Phase Transition of Glycolipid Membranes Studied by Coarse-Grained

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Phase Transition of Glycolipid Membranes Studied by CoarseGrained Simulations Raisa Kociurzynski, Martina Pannuzzo, and Rainer A. Böckmann* Computational Biology, Department of Biology, Friedrich-Alexander University Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany ABSTRACT: Glycolipids are important components of biological membranes. High concentrations of glycolipids are particularly found in lipid rafts, which take part in many physiological phenomena. This different partitioning and interaction pattern of glycolipids in the membrane as compared to those of phospholipids are likely due to their different chemical structures: the polar regions of glycosphingolipids can be even larger than for their hydrophobic moieties, giving rise to a rich conformational landscape. Here we study the influence of glycosphingolipids galactosylceramide (GCER) and monosialotetrahexosylganglioside (GM1) on the structural and thermodynamic properties of a phospholipid (DPPC) bilayer. Using the method of coarse-grained molecular dynamics simulation we show that both glycolipids increase the phase-transition temperature of phospholipid membranes and that the extent of this increase depends on the headgroup size and structure. GM1 shows a strong tendency to form mixed clusters with phospholipids, thereby stabilizing the membrane. In contrast, GCER is dispersed in the membrane. By occupying the interstitial space between phospholipids it causes a tighter packing of the lipids in the membrane.

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INTRODUCTION Lipid rafts are membrane-ordered microdomains which exhibit a different composition and ordering than the surrounding environment and can phase separate and float in an otherwise disordered membrane. These domains carry out a number of important physiological functions: they host signal transduction pathways and influence synaptic transmission, apoptosis, organization of the cytoskeleton, cell adhesion and migration, and TCR activation and are involved in protein and lipid sorting.1,2 Predominant components of lipid rafts are glycolipids as well as sphingomyelin, cholesterol, and transmembrane proteins. Such lipid rafts are known to be resistant toward detergents such as Triton X-100, which might be explained by the ability of cholesterol to cause a closer packing of neighboring acyl chains due to its rigid sterol group.3,4 Depending on the sugar headgroup, glycosphingolipids can be divided in two groups, ceramides and gangliosides. The lipids are referred to as gangliosides if the headgroup contains a sialic acid, a negatively charged sugar group. Glycolipids missing the sialic acid are called ceramides.5 Glycolipid headgroups are covalently attached to either a glycerol or a sphingosine backbone, forming a glycophospho- or glycosphingolipid, respectively. In glycosphingolipids, a long chain of mostly saturated fatty acids is linked to the sphingosine backbone over an amide bond while the sugar headgroup is linked to the hydroxyl group of the backbone. This sugar headgroup can range from a single sugar residue to complex oligosaccharide chains.6 It was observed that pure glycolipid membranes exhibit different phase behavior than pure phospholipid membranes. © 2015 American Chemical Society

Lipid bilayers are known to exist in different phase states and undergo a transition between those states depending on the temperature and the lipid composition. The transition temperature between the gel and the liquid-crystalline phase is defined as the main phase-transition temperature (Tm). The phase transition is a highly cooperative, ordered−disordered transition in which the bilayer undergoes lateral expansion and a decrease in thickness and density. The hydrocarbon chain packing changes from an all-trans configuration below Tm to a state in which the chains have some gauche rotational isomers above Tm.7 The molecular packing in lipid bilayers depends on the lipid components, the temperature, or the ionic composition of the aqueous environment. It has been demonstrated that the physical state of phospholipid acyl chains clearly affects the activity of membrane transport processes and membrane enzymes.8 In addition, Tm can also be altered by the experimental setup. For example, Tm is influenced by the rates of heating and cooling of the scan. The Tm for lipid bilayers determined from heating and cooling scans shows thermal hysteresis9 which increases for increased rates.10 How the presence of glycolipids influences the ordered/ disordered transition is controversially discussed in the literature. It was observed that bilayers composed of pure glycolipids with simple, neutral sugar headgroups have a transition temperature increased by 20−40 °C as compared to that of Received: May 3, 2015 Revised: July 17, 2015 Published: August 12, 2015 9379

DOI: 10.1021/acs.langmuir.5b01617 Langmuir 2015, 31, 9379−9387

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Langmuir pure phospholipids with comparable hydrocarbon chains.11 A possible explanation for this is the large number of hydrogen donor and acceptor groups in the glycolipid headgroups which may stabilize the membrane by intermolecular hydrogen bonds with other glycolipids and water molecules. Furthermore, for bilayers of mixed composition of phospholipids and simple glycolipids an increase in the order of the acyl chains was reported, as compared to pure phospholipid membranes. For example, fluorescence spectroscopy and surface pressure−area measurement studies showed that unsaturated C16:0-GlcCer glycolipids in a POPC bilayer tend to accumulate into highly ordered gel domains and to increase the order of the POPC fluid phase.12 It can therefore be assumed that the increase in order and the accompanying increase in the packing density of the lipid tails will probably result in an increase in Tm for phospholipid bilayers with simple glycolipids, such as glycosylceramide (GCER). The most abundant ganglioside in biological membranes is monosialotetrahexosylganglioside (GM1), which is composed of a large, branched headgroup with four sugar residues and a negatively charged sialic acid. Due to steric hindrances of the bulky GM1 headgroups the area per lipid in pure GM1 membranes is increased. The thereby increased volume for the lipid tails will possibly result in a decreased phase-transition temperature for pure GM1 bilayers as compared to pure phospholipids.11 This is further supported by the observation that an increase in the number of sugar residues in the headgroup of large glycolipids further decreases the phasetransition temperature.11 However, for GM1-phospholipid bilayers a slight increase in Tm could be observed in a number of differential calorimetry studies13−17 Tm was found to increase with increasing GM1 concentration. Above a critical GM1 concentration, an additional transition at higher temperatures was reported. While a few studies claim that GM1 is miscible in phospholipid bilayers at a concentration of up to 70%,16,17 other studies reported a maximum GM1 concentration of 25− 30% mol for the incorporation into a phospholipid membrane.14,15 The latter finding is supported by surface pressure and fluorescence microscopy studies which indicated the most condensed DPPC monolayer at a ratio of 3:1 DPPC/ GM1.18 A number of experimental studies19,20 unveiled the physical and chemical properties of GM1 in pure or mixed bilayers as stabilizing the gel phase, thereby favoring an increase in Tm. On the basis of electron paramagnetic resonance studies it was reported that the incorporation of 22 mol % GM1 molecules in egg yolk phosphatidylcholine (EPC) small unilamellar vesicles (SUVs) increased the order parameter S from 0.59 without GM1 to 0.63. This was explained by strong interactions at the bilayer surface among gangliosides and between gangliosides and phosphatidylcholines (PC).19 Furthermore, it could be shown by steady-state fluorescence polarization that the incorporation of 30% GM1 increases the membrane order and Tm of DPPC and DMPC in multilamellar liposomes.20 Molecular details at atomistic resolution may be gained from molecular dynamics (MD) simulations of glycolipid systems, both concerning the glycolipid function (review in ref 21) as well as their role in membrane microdomain formation (review in ref 22). United atom molecular dynamics simulations revealed that the area per lipid of a DPPC bilayer is decreased from 0.66 to 0.58 nm2 in the presence of 17% GM1 molecules.23 This decrease was partially caused by the replacement of larger-area DPPC molecules with the smaller

ceramide backbone as well as by the condensing effect of GM1 on DPPC.23 The same study predicted an increase in the deuterium order parameter of the hydrocarbon chains in a DPPC bilayer with increasing concentration of GM1. This observation was explained by the strong ability of the GM1 headgroups in forming hydrogen bonds. It was assumed that the increase in lateral interactions among GM1 molecules is responsible for the ordering of a bilayer at higher GM1 concentrations.23 Furthermore, atomistic molecular dynamics simulations on the effect of a single GM1 molecule embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer suggested increased ordering of the water molecules near the glycerol and carboxyl groups of the sialic acid of GM1.24 While there is overall agreement on the effect of GM1 on the Tm of phospholipid bilayers, the related GM1-induced changes in the structural and chemical properties of phospholipid bilayers are debated. For example, fluorescence polarization studies showed that the disorder and hydration of the lipid bilayer region near the exoplasmic surface are enhanced by bovine brain gangliosides.25 Furthermore, single-molecule fluorescence measurements using BODIPY-PC have shown that concentrations below 5 mol % GM1 as well as high concentrations of more than 20 mol % GM1 induce disorder in DPPC membranes. In turn, the order of BODIPY-PCs is highest at 15−20 mol % GM1.26 Apart from these experimental studies, an atomistic molecular dynamics simulation study of a single GM1 molecule revealed an induced local disorder in the arrangement of the surrounding chains and headgroups because the sphingosine chain of GM1 folds up and becomes stacked beneath the sugar residues lying on the surface.24 However, this study also predicted an increased ordering of the GM1 surrounding water molecules, as described above. Here, we comparatively studied the effect of the ceramide GCER bearing a small headgroup and of the ganglioside GM1 with a large headgroup on the phase-transition temperature and the structural properties of a DPPC phospholipid bilayer using coarse-grained (CG) molecular dynamics simulations. These two model systems allow one to discriminate how conformational changes in the glyco/phospholipid headgroup can differently affect the degree of order/disorder of the lipid tails and influence the architecture of the membrane. Overall, important insight into possible functional roles of differently sized glycolipid headgroups on the membrane phase and domain formation is obtained.

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SIMULATION METHODS Coarse-grained simulations were employed to study the phase transition of mixed DPPC/glycolipid membranes and the influence of glycolipids on the membrane structure. Coarse-Grained Simulations. The framework of the Martini parametrization27−29 was used for the coarse-grained representation of lipids (Figure 1) and surrounding water. In contrast to the atomistic approach, the coarse-grained Martini force field models on average four heavy atoms and the associated hydrogens by a single interaction center, speeding up simulations by about 2 orders of magnitude. However, at this reduced resolution, isomers such as glucose and galactose can not be distinguished. Here, we used the polarizable version of the Martini force field,30 which includes charged particles on a spring for the water beads to include polarizability. Initial tests with the standard water model led to a reversed effect for the addition of charged GM1 lipids on the phospholipid phase transition as compared to experiment. Additionally, GM1 9380


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by stiff bonds. The systems were equilibrated for 4 ns at 300 K to let the lipids relax and the remaining water diffuse out of the membrane. Subsequently, the systems were equilibrated for 600 ns at 340 K to allow for cluster formation, cooled down afterward to 260 K, and further equilibrated at 260 K for at least 600 ns. The annealing of the systems was performed between 260 and 360 K with a heating rate of 0.2 K/ns. Since equilibration in the gel phasein particular, possible cluster formationis considerably hampered due to the drastically reduced diffusion, we additionally performed cooling simulations starting from equilibrated fluid-phase structures. For statistics, each simulation was repeated several times by using slightly differing starting conditions. Simulation Parameters. Equilibration simulations were performed using the weak-coupling thermostat and the Berendsen barostat35 with a time constant of 10 or 5 ps. During the heating runs and long equilibrations, the temperature was coupled to a v-rescale thermostat36 with a time constant of 3.0 ps. Temperature coupling was performed separately for DPPC, glycolipids, and water/ions. The Parrinello−Rahman barostat37 was used with semiisotropic pressure coupling and a time constant of 4.0 ps, a compressibility of 4.5 × 10−5 bar−1, and a reference pressure of 1 bar in all heating runs and equilibrations of the liquidcrystalline state. Long-range electrostatic interactions were calculated using the particle mesh Ewald method (PME38). The relative dielectric constant was set to 2.5. The Lennard-Jones potential was cut off between 0.9 and 1.2 nm using the shift function. Bonds were constrained using the LINCS algorithm.39 The short-range neighbor list contained all interaction pairs within a distance of 1.2 nm and was rebuilt every five time steps. Periodic boundary conditions were applied in all three directions. The integration step for the equations of motion was 20 fs for all three systems, and snapshots were saved every 25 ps. Simulations were performed using the GROMACS software package, version 4.6.5.40 The trajectories were analyzed using GROMACS tools and in-house codes. Analysis. The transition temperature was obtained from the area per lipid as a function of temperature, fitted using the Heaviside function. Linear fits were used below and above the phase-transition temperature. The area per lipid was calculated from the lateral extension of the box divided by the number of lipids in one leaflet. The average areas per lipid for the gel and the liquid-crystalline phases were analyzed using GridMATMD.41 The order parameter for carbon atoms along the acyl chains is given by

Figure 1. Sketch of a DPPC molecule and glycolipids used in atomistic and Martini coarse-grained representations. DPPC and GCER have a vanishing net charge (DPPC with ± integer charges on Q0 and Qa, respectively). GM1 has a net charge of −1e, located on coarse-grained bead Qa.

molecules showed a very strong, probably artificially enhanced clustering. In turn, for GCER the phase-transition temperature was shifted similarly for the standard and for the polarizable Martini force field (results not shown). Similarly, adsorption studies of charged peptides on membranes required the polarizable water model for an accurate description.31 The glycolipid headgroups consist of a mono-, di-, or oligosaccharide which is mapped on three beads per monomer.28 Parameters and topology files were obtained from the Martini Web site (http://cgmartini.nl). Refined force field parameters for GM1 were provided by the Martini group, based on the recently published Martini force field for glycolipids.29 The bonded parameters were modified for increased stability and faithfulness to atomistic force fields.32 System Composition and Setup. The following membrane systems were studied: (a) a pure DPPC membrane made up of 336 lipid molecules; (b) a mixed bilayer containing 280 DPPC and 56 GM1 (17% mol, DPPC/GM1); and (c) a mixed bilayer containing 280 DPPC molecules and 56 GCER molecules (17% mol, DPPC/GCER). All systems were assembled with the insane.py script for lipids as symmetrical bilayers with a box size of 10 nm in the x and y directions and 15 nm in the z direction.33,34 NaCl ions were added to all systems at a concentration of 0.1 M (corresponding to 90 Na+Cl− ion pairs), and excess charges were used to neutralize the DPPC/GM1 system. Initial structures were energy-minimized using the steepestdescent algorithm. For minimization, constraints were replaced

SZ =

3 1 ⟨cos2 θZ⟩ − 2 2

(1)

θ describes the angle between the vector along the acyl chain and the membrane normal. The order parameter was calculated separately for every 20 ns over the whole trajectory.

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RESULTS AND DISCUSSION Membrane structural and thermodynamic properties were analyzed form coarse-grained molecular dynamics simulations of pure DPPC bilayers and of mixed phospholipid bilayers with either 17% GM1 (DPPC/GM1) or 17% GCER (DPPC/ GCER) content. The main phase-transition temperature between the gel and the liquid-crystalline phases was determined from both heating and cooling simulations at a rate of 0.2 K/ns. 9381


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Langmuir Transition Temperature and Area per Lipid. One characteristic of the phase transition from the gel to the liquidcrystalline phase is a sharp increase in the area per lipid of the membrane at the transition temperature. In the following text, the Tm values and the area per lipid in the gel phase for the different systems are compared. Although the phase-transition temperature is well-defined only for one-component systems, this expression is also widely used for mixed systems. The systems studied here all show a similar transition upon heating or cooling that does not allow a strict separation of different transition temperatures for the different components. In the following text, the transition temperature always refers to the temperature at which the (mixed) systems undergo a phase transition. In heating simulations (index h), pure DPPC exhibits a Thm of about 305 K, which differs from the experimental value by ∼10 K (315 K42). The hysteresis of ∼40 K as obtained from additional cooling simulations (index c, Tcm = 264.5 K) is considerable. Interestingly, the transition temperature Thm is approximately 10 K lower than the Tm previously determined for DPPC bilayers using the CG Martini force field with the standard water model10 (for comparable rates and system sizes). For lower cooling/heating rates an increased/decreased phase-transition temperature is to be expected as the system is given more time to adapt to the temperature. This and the dependency of the phase-transition temperature on the size of the system and the simulation time were studied by Marrink and colleagues: The true thermodynamic transition temperature (i.e., the phase-transition temperature under equilibrium conditions) is gained by extrapolation to very slow heating and cooling rates as well as to large systems and was reported to be decreased by about 20 K.10 Comparing the two glycosphingolipid systems, the transition temperature Thm was increased by ∼18 K (14 K) for Tcm for the DPPC/GCER, while the addition of GM1 to a DPPC bilayer led to a small increase of Thm by ∼5 K (∼8 K for Tcm). GCER significantly decreased the area per lipid for both the gel and liquid-crystalline states as compared to pure DPPC (Figure 2), and GM1 affected only the lateral area in the liquid-crystalline phase. Possibly, the large and bulky GM1 sugar headgroups prevent the bilayer from assuming a tighter packing in the gel phase through steric hindrance. The condensing effect of GCER in the gel phase is coupled to the small GCER headgroup: the size of the phospholipid headgroup gives rise to comparably large chain−chain distances. For the mixed DPPC/GCER system, GCER tailmediated interactions between the phospholipid chains lead to a tightening of the tails. This also affects the chain order, as shown below. Order Parameter. In agreement with the results for the area per lipid, the order parameter for the DPPC acyl chains of the DPPC/GCER system were increased in the gel and in the liquid-crystalline phase as compared to the system containing pure DPPC (Figure 3). This indicates a closer packing of the DPPC lipid chains induced by GCER molecules. The more dense packing in turn stabilizes the gel phase, resulting in an increased phasetransition temperature Tm (Figure 2). This is in agreement with previous experimental12 and atomistic simulation43 studies that reported an increased phospholipid order when adding glycolipids with small headgroups. Pure simple glycolipid membranes with identical structure, except for the headgroup, were more tightly packed than pure phospholipids.43 The order

Figure 2. Area per lipid in nm2 for all lipids of the systems of pure DPPC (top), DPPC + 17% GCER (middle), and DPPC + 17% GM1 (bottom) using polarizable water as a function of temperature in K. Both cooling (black lines) and heating simulations (blue lines) were performed at a heating rate of 0.2 K/ns. The transition temperature was determined as described in the Methods section using a Heaviside function at the transition point (fit functions as red solid lines). Transition temperatures Tcm and Thm are marked by gray dashed and solid lines, respectively, and those of the mixed DPPC/glycolipid systems, by black lines.

Table 1. Transition Temperatures for the Systems of Pure DPPC and the Mixed Glycolipid Systems Averaged over N Cooling and N Heating Runs at a Cooling/Heating Rate of 0.2 K/ns (Error = Error of the Mean) DPPC DPPC + 17% GM1 DPPC + 17% GCER

N

Tcm(K)

error (K)

Thm(K)

error (K)

6 6 6

264.5 272.1 278.1

1.2 1.1 0.5

305.0 310.9 323.1

0.2 1.0 1.7

parameter for the DPPC acyl chains of the DPPC/GM1 system were slightly decreased in the gel phase but significantly higher in the liquid-crystalline phase as compared to in the pure DPPC membrane (Figure 3). This DPPC order increase and area decrease (see above) in the liquid-crystalline phase induced by GM1 suggest a stabilization of the membrane in this phase relative to a pure DPPC bilayer. Bilayer Thickness. In both membrane phases, the large and bulky GM1 headgroups were protruding out of the DPPC layer as displayed by the density profiles (Figure 4). This CG result is in agreement with previous atomistic simulation studies both on a single GM1 molecule in DMPC or DOPC bilayers24,44 and for GM1 concentrations ranging between 4 and 11% mol GM1.23 These studies reported a stable GM1 conformation where the sialic acid residue (NeuNAc) of the pentasaccharide headgroup extends out of the bilayer whereas the three sugar residues (GalNAc-Gal-Glc) remain on the lipid surface. In contrast, GCER was observed to be fully embedded in the 9382


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Figure 3. Averaged order parameter SZ for the DPPC acyl chains of the pure DPPC, DPPC/GCER, and DPPC/GM1 systems. The order parameter was calculated every 10 ns and averaged over both acyl chains.

DPPC membrane both in the gel and in the liquid-crystalline phase in our CG simulations (Figure 4). Similarly, the chemically similar galactosylceramide (GalCer) was shown in atomistic simulations to only marginally increase the bilayer thickness in the liquid-crystalline phase.45 Arrangement of Glycolipids and Cluster Formation. To investigate possible cluster formation within the membrane, both glycolipid systems were equilibrated for an additional 600 ns at 340 K. Large clusters of 6−28 GM1 molecules were observed for the DPPC/GM1 system (Figure 5A) in the separate leaflets with 1−7 GM1 clusters per leaflet. (See Figure 6 for a snapshot of GM1 clusters in the gel phase.) In contrast, GCER hardly clustered and occurred in aggregates of not more than two to five molecules. Consequently, around 7−14 GCER clusters per leaflet were found (Figure 5B). The cluster size and distribution obtained for GCER is similar to a random distribution as evidenced by the cluster formation of randomly selected DPPC molecules in a pure DPPC bilayer at the same concentration (17%, see Figure 5C). The cluster sizes remained stable for both systems when gradually cooling to 260 K and further equilibration for 600 ns at 260 K. It is apparent that clusters are formed not only between molecules in one leaflet but also among molecules in opposite leaflets, as the calculated average cluster size is almost doubled for the whole bilayer (Figure 5 and Table 2). This result might be explained by the phenomenon of interdigitation of lipids

Figure 5. Average cluster size and the number of clusters during a 600 ns equilibration at 340 K, shown for systems DPPC + 17% GM1 (A), DPPC + 17% GCER (B), and for comparison for randomly selected DPPC molecules (17%) in a simulation of a pure DPPC bilayer (C). Glycolipids/lipids within a distance of 0.65 nm were considered to be in one cluster.

which particularly occurs for lipids with differing chain lengths as is the case for lipids with a sphingosine backbone such as GM1 and GCER.46,47 Clusters within the DPPC/GM1 system contain both GM1 molecules and DPPC molecules in a high fraction. Cluster formation was confirmed in a CG simulation at a reduced GM1 concentration of 7% (>1000 lipids in total, 3.2 μs of simulation time, see Figure 6). One explanation for the interaction of DPPC and GM1 is provided by the attraction of the negatively charged sialic acid moiety of GM1 and the positively charged choline group of DPPC.48 Moreover, the steric hindrance of neighboring GM1 headgroups favors the interstitial placement of DPPC

Figure 4. Density distribution of the different membrane components analyzed for the systems DPPC, DPPC + 17% GM1, and DPPC + 17% GCER for the gel (left) and the liquid-crystalline (right) phase. 9383


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Solvent-Accessible Surface (SAS). The solvent-accessible surface (SAS) area for the DPPC bilayer is significantly increased in the gel phase upon addition of GM1 (Figure 8(a)). One reason is the comparably large surface area of the GM1 headgroups (Figure 8(b)). However, in the liquid-crystalline phase the total SAS area for the DPPC/GM1 system is similar to that of pure DPPC. This is related to the decreased area per DPPC molecule in the DPPC/GM1 system as compared to that for pure DPPC (Figure 8(b), blue and black lines). This probably reflects the shielding of DPPC molecules from the surrounding water by the large GM1 headgroups. In contrast, the addition of GCER to DPPC decreased the SAS area of the whole bilayer both in the gel and in the liquidcrystalline phase (Figure 8(a)). GCER molecules are less accessible to water as compared to DPPC, and they are subject to shielding by DPPC. Contacts between lipid headgroups and water may disrupt intermolecular contacts in the membrane, thus resulting in a destabilization and therefore a decrease in Tm. Thus, a decrease in the SAS area, as reported above for GCER, probably stabilizes the membrane and contributes to the overall increase in Tm.

Figure 6. Left: Top view of cluster formation in a DPPC/GM1 system at 7% GM1 content after 3.2 μs of simulation. The clusters were formed in the liquid-crystalline phase. Only the P−N dipole is shown for the DPPC molecules (ball and stick). GM1 glycolipids are emphasized by a green surface representation. The largest cluster contains 14 GM1 lipids and 21 DPPC lipids in the interstitial area between the GM1 molecules. Right: Side view of a representative GM1/DPPC cluster. DPPC molecules occupying the interstitial area between GM1 lipids are highlighted in blue.

molecules. The observed mixture of phospholipids and gangliosides in clusters is supported by experimental studies that unveiled a maximum lipid tail order for a GM1/DPPC ratio of ∼1:3.18,26 Contacts. Glycolipid headgroups can exhibit a large number of hydrogen bonds due to their ability to act both as hydrogen donors and as hydrogen receptors. However, (atomistic) hydrogen bonds cannot be detected using a coarse-grained approach. Here, we focus instead on the number of intermolecular contacts (i.e., contacts within 0.5 nm of the glycolipids). For the glycolipid systems, the number of contacts both between the glycolipids and between glycolipids and DPPC molecules decreases with increasing temperature (Figure 7). This is explained by the increasing distance between the molecules at higher temperature. While the number of water contacts of GM1 molecules decreases slightly for increasing temperature, a dramatic increase in the number of contacts to water is seen for GCER upon phase transition, coupled to a temporarily strong decrease in the number of contacts to DPPC but also to GCER molecules. The sudden jump in the number of contacts between GCER and water molecules reflects the deep embedding of GCER in the DPPC bilayer in the gel phase, in line with the results obtained for the area per lipid and the solvent accessibility of the lipids (see below).

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SUMMARY AND CONCLUSIONS Previous experimental DSC studies showed that glycolipids increase the main phase-transition temperature (Tm) of phospholipid bilayers.16 The increase in Tm was attributed to the ability of glycolipid headgroups to form a large number of hydrogen bonds. Here, we studied the molecular properties of phospholipid membranes with 17% glycolipid content for glycosphingolipids GM1 and GCER with different headgroups using coarse-grained molecular dynamics simulations. Both GM1 and GCER increased the Tm of a DPPC bilayer. The shift was much larger for GCER, which has a comparingly small headgroup. GCER (one sugar residue) dissolved in the phospholipid bilayer and showed a condensing effect on DPPC resulting in increased order of the acyl chains, in particular for the gel phase. Thereby, the addition of GCER promotes a stabilization of the lipid gel phase. Differently, GM1 molecules showed cluster formation in a DPPC bilayer. The highly dynamic clusters involved a large fraction of phospholipids as well, forming fluctuating nanoassemblies.49 The formation of distinct phases probably requires an increased connectivity that may be achieved by

Table 2. Membrane Propertiesa SZ area per lipid (Å2) tilt angle (deg) average cluster size (molecules)

SAS (nm2)

gel liquid-crystalline gel liquid-crystalline gel liquid-crystalline whole bilayer upper leaflet lower leaflet gel liquid-crystalline

DPPC (PW)

DPPC + 17% GM1

DPPC + 17% GCER

0.95 0.38 47.38 (0.28) 72.97 (1.27)

0.93 0.38 47.75 (0.22) 68.11 (1.20) 147.28 (1.53) 137.31 (2.93) 35.12 (16.4) 11.42 (5.51) 18.02 (8.26) 515 (9.3) 892 (26.7)

0.84 0.4 46.23 (0.23) 68.08 (1.16) 151.31 (1.92) 139.00 (3.04) 6.93 (3.21) 3.15 (0.78) 3.25 (1.01) 390 (7.4) 779 (25.1)

434 (8.4) 885 (26.0)

a

Structural observables were determined for both the gel and the liquid-crystalline phase (initial 100 ns and last 100 ns of the simulation, respectively). Order parameter SZ describes the average SZ of both hydrocarbon chains for the DPPC lipids in all three systems. The average tilt angle refers to the headgroups of GM1 and GCER (Methods section). The average cluster size was calculated on the last 100 ns of 600-ns-long equilibrations at 340 K (SAS = solvent assessible surface; standard deviation given in parentheses). 9384


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Figure 7. Number of glycolipid−glycolipid, glycolipid−DPPC, and glycolipid−water contacts for systems DPPC/GM1 (a) and DPPC/GCER (b) for heating simulations (PW = polarizable water).

Figure 8. Solvent-accessible surface (SAS) area for DPPC, DPPC + 17% GM1, and DPPC + 17% GCER analyzed for heating simulations (a). The area per residue for the gel (b) and the liquid-crystalline (c) phase is also shown. Residues 141−168 and 309−336 represent GM1 (cyan) or GCER (red) molecules, respectively.

the addition of cholesterol.49 It remains to be shown how the addition of peptides or proteins and specific lipids or sterols influences GM1-rich membranes as described here and contributes to more stable membrane-ordered assemblies.1 The phase-transition temperature Tm was slightly increased for DPPC/GM1, albeit a condensing effect for the mixed gel phase is missing and also the acyl chain order in the gel phase was even decreased for the DPPC/GM1 system. However, the bulky GM1 headgroups and the embedding of DPPC in GM1 clusters decreased the overall water accessibility of DPPC, resulting in a stabilization of the gel phase. Interestingly, the standard nonpolar coarse-grained water in the Martini force field results in a decreased Tm (data not shown), thus underlining the crucial role of water and its proper description in studies on membrane phase transitions. In turn, the results obtained with the polarizable Martini force field are qualitatively in very good agreement with experiment. In summary, there is no simple relationship between the number of sugar residues in glycosphingolipid headgroups and their effect on the main phase transition of phospholipid membranes. The physicochemical properties of both the glycolipid head and chain regions determine the differential interaction pattern with the phospholipids.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the German Science Foundation (DFG) within the Research Training Group 1962 − Dynamic Interactions at Biological Membranes. M.P. was supported by a scholarship within the Programme to Promote Equal Opportunities for Women in Research and Teaching (FFL) of the Friedrich-Alexander University of Erlangen-Nürnberg. We thank the Marrink Group for sharing the glycolipid Martini parameters. Computer time was provided by the Computing Center of the Friedrich-Alexander University of ErlangenNürnberg (RRZE).

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REFERENCES

(1) Lingwood, D.; Simons, K. Lipid rafts as a membrane-organizing principle. Science 2010, 327, 46−50. (2) Munro, S. Lipid rafts: elusive or illusive? Cell 2003, 115, 377− 388. (3) Owicki, J.; McConnell, H. Lateral diffusion in inhomogeneous membranes. Model membranes containing cholesterol. Biophys. J. 1980, 30, 383−397. (4) Sankaram, M.; Thompson, T. Interaction of cholesterol with various glycerophospholipids and sphingomyelin. Biochemistry 1990, 29, 10670−10675. (5) van der Wouden, J. M.; Maier, O.; Slimane, T.; van Ijzendoorn, S. Membrane dynamics and cell polarity the role of sphingolipids. J. Lipid Res. 2003, 44, 869−877.

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Phase Transition of Glycolipid Membranes Studied by Coarse-Grained

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