A Ring to Rule Them All: The Effect of ... - ACS Publications

Article pubs.acs.org/JPCB

A Ring to Rule Them All: The Effect of Cyclopropane Fatty Acids on the Fluidity of Lipid Bilayers David Poger*,† and Alan E. Mark†,‡ †

School of Chemistry and Molecular Biosciences and ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia S Supporting Information *

ABSTRACT: Cyclopropane fatty acids are widespread in bacteria. As their concentration increases on exposure to hostile environments, they have been proposed to protect membranes. Here, the effect of cyclopropane and unsaturated fatty acids, both in cis and trans configurations, on the packing, order, and fluidity of lipid bilayers is explored using molecular dynamics simulations. It is shown that cyclopropane fatty acids disrupt lipid packing, favor the occurrence of gauche defects in the chains, and increase the lipid lateral diffusion, suggesting that they enhance fluidity. At the same time, they generally induce a greater degree of order than unsaturated fatty acids of the same configuration and limit the rotation about the bonds surrounding the cyclopropane ring. This indicates that cyclopropane fatty acids may fulfill a dual function: stabilizing membranes against adverse conditions while simultaneously promoting their fluidity. Marked differences in the effect of cis- and trans-monocyclopropanated fatty acids were also observed, suggesting that they may play alternative roles in membranes.

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INTRODUCTION Biological membranes consist of a lipid matrix wherein other membrane components are embedded. Lipids are multifarious molecules and may include saturated, unsaturated, straight, branched, cycloalkylated, or functionalized (e.g., hydroxylated and brominated) fatty acids.1 Among the various branched fatty acids, some incorporate a cyclopropane ring in their hydrocarbon chain. Cyclopropane fatty acids are broadly distributed in a variety of organisms, in particular bacteria such as Escherichia coli, Streptococcus, and Salmonella, where they are derived from the corresponding cis-unsaturated fatty acids by in situ methylenation of the double bond.2 While the cyclopropane fatty acid synthesis pathway in bacteria has been the most extensively examined in E. coli, it seems that natural cyclopropane fatty acids generally only occur with a cis configuration about the cyclopropane moiety. Although trans-cyclopropane fatty acids are much less widespread, they are common in the cell envelope of Mycobacterium tuberculosis where they are found alongside cis-cyclopropane fatty acids.3 Fatty acid cyclopropanation occurs in bacteria in response to exposure to adverse environmental conditions.2,4 Specifically, an increase in the concentration of cyclopropane fatty acids in the membrane of a number of bacteria has been associated with tolerance to high osmotic pressure,5−8 high temperature,5,9 low pH,10−14 nutrient deprivation,15,16 and high alcohol concentrations.12,14,17,18 Cyclopropane fatty acids have also been shown to play a role in pathogenesis. Cyclopropanation of mycolic acids, a major component of the cell envelope in M. tuberculosis, is correlated with the persistence of the pathogen. The presence of cyclopropanated mycolic acids also modulates the innate immune response of the host.19,20 The stereochemistry of the cyclopropane ring is critical. It is trans-cyclopropanated mycolic © 2015 American Chemical Society

acids that play a role in regulating and restricting the virulence of M. tuberculosis.21 Although significant variations in the membrane content of cyclopropane fatty acids have been identified in a multitude of physiological situations, little is known regarding the actual role these fatty acids play in membranes. The presence of cyclopropane fatty acids is generally assumed to enhance the chemical and physical stability of membranes. For example, double bonds are particularly sensitive to oxidation. Their methylenation has been hypothesized to improve resistance to superoxide, singlet oxygen, ozonolysis, and oxidative stress in general.2 It has also been suggested that cyclopropane fatty acids may reduce the fluidity of membranes, thereby limiting their permeability to undesirable compounds ranging from a single proton12 to butan-1-ol17 and possibly even some antibiotics.22 Experimentally, cyclopropane fatty acids have been shown to decrease the mobility of reporter molecules within a membrane and to increase the tightness of packing within lipid bilayers.23−25 The extent to which cis-cyclopropane fatty acids modify the packing of lipids within membranes is intermediate between the levels obtained by cis- and trans-unsaturated fatty acids.23 In general, lipid bilayers containing cyclopropane fatty acids display greater chain order than their unsaturated analogues.24,26−29 Like unsaturated fatty acids, cis-cyclopropane fatty acids tend to be less ordered than their trans isomers.30 Based on nuclear magnetic resonance (NMR) relaxation measurements, it has been proposed that the cyclopropane moiety behaves like a Received: January 29, 2015 Revised: March 23, 2015 Published: March 24, 2015 5487

DOI: 10.1021/acs.jpcb.5b00958 J. Phys. Chem. B 2015, 119, 5487−5495

Article

The Journal of Physical Chemistry B

Figure 1. Structures of the lipids investigated: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; PePPC, 1-palmitelaidoyl-2-palmitoyl-sn-glycero-3phosphocholine; PoPPC, 1-palmitoleoyl-2-palmitoyl-sn-glycero-3-phosphocholine; PlePPC, 1-palmitolinoelaidoyl-2-palmitoyl-sn-glycero-3-phosphocholine; PloPPC, 1-palmitolinoleoyl-2-palmitoyl-sn-glycero-3-phosphocholine; cp(c)PPPC, 1-[(9R,10S)-9,10-methanopalmitoyl]-2-palmitoyl-snglycero-3-phosphocholine; cp(c)2PPPC, 1-[(9R,10S,12R,13S)-9,10:12,13-dimethanopalmitoyl]-2-palmitoyl-sn-glycero-3-phosphocholine; cp(t)PPPC, 1-[(9S,10S)-9,10-methanopalmitoyl]-2-palmitoyl-sn-glycero-3-phosphocholine; cp(t) 2 PPPC, 1-[(9S,10S,12S,13S)-9,10:12,13dimethanopalmitoyl]-2-palmitoyl-sn-glycero-3-phosphocholine.

cyclopropane fatty acids enhanced the fluidity of lipid bilayers by altering lipid packing and favoring the formation of gauche conformers in the acyl chains while increasing the degree of order along the chains and restricting the rotation about the bonds flanking the cyclopropane moiety. The simulations suggest that the impact of cyclopropane fatty acids on the fluidity is partly due to a steric hindrance introduced by the cyclopropane moiety.

barrier and prevents the propagation of motion from one end of the acyl chain to the other, and postulated that this gives rise to the properties of cyclopropane-fatty-acid-containing membranes.28 However, exactly how the effect of cyclopropane fatty acids compares to their unsaturated counterparts remains unclear.27,30 For example, the effect of cyclopropane fatty acids on the gel-to-liquid-crystalline phase transition varies with lipid composition. Specifically, the phase transition for phospholipid bilayers wherein both the sn-1 and sn-2 fatty acids are identical and cyclopropanated or unsaturated occurs at a higher temperature for cyclopropane-containing bilayers, suggesting that they have a lower degree of fluidity.30−32 In contrast, for phospholipid bilayers in which only the sn-2 tail is cyclopropanated, the phase transition temperature is decreased with respect to the related sn-2-unsaturated phospholipid bilayer, indicating a higher degree of fluidity.26,27 In this study, we examine the relative effect of fatty acids containing either cyclopropane rings or double bonds, both in cis and trans configurations, on the fluidity of a lipid bilayer and on the order of the lipid chains using molecular dynamics simulation. The fluidity of a bilayer is a collective property which cannot be easily defined. In fact, it is generally characterized through a collection of properties. Here, we investigated both structural (lipid packing, bilayer thickness, and conformation of the unsaturated and cyclopropanated chains) and dynamical (lateral diffusion of lipids) properties to capture the degree of fluidity of the lipid bilayers. Lipid order was determined through the average relative orientation of carbon− hydrogen and carbon−carbon bonds in the fatty acids with respect to the normal to the bilayer. It was found that

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METHODS Model Systems. Nine different systems were examined. Each consisted of preassembled bilayer of 288 lipids and 12960 water molecules (water-to-lipid ratio of 45) to ensure a fully hydrated state. The lipid molecule was either DPPC (1,2dipalmitoyl-sn-glycero-3-phosphocholine; 16:0/16:0) or a derivative of DPPC in which the sn-1 fatty acid chains included one double bond (lipids named PoPPC and PePPC), two double bonds (PloPPC and PlePPC), one methylene bridge (cp(c)PPPC and cp(t)PPPC), or two methylene bridges (cp(c)2PPPC and cp(t)2PPPC). The system consisting of a pure DPPC bilayer was taken from a previous study.33 All other lipid bilayers were generated by modifying the sn-1 palmitoyl tails in an equilibrated DPPC bilayer obtained after 400 ns of simulation.33 The double bonds and the methylene bridges were constructed in an all-cis (PoPPC, PloPPC, cp(c)PPPC, and cp(c)2PPPC) or all-trans (PePPC, PlePPC, cp(t)PPPC, and cp(t)2PPPC) configuration. In all cases, the bilayers contained a single lipid species. In the case of monounsaturated (lipids named PoPPC and PePPC) and monocyclopropanated (cp(c)PPPC and cp(t)PPPC) chains, the modification was included between carbons 9 and 10. In diunsaturated (PloPPC and PlePPC) and dicyclopropanated 5488


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The Journal of Physical Chemistry B (cp(c)2PPPC and cp(t)2PPPC) chains, the second modification was added between carbons 12 and 13. The structures and the names of the lipids are given in Figure 1. Simulation Parameters. All simulations were performed using the GROMACS package, version 3.3.334 in conjunction with the GROMOS 54A7 united-atom force field.35 Parameters for the unsaturated lipids (PoPPC, PePPC, PloPPC, and PlePPC) were derived from those of POPC (2-oleoyl-1palmitoyl-sn-glycero-3-phosphocholine; 16:0/18:1c9). Parameters for the cyclopropanated acyl chains were obtained from the Automated Topology Builder (ATB).36 They were based on (1R,2S)-1,2-dipropylcyclopropane and (1R,2R)-1,2-dipropylcyclopropane for the methylene bridges in a cis (cp(c)PPPC and cp(c)2PPPC) and trans (cp(t)PPPC and cp(t)2PPPC) configuration, respectively. Each system was simulated under periodic boundary conditions in a rectangular box. The temperature of the system was maintained by coupling to an external temperature bath at the reference temperature of 323 K with a coupling constant of 0.1 ps using a Berendsen thermostat.37 This temperature is above the gel-to-fluid-phase-transition temperature (Tm) of a pure DPPC bilayer (314.4 K).38,39 To our knowledge, Tm has not been measured systematically for pure bilayers of lipids analogous to DPPC in which the sn-1-palmitoyl chain contains one or two double bonds or cyclopropane rings. However, studies on related phosphatidylcholine bilayers in which the tails included one or two double bonds in a cis or trans configuration, or a single methylene bridge in a cis or trans configuration, have shown that the chain modifications lower the Tm compared to the corresponding saturated, noncyclopropanated lipid bilayer.40 Therefore, it is reasonable to assume that all of the lipid bilayers investigated are in a liquid-crystalline phase at 323 K. Lipids and water were coupled independently to the heat bath. The pressure was kept at 1 bar in the lateral and normal directions with respect to the bilayer by weakly coupling to an anisotropic Berendsen barostat37 using an isothermal compressibility of 4.6 × 10−5 bar−1 and a coupling constant of 1 ps. The length of all covalent bonds within lipids were constrained using the LINCS algorithm.41 The geometry of the simple point charge (SPC) water molecules42 was constrained using SETTLE.43 A 2-fs time step was used. Nonbonded interactions were evaluated using a twin-range cutoff scheme: interactions falling within the 0.8-nm short-range cutoff were calculated every step whereas interactions falling within the 1.4-nm long-range cutoff were updated every 10 fs, together with the pair list. A reaction-field correction44 was applied to account for the truncation of the electrostatic interactions beyond the long-range cutoff using a relative dielectric permittivity constant of 62, as appropriate for SPC water.45 Each system was first energy-minimized and then simulated at 50 K for 10 ps. The temperature was increased gradually over 180 ps until the final simulation temperature of 323 K was reached. Whether a given system had reached equilibrium was determined by examining the time evolution of the potential energy and the area per lipid. Once the systems were equilibrated, data were collected for 200 ns. Each system was simulated three times with different initial velocities. An overview of the simulations performed is given in Table 1.

Table 1. Overview of the Systems Simulated lipida

nature of the sn-1 tailb

nature of the sn-2 tail

total time (ns)c

DPPCd PoPPC PePPC PloPPC PlePPC cp(c)PPPC cp(t)PPPC cp(c)2PPPC cp(t)2PPPC

16:0 16:1c(9) 16:1t(9) 16:2c(9,12) 16:2t(9,12) 16:1cp(c)(9) 16:1cp(t)(9) 16:2cp(c)(9,12) 16:2cp(t)(9,12)

16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0

400 450 450 450 450 450 450 450 450

a

The names of the lipids are given in Figure 1. bcp(c), ciscyclopropane-1,2-diyl; cp(t), trans-cyclopropane-1,2-diyl. cEach system was simulated three times and analyzed over the last 200 ns of each individual simulation. dFrom ref 33.

lipid diffusion. In this work, the packing of lipids was estimated using the area per lipid, AL. AL was calculated as the lateral area of the simulation box divided by the number of lipids in each leaflet over the last 200 ns of the individual simulations. The mean value for each system is listed in Table 2. Double bonds in a trans configuration had no effect on AL compared to that of DPPC whereas AL increased with the number of cis double bonds. This indicates that unlike trans double bonds, cis double bonds enhance the fluidity of lipid bilayers by disrupting the packing of the lipids, in line with previous observations.46−48 The presence of one or two cyclopropane rings led to an increase in AL. The addition of a single methylene bridge had the same effect on AL, irrespective of its configuration. The increase of AL was equivalent to that of the two double bonds in PloPPC. The incorporation of a second cis-cyclopropane ring in cp(c)2PPPC resulted in a further increase in AL. In contrast, the effect of trans bridges was not cumulative as the values of AL for cp(t)PPPC and cp(t)2PPPC are similar. The simulations suggest that cyclopropane fatty acids lead to a greater increase in fluidity than unsaturated chains, in agreement with previous NMR measurements on phospholipid bilayers in which only the sn-2 chain contained a cyclopropane ring.26,27 Furthermore, although the inclusion of one methylene group caused an increase of AL, such as for unsaturated fatty acids, only in the case of cis methylene bridges did adding a second have a cumulative effect. This suggests that multiple trans methylene bridges can pack against each other, limiting the effect of any defects. The thickness DHH of the lipid bilayers was calculated as the distance between the two maxima of the electron density of the whole systems along the bilayer normal (z-axis) over the last 200 ns of the individual simulations (Table 2). In addition, a hydrophobic thickness DCC of the bilayers defined as the distance between the two peaks corresponding to carbon 2 in each of the acyl chains was computed. The total electron density profiles of the hydrated bilayers are displayed in Figure 2 along with the contributions of carbon 2. The presence of cyclopropane rings or trans double bonds did not change DHH or DCC while the presence of cis double bonds resulted in a decrease in both thickness measures. Again, the effect was greater for PloPPC than PoPPC. That any increase in AL due to the inclusion of cyclopropane fatty acids was not accompanied by a thinning of the whole bilayer or its hydrophobic core indicates that the increase in AL is mostly due to packing defects. This is also indicated by the variation in the mean effective length, S of each tail (Table 2). S was defined as the distance between carbons 1

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RESULTS AND DISCUSSION The fluidity of a lipid bilayer is a collective property that is often defined in terms of changes in lipid packing, the bilayer thickness, the conformational preference of the fatty acids, chain order, and 5489


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The Journal of Physical Chemistry B Table 2. Properties of the Bilayers Investigateda S (nm) bilayer b

DPPC PoPPC PePPC PloPPC PlePPC cp(c)PPPC cp(t)PPPC cp(c)2PPPC cp(t)2PPPC

AL (nm2)

DHH (nm)

DCC (nm)

sn-1

sn-2

+ L (μm2·s−1)

0.623 (0.008) 0.640 (0.007) 0.616 (0.007) 0.657 (0.008) 0.618 (0.009) 0.658 (0.006) 0.654 (0.006) 0.675 (0.006) 0.652 (0.007)

3.59 (0.05) 3.46 (0.02) 3.59 (0.03) 3.37 (0.03) 3.52 (0.04) 3.53 (0.04) 3.53 (0.03) 3.52 (0.04) 3.62 (0.01)

2.72 (0.04) 2.65 (0.02) 2.77 (0.04) 2.50 (0.01) 2.77 (0.01) 2.66 (0.03) 2.69 (0.05) 2.70 (0.02) 2.79 (0.01)

1.53 (0.19) 1.43 (0.22) 1.56 (0.17) 1.25 (0.21) 1.56 (0.18) 1.51 (0.20) 1.52 (0.21) 1.42 (0.20) 1.57 (0.22)

1.54 (0.18) 1.52 (0.18) 1.54 (0.19) 1.51 (0.18) 1.53 (0.18) 1.52 (0.19) 1.52 (0.19) 1.50 (0.19) 1.53 (0.19)

2.4 (0.1) 2.8 (0.1) 1.9 (0.1) 2.9 (0.3) 2.2 (0.2) 3.2 (0.1) 2.6 (0.2) 2.6 (0.3) 2.8 (0.2)

a

AL, area per lipid; DCC, hydrophobic thickness; DHH, bilayer thickness; + L , lateral diffusion coefficient of lipids; S , effective length of the acyl chain. The numbers in parentheses are the standard deviations of the means. bFrom ref 33.

The conformational restriction of the sn-1 chain imposed by the presence of a cyclopropane ring was further highlighted by the comparison of the distributions of the dihedrals that immediately precede or follow either the double bond(s) or the cyclopropane group(s). The distribution of these dihedrals is shown in Figure 3. The dihedrals themselves ϕ9 and ϕ11 in PoPPC, PePPC, cp(c)PPPC, and cp(t)PPPC and ϕ9, ϕ11, ϕ12, and ϕ14 in PloPPC, PlePPC, cp(c)2PPPC, and cp(t)2PPPC are defined in panels A and B of Figure 3 for unsaturated and cyclopropanated chains, respectively. As can be seen, the profiles obtained for the dihedrals ϕ9, ϕ11, ϕ12, and ϕ14 in PoPPC, PePPC, PloPPC, and PlePPC are virtually identical. The rotation was relatively free about the bonds that flank the double bonds as |ϕ| varied within 60−180°. The nearly free rotation between the two double bonds in PloPPC (dihedrals ϕ11 and ϕ12) contributed to fluidifying the bilayer and likely accounted for the largest decrease of S in the sn-1 tail among all of the unsaturated systems. Overall, the arrangement of the atoms C− C−CC corresponding to these dihedrals was anticlinal, in relative agreement with previous simulation studies of cis-monoand cis,cis-diunsaturared lipid bilayers.52,53 In the cyclopropanated fatty acids, the range of variations of the dihedrals was however limited (120° ≤ |ϕ| ≤ 180° in general) and the arrangement of the atoms was clearly shifted toward an antiperiplanar (trans) orientation, irrespective of the number and the configuration of the methylene bridges (a minor form in which the atoms were in a gauche− arrangement (ϕ ≈ −60°) also existed in cp(t)PPPC and cp(t)2PPPC). This indicates that a methylene bridge induces more conformational hindrance and enhances a greater separation between the two CH2 groups in β and β′ of the cyclopropane moiety (that is between carbons 7 and 12 in cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC and also carbons 10 and 15 in cp(c)2PPPC and cp(t)2PPPC) than a double bond. This is consistent with the lower degree of bending of the cyclopropanated sn-1 chains estimated earlier with S . The fluidity of the bilayers was also characterized in terms of the magnitude of the order parameter SCH along the acyl chains. SCH measures the relative orientation of the C−H bond with respect to the bilayer normal calculated as

Figure 2. Electron density profiles along the bilayer normal in the simulations of (A) hydrated PoPPC, PePPC, PloPPC, and PlePPC bilayers and (B) hydrated cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC bilayers. The profile calculated from the simulations of a hydrated DPPC bilayer is plotted in black in each panel as a reference. In each panel, the total electron density profile and the contribution from carbon 2 in the sn-1 and sn-2 acyl chains are shown and indicated as “total” and “C2 acyls”, respectively.

and 16 of a given tail. The change in the relative value of S calculated for the sn-1 chain of DPPC was in agreement with previous estimates from NMR.S did not change for the sn-2 chain in any of the systems. It only decreased significantly for the sn-1 chain of PloPPC. This shows that cis double bonds cause the thinning of bilayers by promoting flexibility within the lipid chains, favoring their overall bending. This compares well with a previous analysis of the bending rigidity of polyunsaturated lipid bilayers.51 The two cis-cyclopropane rings in the sn-1 tail of cp(c)2PPPC reduced its effective length to an extent comparable to that of the cis-monounsaturated chain in PoPPC without a change in DHH or DCC. This suggests that the enhanced fluidity induced by cyclopropane fatty acids is not associated with as much bending of the tails as in unsaturated chains. This is probably a consequence of the steric constraints inherent to the methylene bridges.

SCH =

1 ⟨3 cos2 β − 1⟩ 2

where β is the angle between a C−H bond and the normal to the bilayer. The angular brackets denote an ensemble average over all of the lipids for each frame of the simulation. The variation in |SCH| for the main-chain carbons 2−15 in the sn-1 and sn-2 chains is shown in Figure 4 along with those obtained for the palmitoyl 5490


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Figure 4. Carbon−hydrogen bond order parameter |SCH| profiles of the sn-1 and sn-2 fatty acyl chains of hydrated PoPPC, PePPC, PloPPC, PlePPC, cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC and cp(t)2PPPC bilayers calculated from the simulations. The |SCH| profiles of the sn-1 and sn-2 palmitoyl chains calculated from the simulations of a hydrated DPPC bilayer are plotted in each panel as a reference (black lines). The positions of the double bonds in PoPPC, PePPC, PloPPC and PlePPC and cyclopropanes in cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC and cp(t)2PPPC are shown by gray dashed lines. The |SCH| values are averaged over all of the sn-1 and sn-2 acyl chains in the bilayers and over the three simulations. Error bars are mostly within the size of the symbols (and thus not visible).

Figure 3. Rotation about the carbon−carbon bonds flanking the double bonds and the cyclopropane groups in the sn-1 acyl chain in hydrated PoPPC, PePPC, PloPPC, PlePPC, cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC bilayers. (A,B) Definition of the torsion angles ϕ in (A) the unsaturated fatty acids (in PoPPC, PePPC, PloPPC, and PlePPC) and (B) cyclopropanated fatty acids (in cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC). (C) Distribution of the dihedral angles calculated from the simulations. In PloPPC, PlePPC, and cp(t)PPPC, the profiles for ϕ10 and ϕ11 almost perfectly overlap.

phosphocholine bilayer.58 The impact of the presence of cyclopropane rings on the degree of order within the sn-1 chain varied depending on the number and the configuration of the rings. The cis methylene bridge at position 10′ in cp(c)PPPC and cp(c)2PPPC led to a local dip at position 10 in the |SCH| profiles. Overall, however, the degree of order was greater in the case of cp(c)PPPC and cp(c)2PPPC than in PoPPC and PloPPC, consistent with the results from NMR measurements on cismonocyclopropanated lipid bilayers.26,28 Interestingly, the second methylene bridge at position 13′ in cp(c)2PPPC had almost no effect on the sn-1 chain order. The trans-cyclopropane ring in cp(t)PPPC reduced the order in the distal section of the chain past the ring (carbons 11−15) in a way reminiscent of that of methyl-branched fatty acids.33 The two trans-cyclopropane rings in cp(t)2PPPC had an effect analogous to that of the two trans double bonds in PlePPC, but the increase in order was significantly larger. The degree of order induced by ciscyclopropane fatty acids compared to the corresponding unsaturated fatty acids suggests that cyclopropanation may enable bacterial membranes to remain in a functional fluid phase while at the same time increasing the rigidity of the acyl chains, in particular in the region surrounding the methylene bridge. This might help mitigate the effects of high temperatures and other

chains in DPPC from simulations performed using the same force field.33 The latter are in good agreement with experiment.50,54 As can be seen, modifications within the sn-1 chain affected the order of this chain only. In general, the degree of order within the sn-2 chain was slightly lower than that observed for DPPC (by less than 0.02). The exception was cp(c)2PPPC which was significantly less ordered than DPPC (|SCH| decreased by 0.03−0.05). The presence of one or more cis double bonds dramatically diminished the level of order along the sn-1 chain. In contrast, trans double bonds resulted in enhanced order locally and a moderate overall increase in order of the whole bilayer compared to that of DPPC. This is in accordance with NMR studies of related cis- and trans-monounsaturated and cis,cisdiunsaturated 18-carbon chains.26,55−57 Little experimental data on polyunsaturated chains are available. However, the increase in |SCH| for carbon 13 in the second double bond found in this work is similar to that observed previously in a |SCH| profile calculated from a simulation of a 2-linoleoyl-1-palmitoyl-sn-glycero-35491


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Figure 5. Carbon−carbon bond order parameter SCC profiles of the sn-1 and sn-2 fatty acyl chains of (A) hydrated PoPPC, PloPPC, cp(c)PPPC, and cp(c)2PPPC bilayers and (B) hydrated PePPC, PlePPC, cp(t)PPPC, and cp(t)2PPPC bilayers. The SCC profiles of the sn-1 and sn-2 palmitoyl chains calculated from the simulations of a hydrated DPPC bilayer are plotted in each panel as a reference (in black). The positions of the double bonds in PoPPC, PePPC, PloPPC, and PlePPC and methylene bridges in cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC are shown by gray dashed lines. The SCC values are averaged over all of the sn-1 and sn-2 acyl chains in the bilayers and over the three simulations. Error bars are mostly within the size of the symbols.

− rxy(t0)||2⟩t0 is the mean squared displacement (MSD) of the center of mass of the phosphate of a lipid projected onto the bilayer plane over the time t and computed over all possible starting times t0 < t. The drift of the leaflets of the bilayers during the simulations was corrected for by subtracting the motion of the center of mass of the leaflet from the MSD of each lipid. In practice, + L was calculated by averaging the MSD over all of the lipid molecules in the bilayer. + L was then derived from the slope of the linear regime of the time evolution of the average MSD. The evolution of the MSD as a function of the averaging time calculated for the unsaturated and cyclopropanated bilayers is given in Figure 6. It can be seen that the MSD varies linearly from an averaging time of about 50 ns to 250−300 ns so the slope was estimated over the time interval 50 ns ≤ t ≤ 250 ns for all simulations. The values of + L obtained from the simulations are

environmental insults and reduce the permeability of the membranes to potentially toxic compounds. The degree of order within the tails was further examined using the order parameter SkCC of the carbon−carbon bonds: k SCC =

1 ⟨3 cos2 αk − 1⟩ 2

where αk is the angle between the bond connecting the atoms Ck−1 and Ck and the bilayer normal. The SCC profiles of the sn-1 and sn-2 chains are given in Figure 5. The variations of SkCC are in agreement with both NMR and previous simulation studies.50,59−61 There is an “odd−even” effect whereby S2CC>S3CC, S4CC > S5CC, etc. This is clearly more marked for the sn-1 chain than for the sn-2 chain. The odd−even “zig-zag” profile is associated with the occurrence of gauche defects and a tilt of the chain with respect to the bilayer normal.60 From Figure 5, it appears that the presence of double bonds or cyclopropanes in the sn-1 chain had a significant effect on the SCC profile of the sn-1 chain but almost none on the SCC profile of the sn-2 chain. Interestingly, the SCC profiles were similar for the chains of the same configuration, regardless of the number of double bonds and rings. However, the magnitude of the variations for the sn-1 chain were larger when it was in a cis configuration. This indicates that cis double bonds and cyclopropane moieties promote the formation of gauche defects. This preference for gauche rotamers is characteristic of the fluidification of a lipid bilayer.50,62,63 This is particularly clear in the case of PloPPC and cp(c)2PPPC which displayed the most pronounced odd−even effect. Finally, the relative effect of cyclopropanation on the dynamics of a lipid bilayer was characterized through the lateral diffusion of lipids + L . + L was calculated using the Einstein relation: +L =

⎡1 ⎤ 1 lim ⎢ ⟨||rxy(t0 + t ) − rxy(t0)||2 ⟩t0 ⎥ ⎦ 2n f t →∞⎣ t Figure 6. Time evolution of the mean squared displacement (MSD) of the center of mass of the phosphate group of the lipid molecules in the simulations of hydrated PoPPC, PePPC, PloPPC, PlePPC, cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC bilayers.

where nf is the number of translational degrees of freedom (nf = 2) and rxy(t) is the position of the center of mass of the phosphate group of a lipid molecule in the bilayer plane at time t. ⟨||rxy(t+t0) 5492


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The Journal of Physical Chemistry B given in Table 2. Overall, + L was in the range 1.9−3.2 μm2·s−1 for all systems. To our knowledge, there is no lateral diffusion coefficient reported in the literature for the lipids examined here other than DPPC. In this case, there is a reasonable agreement with some experimental estimates using fluorescence (1.7−3.2 μm2·s−1)64 and NMR (5.5−9 μm2·s−1)65 spectroscopy, but the values from the simulations are generally lower than the experimental value. This may be due in part to the removal of collective motions within the bilayer in the simulations. However, experimental values of + L can also vary greatly (over 3 orders of magnitude), not only between the techniques used but also between different studies using the same experimental technique.66 In the simulations, cis double bonds increased the diffusion rate of lipids compared to DPPC. There was no substantial difference between PoPPC and PloPPC. In contrast, trans double bonds decreased + L , the effect being larger for PePPC than for PlePPC. This indicates that unlike trans-unsaturated fatty acids, cis-unsaturated fatty acids enhance the fluidity of lipid bilayers by increasing the mobility of lipids. The variation of + L between DPPC, PoPPC, and PloPPC is in good agreement with estimates from NMR experiments and simulations on homologous systems wherein the acyl tails are 18 carbons long.67,68 Therefore, although the values of + L obtained from the simulations may appear low, the trend in the variations of + L was correctly reproduced. The presence of the cyclopropane rings led to an increase in + L . The greatest effect was for cp(c)PPPC. The changes for cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC were similar, irrespective of the number and the configuration of the rings and, in magnitude, equivalent to the effect observed for PoPPC and PloPPC. The simulations suggest that cyclopropane fatty acids lead to an increase in fluidity that is analogous to or greater than cis-unsaturated fatty acids. This is consistent with the looser packing of the lipids caused by the steric constraints of the cyclopropane rings. According to the free-volume theory69 applied to lipid bilayers,70 the diffusion of a lipid in the plane of a bilayer is limited by the probability of the formation of a local free area that is greater than a critical size. Therefore, as the methylene bridges in cyclopropane fatty acids promote the occurrence of packing defects between lipids, the occurrence of an adjacent vacant free area large enough to allow a lipid to move into may be increased, thereby enhancing the diffusion of lipids.

However, the trans-monocyclopropanated chain was less ordered than the corresponding saturated and trans-unsaturated chains and behaved in a fashion reminiscent of some monomethyl-branched fatty acids.33 The simulations suggest that cyclopropane fatty acids that are in a cis configuration, which is the most common across bacteria, may fulfill a dual role in bacterial membranes. They appear to increase fluidity in the plasma membrane while simultaneously inducing a more ordered state within the hydrocarbon chains as compared to unsaturated fatty acids. This could explain how cyclopropane fatty acids can enhance the stability of the membrane against adverse conditions (such as high osmotic pressure5−8 and high temperature5,9), and at the same time reduce its permeability against toxic compounds. Furthermore, the fact that cis- and trans-cyclopropane fatty acids have distinct ordering effects would indicate that the specific role of trans-cyclopropanation of mycolic acids in M. tuberculosis21 may be mediated through different membrane properties.

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ASSOCIATED CONTENT

S Supporting Information *

Topology files of PoPPC, PePPC, PloPPC, PlePPC, cp(c)PPPC, cp(t)PPPC, cp(c)2PPPC, and cp(t)2PPPC (pdf). This material is available free of charge via the Internet at http://pubs.acs.org. These files also available on the Automated Topology Builder and Repository (http://compbio.biosci.uq.edu.au/atb).36

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61 (0)7 3365 7562. Fax: +61 (0)7 3365 3872. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded from the National Health and Medical Research Council (Project Grant APP1044327) with the assistance of high-performance computing resources provided by the National Computational Infrastructure National Facility and iVEC located at iVEC@Murdoch through the National Computational Merit Allocation Scheme supported by the Australian Government (Projects m39 and m72).

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CONCLUSION In summary, the effect of cis- and trans-cyclopropane fatty acids on the fluidity of a lipid bilayer was investigated and compared with that of unsaturated and saturated (DPPC) lipids. It has been claimed that cyclopropane fatty acids reduce the fluidity of a lipid bilayer by modulating lipid packing and chain order.23−29 However, from previous studies it was unclear whether the effects of cyclopropane fatty acids were similar to unsaturated fatty acids or the degree to which the stereochemistry of the cyclopropane group influenced their effect on membranes.27,30 In this study, it was found that cyclopropane fatty acids tend to promote the fluidity of lipid bilayers by interfering with lipid packing, enhancing the formation of gauche defects and increasing the lipid diffusion. This was irrespective of the configuration of the methylene bridge and, in the case of cis-cyclopropane fatty acids, this was to a greater extent than unsaturated chains. Our results show that this effect originates partly from the steric restraints caused by the methylene bridges. Cyclopropane fatty acids were in general more ordered than the analogous unsaturated chains.

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