is considered as a reference molecule for comparison with chloroform. But only one simulation was performed, so neither concentration dependencies of the calculated properties nor features specific to the nonpolar compounds were reported. We can state that computer simulations of the nonpolar organic solvents inside the lipid bilayer are not available in the literature. In the present work, we have studied the effect of benzene on the equilibrium properties of lipid bilayer by means of molecular dynamics simulations. We used benzene as a model volatile organic molecule, which is also an abundant environmental pollutant. We aimed to analyze alteration of the structure of the membrane after addition of gradually increasing concentration of benzene. Special care was taken to investigate a wide range of concentrations, including the “saturated” regime, when the amount of benzene dissolved in water exceeds the solubility limit. This allowed us to take into consideration the case when the system was supposed to be in close contact with the condensed phase of benzene, as well as when it was exposed to the vapor. The results provide a glimpse of the processes leading to the structural degeneration and rupture of the cellular membrane.
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COMPUTATIONAL METHODOLOGY Molecular dynamics simulations were carried out for the system that included lipid bilayer consisting of dimyristoylphosphatidylcholine (DMPC) molecules and water. It is not a trivial task to reproduce experimentally measured properties of lipid membranes. The equilibrium in the system depends on the fine features of the numerous intra- and intermolecular interactions, inspiring many research groups to develop special force fields for lipid simulations. We used the recent CHARMM36 force field for lipids,27 which represented a significant improvement in this area.28 We also used the CHARMM General force field29 for benzene and TIP3P30 water model modified for CHARMM force field.31 Lipid membrane (with benzene molecules inside it) and water were separately coupled to Nose−Hoover32 thermostat with reference temperature of 303 K and a time constant of 1.0 ps. The system was also affected by semiisotropic Parrinello− Rahman33 barostat with reference pressure of 1.0 atm, time constant of 5.0 ps, and isothermal compressibility of 4.5 × 10−5 bar−1 in both lateral and normal directions. The lengths of all covalent bonds involving hydrogen atoms were kept fixed via LINCS34 algorithm, which made it possible to use time step of 2 fs. The cut-off distance of long-range interactions was 1.2 nm, with Van der Waals forces being switched to zero between 1.0 and 1.2 nm. The long-range part of the electrostatic potential was treated via PME35 method. In all simulations, we used GROMACS-5.0.536 program package obtaining input files with the aid of CHARMM-GUI37 web tool. Because the original CHARMM36 force field was developed and tested using CHARMM software, it was vital to ensure the applicability of our computational environment in the context of the present problem. To achieve this, we calculated equilibrium properties of DMPC lipid bilayer in pure water. The initial configuration of the membrane consisted of 72 lipid molecules in disordered state and 1848 water molecules was borrowed from the web page of Dr. R. Pastor, available at http://www.lobos.nih.gov/ mbs. The system was equilibrated for 40 ns, after which simulation continued for 100 ns. The equilibrium value of the average membrane area per lipid was found to be AL = 59.7 ± 1.3 Å2, and lateral diffusion coefficient of the lipid molecule was
Figure 1. Deuterium order parameters from MD simulations and experiment.42 Change of the benzene content from 0.00 to 3.56 molecules per lipid is indicated by an arrow.
After we confirmed the reproducibility of experimental results, we started to introduce the desired number of benzene molecules in the equilibrium configuration of the membrane. We placed the molecules at the random points and with random orientations inside the hydrophobic region occupied by DMPC tails. The geometry of the system was optimized, and the equilibrium was reached in 3 steps. First, we performed short (10 ps) relaxation keeping volume fixed. Second, we allowed the box to expand over the period of 50 ps in the constant pressure (NPT) simulation with a Berendsen barostat.43 Finally, the long NPT simulation with regular parameters was run, which was then divided into two parts (relaxation and data production). We repeated this procedure for different amounts of benzene in the system, namely, 12, 24, 36, 48, 60, 94, 128, 192, and 256 molecules. Further, we will denote the benzene content inside the bilayer as xbenz, which is equal to the number of benzene molecules per one lipid molecule. In order to test reliability of the results, two simulations with independent starting configurations were performed for each benzene concentration. To prove the independence of our results on the initial configuration, we created a box containing 12 benzene molecules located randomly in the water region. During MD simulation, these molecules moved to the hydrophobic membrane interior due to the passive diffusion, and the resulting density profile of benzene coincided with the profile obtained previously within the limits of statistical uncertainty (see Figure S2 in the Supporting Information). Relatively small models of lipid membranes available in the computer simulations often give rise to large fluctuations of instantaneous values of dynamical variables. Special attention should be given to checking for convergence when averaging them over an ensemble. Therefore, we conducted 100 ns simulations for the cases of xbenz = 0.17 and xbenz = 3.56. We chose AL, SCD, and benzene distribution as the representative 15007
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profile along the normal to the bilayer plane (see Figure 3). Increase in benzene concentration is followed by the dramatic
parameters describing current configuration of the lipid bilayer and monitored their time dependence. Any visible signs of nonequilibrium behavior vanished during first 20 ns of the MD trajectory. We rejected this part and estimated the statistical errors by block-averaging method.44 In both systems, averaging over the period of at most 30 ns was sufficient to achieve reasonable statistical accuracy (see Figure S1 in the Supporting Information). On the basis of these results, we set the length of the MD trajectories to 50 ns in all subsequent simulations and used only the last 30 ns to perform data sampling and analysis. We also tested the impact of system size on the equilibrium properties of the lipid bilayer. We constructed a large box consisting of 288 lipid molecules via stacking four small boxes in the bialyer plane and relaxing the system in NPT simulation until it lost its periodicity. We calculated membrane surface area and benzene partitioning for xbenz = 0, 1.31, and 3.56. The values obtained with large box were very close to the results obtained in regular calculations (see Results and Discussion), thus validating the use of small box, which was crucial to produce numerous simulations over a wide range of benzene concentrations.
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Figure 3. Mass density profiles for different moieties. Increase in the benzene content from 0.00 to 3.56 molecules per lipid is indicated by arrows.
RESULTS AND DISCUSSION Accumulation of Benzene Inside the Bilayer. In the beginning of our MD simulations benzene molecules were placed inside the hydrophobic part of the lipid bilayer and, for the most part, remained there during the simulation. Even with the maximum number of benzene molecules in the simulation box, we observed only 1−2 of them residing in water at any specific time. Resulting structures are presented in Figure 2 for the cases of pure water, maximum concentration of benzene (xbenz = 3.56), and some intermediate concentration (xbenz = 0.5). Equilibrium distribution of benzene and other moieties inside the lipid bilayer can be described by the mass density
growth of the benzene peak and membrane swelling, which corresponds to the shift of water and phosphorus profiles from the center of bilayer. The essential feature of mass density profile of benzene shown in Figure 3 is the distinct three-peak structure. The side peaks reflect dissolution of the benzene in two leaflets of the membrane, with benzene molecules located between acyl chains. The central peak occurs when benzene molecules infiltrate the interstice between the leaflets. The relative height of the central peak increases with the benzene concentration. It can be seen in Figure 3 that for the systems containing a large amount of benzene, mass density of lipid tails has a deep well near the center of bilayer. Two membrane leaflets become divided by the film of almost pure benzene. It means that the boundary between lipid monolayers can serve as a condensation nucleus for the new phase. Condensation occurs when the concentration of benzene in the surrounding water reaches the solubility limit. By dividing lipid monolayers, benzene disturbs the stability of bilayer and facilitates the rupture of the membrane. The same effect was confirmed experimentally by Mishima et al.,2 who observed the rupture of DMPC vesicles when the amount of carbon tetrachloride in water exceeded the limit of solubility. Our simulations provide direct illustration of this effect for the case of benzene. Main conclusions can be extrapolated to some extent to other organic nonpolar solvents. In order to describe numerically the growth of benzene film between membrane leaflets, we used a simple computational trick. We performed least-squares fit of the benzene density by three Gaussian functions (see Figure S2 in the Supporting Information). The coefficients of this fit correspond to the number of benzene molecules inside the central region and inside the monolayers. The results are shown in Figure 4 as a function of the total benzene content. With a small amount of benzene molecules, they are distributed almost equally between central and side regions (within the large relative error). However, when the number of molecules exceeds some value, the partitioning between central and side regions starts to
Figure 2. Typical configurations of the lipid bilayer with 0.00 (a), 0.50 (b), and 3.56 (c) benzene molecules per lipid. Phosphatidylcholine groups are shown with pink, myristoyl tails with cyan, and benzene molecules with yellow. 15008
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Figure 5. Dependence of the membrane surface area per lipid on the benzene content. Filled markers correspond to the total amount of benzene (results for two independent sets of simulations are presented). Open squares mean effective number of benzene molecules contained inside the leaflets. Red diamonds represent the results obtained with large simulation box.
Figure 4. Benzene content in the leaflets and between them. Circles and squares denote two independent sets of simulations. Red crosses indicate the results obtained with large simulation box.
diverge. The population of monolayers approaches saturation and the slope of the corresponding curve becomes more shallow, while the population of interlayer boundary increases even faster. It is interesting to compare the point where curves in Figure 4 begin to diverge with the equilibrium content of benzene at the limit of solubility in water. In this case, the number of benzene molecules in the lipid bilayer is satur xbenz = swatP
the direction normal to the bilayer plane. The sign of the latter effect depends on the ordering of the lipid chains and is hard to be predicted a priori. In the present work, we define the thickness of the bilayer as a distance δP−P between the peaks of the mass density of phosphorus atoms (see Figure 3). Lipid chain ordering can be characterized by the tilt angle θt, which is the angle between the bilayer normal and the vector connecting the first and the last atoms of the acyl chain. One more useful quantity is the average deuterium order parameter SCD defined as
NH2O m H2O Vlip Nlip mbenz VH2O
(1)
where swat is the benzene solubility limit, P is oil−water partition coefficient of benzene, NH2O and Nlip represent the number of water and lipid molecules inside the simulation box, mH2O and mbenz are the masses of water and benzene molecules, Vlip and VH2O mean average volumes occupied by lipids and water. Substituting reference values at 25 °C (swat = 0.178 mass %45 and log P = 2.1346) in eq 1, one can obtain xsatur benz ≈ 1.4, which is in remarkable agreement with Figure 4. This coincidence strengthens our interpretation of the observed phenomena and implicitly demonstrates the validity of the chosen force field for the description of phase equilibrium in the complex heterogeneous system which we consider in this work. Changes in the Structure and Viscosity of the Lipid Bilayer. One of the main consequences of the accumulation of benzene is membrane swelling. It can be described in terms of the increase of the surface area per lipid molecule AL, which is shown in Figure 5. It should be noted that if we plot along the x-axis not the total number of benzene molecules but an effective number contained inside the leaflets, then the dependence become linear with the slope 15.8 Å2 (molecule per lipid)−1. This indicates that benzene and lipid tails inside the monolayer mix like two ideal liquids, whereas the excess of benzene between the monolayers does not influence the expansion of the bilayer in lateral directions. Alteration of the membrane thickness under the action of benzene is expected to be governed by two different factors. First, the growing layer of benzene moves the membrane leaflets apart. Second, the thickness of monolayers changes following the change in the averaged size of lipid molecule in
⟨3cos2 θ − 1⟩ (2) 2 where θ is the angle between C−H bond and the bilayer normal, and ⟨...⟩ means averaging over MD trajectory and over all carbon atoms in the acyl chain. The dependence of δP−P, θt, and SCD on the amount of benzene is presented in Figure 6. It can be clearly seen that the resulting effect in this case is hidden to a considerable extent by the statistical fluctuations of large magnitude. The deviation from the pure water certainly exceeds standard error only for the samples with very high concentration of benzene. The membrane thickness shows a slight upward trend, which can be attributed to the accumulation of benzene between the leaflets. The impact of chain reordering is opposite. The average value of the tilt angle increases (if we exclude one outlier), implying stronger deviation of the lipid tails from the bilayer normal. The absolute value of SCD slightly decreases, also indicating the formation of a less-ordered structure. However, a slight thinning of the leaflets cannot overcome the upward trend in membrane thickness. It is interesting to compare the behavior of an average order parameter with the detailed picture of SCD for every carbon atom in acyl chain shown in Figure 1. It can be seen that statistical fluctuations occur mainly in the part of the chain adjacent to the headgroup (left half of Figure 1). The other part of the curve decreases almost monotonically. Lesser statistical uncertainties originate from the higher fluidity of the inner membrane region, where the acyl chains can move freely. The presence of a low-molecular-weight component (benzene) SCD =
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Figure 7. Translational (lateral) and rotational diffusion coefficients of lipid molecules as a function of the benzene content. Squares and circles denote two independent sets of simulations. Figure 6. Dependence of the membrane thickness δP−P, tilt angle θt, and mean deuterium order parameter SCD on the benzene content. Squares and circles denote two independent sets of simulations.
ion pair (see Figure 8). Distance between phosphorus atoms exhibits broader, double-peaked distribution. Here we make no distinction between these peaks and assume that every pair of phosphorus atoms separated by the distance less than 10 Å corresponds to the first coordination sphere. Presence of benzene changes RDFs (see Figure 8). While P−P distribution, especially the first peak, decreases, the peak of N−N RDF becomes even higher. Taking into account this anomalous behavior and the fact that the positions of minima and maxima of the RDF remains the same with and without benzene, one may conclude that despite the overall expansion of the membrane, this process is not homogeneous. The net of mutual connections between charged head groups maintains the bond length but changes the coordination number. This is illustrated in the top of Figure 8. Lipid head groups are presented as rigid spheres with diameter equal to the closest approach distance between phosphorus atoms (depicted as small circles inside the spheres). All head groups separated by 10 Å or less are connected with lines, so chains and clusters of connected head groups are visible. After addition of benzene these chains become longer and the size of clusters decreases. The increase of the membrane surface area is related to the growth of “holes” between the connected fragments (filled area in Figure 8). These observations have relation to the problem of passive transport of protons and ions across the lipid membrane. Passive transport of charged and hydrophilic molecules through the hydrophobic region of the lipid bilayer proceeds via the preliminary formation of water pathways due to the fluctuations of lipid tails. Although these events are extremely rare under normal conditions, structural degradation of lipid membrane after addition of benzene can favor spontaneous formation of the hydrophilic pores greatly
further facilitates relaxation, causing much better convergence for the averaging over the same ensemble. Structural changes in the lipid bilayer are accompanied by the changes in dynamical properties, such as microviscosity, which depends on the local mobility of lipids. Neglecting flip-flop transitions, we assume that relaxation of membrane structure involves translational and rotational degrees of freedom of individual lipid molecules. We describe these kinds of motions with lateral DL and rotational Drot diffusion coefficients. We extracted the value of DL from the linear fit of the time dependence of mean square displacement of lipids in the bilayer plane, whereas for the estimation of Drot, we used the vector connecting carbon and oxygen atoms of the carbonyl group. The results are presented in Figure 7. It can be seen that benzene has a greater effect on the microviscosity than on the static properties. For the largest benzene content, lateral diffusion of lipids is accelerated by almost an order of magnitude, and rotational diffusion becomes twice as fast as in pure water. This dramatic increase is evident despite large fluctuations. Increased fluidity is therefore expected to play an important role in the dysfunction of cellular membrane under the influence of benzene, which is in good agreement with experimental observations.14 Another interesting feature of the membrane swelling concerns an ordering of the lipid head groups originating from the interactions between oppositely charged groups of the phosphatidylcholine moieties. Radial distribution function in the bilayer plane (RDF) between phosphorus and nitrogen atoms has a sharp peak at 4 Å that indicates the formation of 15010
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this angle gradually decreases with increasing benzene content (see Figure 8), indicating more vertical orientation of the head groups. The direction of this change is the same as reported in ref 25 for chloroform and CCl4 in the bilayer consisted of unsaturated lipids. Comparison of Benzene with Other Solvents. A nonpolar, hydrophobic molecule of benzene has no permanent dipole moment. This determines preferential accumulation of benzene between the membrane leaflets. “Amphiphilic” compounds (such as alcohols or acetone24) accumulate primarily near the boundary between the hydrophobic and hydrophilic parts of the lipid bilayer, where their polar groups interact with polar lipid head groups and water. The same is the case for the hydrophobic molecules possessing significant permanent dipole moment. In ref 25, this difference is illustrated through the example of chloroform and carbon tetrachloride (CCl4). Although these molecules have a similar chemical structure, they are distributed inside the membrane in a qualitatively different manner due to the large difference in their dipole moments. If the substance tends to accumulate near the hydrophobic/hydrophilic interface, then this process can lead to a decrease in the membrane thickness.24 In the case of benzene, we observed the opposite effect. The growing layer of benzene monotonically increases the thickness of hydrophobic region. On the one hand, this should prevent the membrane from losing the barrier function against passive transport of ions. On the other hand, the benzene layer moves membrane leaflets apart, thus promoting mechanical rupture of the bilayer. In assessing the results reported in the present work, as well as presented in the literature, it is important to take into account uncertainties emerging from the averaging over MD trajectories of finite length. Estimation of these errors was the main reason for doing systematic analysis of the influence of benzene on the membrane properties, which included many data points and covered a wide range of concentrations. Different properties behaved differently in this respect. Surface area per lipid (see Figure 5) and benzene partitioning (see Figure 4) showed very clear dependence. Lateral and rotational diffusion coefficients (see Figure 7) suffered from statistical fluctuations, which were, however, not able to mask the main effect due to its large magnitude. Lastly, the mean values of SCD, δP−P, and θt were comparable with fluctuations. In this case, the direction and the value of the impact of benzene can be confirmed only if the benzene content exceeds the saturation limit xsatur benz ≈ 1.4. We conclude that for a similar numerical experiment, one can not be sure of the results if only one (or very few) data points are presented and the system size and trajectory length are comparable with that used in the present work. Unfortunately, it is often the case in the studies on the accumulation of small molecules inside the lipid membranes, which were cited above. Moreover, among the molecules represented in these studies, only CCl4 can be considered as an analogue of benzene. For this compound, an increase in acyl chain ordering for liquid phase was reported,25 which contradicts our observations of the changes in the deuterium order parameter SCD and tilt angle θt. The question remains whether this discrepancy originates from the difference between the systems or it is just a consequence of a random fluctuation in the numerical experiment. Apart from the practical importance of its physiological effects, we used benzene as a typical example of nonpolar organic molecule. Accumulation of nonpolar molecules within
Figure 8. Changes of the headgroup arrangement upon benzene accumulation. (a) Schematic representation of the headgroup positions for pure water (left) and maximum benzene content (right), see explanation in the text. (b) Phosphorus−phosphorus (left) and phosphorus−nitrogen (right) radial distribution functions in the bilayer plane. Shift of the main peak after addition of benzene is denoted by arrow. (c) Average angle of the P−N vector relative to the bilayer normal.
accelerating the passive transport, as is the case for DMSO16 and ethanol.23 Growing “holes” between the lipid head groups seem to be well suited to serve as possible sites for these pores. However, we have not detected any additional water clusters inside the hydrophobic region during MD simulations even with the presence of the large number of benzene molecules. This conclusion is confirmed by the unchanged profile of the water mass density in Figure 3. In our opinion, the possibility of the formation of hydrophilic pores inside the membrane at the high concentrations of benzene is still open to question. Although similar events were observed via brute force MD simulations for polar solvents,16,23 in the case of nonpolar solvent they can be far more rare but still capable of providing the way for passive transport of ions. This provides an explanation for the increase in the passive proton flux across the liposomal membrane that has been measured experimentally.14 Arrangement of the head groups can also be characterized by the angle θPN between the vector connecting phosphorus and nitrogen atoms and the bilayer normal. The average value of 15011
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also thank Prof. V. Livshits for careful reading and discussion of the manuscript.
lipid membrane does not require any specific interactions besides the hydrophobic attraction. We suggest that the main conclusions of the present work can be extrapolated to the other compounds from this group. There are some experimental indications that this hypothesis is valid. First of all, the same toxic effect on living bacteria was reported for different organic solvents.47 Moreover, the changes in physicochemical properties of liposomes were proved to be dependent only on the oil−water partition coefficient of the particular solvent but not on the chemical structure of the molecule.14 We cannot, however, rule out the possibility that the chemical nature of the solvent may affect the details of lipid chain ordering, which showed moderate and ambiguous dependence in the case of benzene.
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(1) Dreiem, A.; Myhre, O.; Fonnum, F. Involvement of the Extracellular Signal Regulated Kinase Pathway in HydrocarbonInduced Reactive Oxygen Species Formation in Human Neutrophil Granulocytes. Toxicol. Appl. Pharmacol. 2003, 190, 102−110. (2) Mishima, K.; Watanabe, H.; Kaneko, S.; Ogihara, T. Membrane Disordering Induced by Chloroform and Carbon Tetrachloride. Colloids Surf., B 2003, 28, 307−312. (3) Sikkema, J.; de Bont, J.; Poolman, B. Mechanisms of Membrane Toxicity of Hydrocarbons. Microbiol. Rev. 1995, 59, 201−222. (4) Seeman, P.; Kwant, W.; Sauks, T.; Argent, W. Membrane Expansion of Intact Erythrocyte by Anesthetics. Biochim. Biophys. Acta, Biomembr. 1969, 183, 490−498. (5) Engelke, M.; Diehl, H.; Tahti, H. Effects of Toluene and nHexane on Rat Synaptosomal Membrane Fluidity and Integral Enzyme Activities. Pharmacol. Toxicol. 1992, 71, 343−347. (6) Turkyilmaz, S.; Chen, W.-H.; Mitomo, H.; Regen, S. L. Loosening and Reorganization of Fluid Phospholipid Bilayers by Chloroform. J. Am. Chem. Soc. 2009, 131, 5068−5069. (7) Wilbur, S.; Bosch, S. Interaction Profile for: Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX); Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services, Public Health Service: Atlanta, GA, 2004. (8) Sardessai, Y.; Bhosle, S. Industrial Potential of Organic Solvent Tolerant Bacteria. Biotechnol. Prog. 2004, 20, 655−660. (9) Darracq, G.; Couvert, A.; Couriol, C.; Amrane, A.; Le Cloirec, P. Removal of Hydrophobic Volatile Organic Compounds in an Integrated Process Coupling Absorption and Biodegradation − Selection of an Organic Liquid Phase. Water, Air, Soil Pollut. 2012, 223, 4969−4997. (10) Kobayashi, H.; Uematsu, K.; Hirayama, H.; Horikoshi, K. Novel Toluene Elimination System in a Toluene-Tolerant Microorganism. J. Bacteriol. 2000, 182, 6451−6455. (11) Gao, Y.; Dai, J.; Peng, H.; Liu, Y.; Xu, T. Isolation and Characterization of a Novel Organic Solvent-Tolerant Anoxybacillus sp. PGDY12, a Thermophilic Gram-Positive Bacterium. J. Appl. Microbiol. 2011, 110, 472−478. (12) Azmatunnisa, M.; Rahul, K.; Lakshmi, K.; Sasikala, C.; Ramana, C. Lysinibacillus Acetophenoni sp. nov., a Solvent-Tolerant Bacterium Isolated from Acetophenone. Int. J. Syst. Evol. Microbiol. 2015, 65, 1741−1748. (13) Lăzăroaie, M. Multiple Responses of Gram-Positive and GramNegative Bacteria to Mixture of Hydrocarbons. Brazilian Journal of Microbiology 2010, 41, 649−667. (14) Sikkema, J.; de Bont, J.; Poolman, B. Interactions of Cyclic Hydrocarbons with Biological Membranes. J. Biol. Chem. 1994, 269, 8022−8028. (15) Notman, R.; Noro, M.; O’Malley, B.; Anwar, J. Molecular Basis for Dimethylsulfoxide (DMSO) Action on Lipid Membranes. J. Am. Chem. Soc. 2006, 128, 13982−13983. (16) Gurtovenko, A. A.; Anwar, J. Modulating the Structure and Properties of Cell Membranes: The Molecular Mechanism of Action of Dimethyl Sulfoxide. J. Phys. Chem. B 2007, 111, 10453−10460. (17) Dabkowska, A. P.; Foglia, F.; Lawrence, M. J.; Lorenz, C. D.; McLain, S. E. On the Solvation Structure of Dimethylsulfoxide/Water around the Phosphatidylcholine Head Group in Solution. J. Chem. Phys. 2011, 135, 225105. (18) Hughes, Z. E.; Mark, A. E.; Mancera, R. L. Molecular Dynamics Simulations of the Interactions of DMSO with DPPC and DOPC Phospholipid Membranes. J. Phys. Chem. B 2012, 116, 11911−11923. (19) Malajczuk, C. J.; Hughes, Z. E.; Mancera, R. L. Molecular Dynamics Simulations of the Interactions of DMSO, Mono- and Polyhydroxylated Cryosolvents with a Hydrated Phospholipid Bilayer. Biochim. Biophys. Acta, Biomembr. 2013, 1828, 2041−2055. (20) Hughes, Z. E.; Malajczuk, C. J.; Mancera, R. L. The Effects of Cryosolvents on DOPC-β-Sitosterol Bilayers Determined from
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CONCLUSIONS The majority of published studies on the computer simulations of accumulation of small molecules inside the lipid bilayer concern either “amphiphilic” compounds (such as dimethyl sulfoxide, alcohols and acetone) or hydrophobic molecules possessing large permanent dipole moment (chloroform). Therefore, a key contribution of our research is a systematic analysis of the influence of varying concentrations of nonpolar organic solvent on the model lipid membrane. This connects our numerical experiment to the problem of the toxicity of aromatic compounds and provides a direct link to the experimental studies devoted to the bactericidal action of high concentrations of organic solvents against microorganisms. Molecular dynamics simulations allowed for a numerical description of the membrane swelling and increase in fluidity, which is responsible for the loss of biologically relevant functions. Of particular interest is an observation of the accumulation of benzene between the membrane leaflets, which leads to the formation of a new phase if the amount of benzene exceeds the solubility limit. This behavior appears to be typical for the nonpolar organic compounds.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b09420. Results of the convergence tests for surface area, chain order parameter, and benzene distribution over the 100 ns MD trajectories have been provided; a comparison of benzene density profiles obtained with different types of initial configurations and an example of curve fitting have also been included (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation, contract no. 15-13-00163. The computational facilities were provided by the Joint Supercomputer Center of the Russian Academy of Sciences. The authors are grateful to Prof. J. Klauda and Dr. R. Pastor for their advice during the preparation of the initial configuration of the membrane and simulation setup. We 15012
DOI: 10.1021/acs.jpcb.5b09420 J. Phys. Chem. B 2015, 119, 15006−15013
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The Journal of Physical Chemistry B Molecular Dynamics Simulations. J. Phys. Chem. B 2013, 117, 3362− 3375. (21) Terama, E.; Ollila, O. H. S.; Salonen, E.; Rowat, A. C.; Trandum, C.; Westh, P.; Patra, M.; Karttunen, M.; Vattulainen, I. Influence of Ethanol on Lipid Membranes: From Lateral Pressure Profiles to Dynamics and Partitioning. J. Phys. Chem. B 2008, 112, 4131−4139. (22) Gurtovenko, A. A.; Anwar, J. Interaction of Ethanol with Biological Membranes: The Formation of Non-Bilayer Structures within the Membrane Interior and their Significance. J. Phys. Chem. B 2009, 113, 1983−1992. (23) Thind, R.; O’Neill, D.; Del Regno, A.; Notman, R. Ethanol Induces the Formation of Water-Permeable Defects in Model Bilayers of Skin Lipids. Chem. Commun. 2015, 51, 5406−5409. (24) Posokhov, Y.; Kyrychenko, A. Effect of Acetone Accumulation on Structure and Dynamics of Lipid Membranes Studied by Molecular Dynamics Simulations. Comput. Biol. Chem. 2013, 46, 23−31. (25) Reigada, R. Influence of Chloroform in Liquid-Ordered and Liquid-Disordered Phases in Lipid Membranes. J. Phys. Chem. B 2011, 115, 2527−2535. (26) Vorobyov, I.; Bennett, W.; Tieleman, D.; Allen, T.; Noskov, S. The Role of Atomic Polarization in the Thermodynamics of Chloroform Partitioning to Lipid Bilayers. J. Chem. Theory Comput. 2012, 8, 618−628. (27) Klauda, J. B.; Venable, R. M.; Freites, A.; O’Connor, J. W.; Tobias, D. J.; Mondragon-Ramirez, C.; Vorobyov, I.; MacKerell, A. D., Jr.; Pastor, R. W. Update of the CHARMM All-Atom Additive Force Field for Lipids: Validation on Six Lipid Types. J. Phys. Chem. B 2010, 114, 7830−7843. (28) Venable, R. M.; Brown, F. L.; Pastor, R. W. Mechanical Properties of Lipid Bilayers from Molecular Dynamics Simulation. Chem. Phys. Lipids 2015, DOI: 10.1016/j.chemphyslip.2015.07.014. (29) Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; et al. CHARMM General Force Field: A Force Field for Drug-Like Molecules Compatible with the CHARMM all-Atom Additive Biological Force Fields. J. Comput. Chem. 2009, 31, 671−690. (30) 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. (31) Durell, S.; Brooks, B.; Ben-Naim, A. Solvent-Induced Forces between Two Hydrophilic Groups. J. Phys. Chem. 1994, 98, 2198− 2202. (32) Nosé, S. A. Molecular Dynamics Method for Simulations in the Canonical Ensemble. Mol. Phys. 1984, 52, 255−268. (33) Parrinello, M.; Rahman, A. Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method. J. Appl. Phys. 1981, 52, 7182−7190. (34) Hess, B.; Bekker, H.; Berendsen, H.; Fraaije, J. LINCS: A Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463−1472. (35) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An Nlog(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. (36) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A.; Berendsen, H. GROMACS: Fast, Flexible and Free. J. Comput. Chem. 2005, 26, 1701−1718. (37) Jo, S.; Kim, T.; Iyer, V.; Im, W. CHARMM-GUI: A Web-based Graphical User Interface for CHARMM. J. Comput. Chem. 2008, 29, 1859−1865. (38) Kučerka, N.; Nieh, M.-P.; Katsaras, J. Fluid Phase Lipid Areas and Bilayer Thicknesses of Commonly Used Phosphatidylcholines as a Function of Temperature. Biochim. Biophys. Acta, Biomembr. 2011, 1808, 2761−2771. (39) Kučerka, N.; Liu, Y.; Chu, N.; Petrache, H.; Tristram-Nagle, S.; Nagle, J. Structure of Fully Hydrated Fluid Phase DMPC and DLPC Lipid Bilayers Using X-ray Scattering from Oriented Multilamellar Arrays and from Unilamellar Vesicles. Biophys. J. 2005, 88, 2626− 2637.
(40) Almeida, P.; Vaz, W.; Thompson, T. Lateral Diffusion in the Liquid Phases of Dimyristoylphosphatidylcholine/Cholesterol Lipid Bilayers: a Free Volume Analysis. Biochemistry 1992, 31, 6739−6747. (41) Filippov, A.; Orädd, G.; Lindblom, G. Influence of Cholesterol and Water Content on Phospholipid Lateral Diffusion in Bilayers. Langmuir 2003, 19, 6397−6400. (42) Douliez, J.-P.; Léonard, A.; Dufourc, E. J. Restatement of Order Parameters in Biomembranes: Calculation of C-C Bond Order Parameters from C-D Quadrupolar Splittings. Biophys. J. 1995, 68, 1727−1739. (43) Berendsen, H.; Postma, J.; van Gunsteren, W. F.; DiNola, A.; Haak, J. Molecular Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684−3690. (44) Hess, B. Determining the Shear Viscosity of Model Liquids from Molecular Dynamics Simulations. J. Chem. Phys. 2002, 116, 209−217. (45) Shiu, W.-Y.; Ma, K.-C. Temperature Dependence of PhysicalChemical Properties of Selected Chemicals of Environmental Interest. I. Mononuclear and Polynuclear Aromatic Hydrocarbons. J. Phys. Chem. Ref. Data 2000, 29, 41. (46) Sangster, J. Octanol-Water Partition Coefficients of Simple Organic Compounds. J. Phys. Chem. Ref. Data 1989, 18, 1111−1227. (47) Kieboom, J.; Dennis, J. J.; Zylstra, G. J.; de Bont, J. A. Active Efflux of Organic Solvents by Pseudomonas Putida S12 Is Induced by Solvents. J. Biol. Chem. 1998, 180, 6769−6772.
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DOI: 10.1021/acs.jpcb.5b09420 J. Phys. Chem. B 2015, 119, 15006−15013
