Characterization of Symmetric and Asymmetric Lipid Bilayers

3346

J. Phys. Chem. B 2008, 112, 3346-3356

Characterization of Symmetric and Asymmetric Lipid Bilayers Composed of Varying Concentrations of Ganglioside GM1 and DPPC Ronak Y. Patel and Petety V. Balaji* School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India ReceiVed: July 28, 2007; In Final Form: December 14, 2007

Gangliosides are a group of structurally diverse, sialic acid containing glycosphingolipids embedded into the membrane via their hydrophobic ceramide moiety. To gain atomic level insights into the structural perturbations caused by Galβ3GalNAcβ4(NeuAcR3)Galβ4Glc1Cer (GM1), molecular dynamics (MD) simulations of a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer containing GM1 at five different concentrations have been performed. Biological membranes contain GM1 only on the exoplasmic leaflet. However, vesicles prepared in the laboratory contain GM1 in both the leaflets albeit unequally. Hence, simulations were performed with GM1 present in only one (asymmetric bilayers) or in both of the leaflets (symmetric bilayers) of the bilayer. In symmetric bilayers, there is a decrease in surface area, an increase in deuterium order parameter, and an increase in peak-to-peak distance of DPPC with increasing concentration of GM1. Thus, the overall area of the lipid bilayer decreases (condensation effect) and the thickness increases with increasing concentrations of GM1. Even in asymmetric systems, decrease in surface area and increase in deuterium order parameter of hydrocarbon chains of DPPC are observed. However, the decrease in bilayer area and the increase in bilayer thickness are not as much as in the symmetric bilayer.

Introduction Gangliosides are a group of structurally diverse, sialic acid containing glycosphingolipids. They are embedded into the membrane via the hydrophobic ceramide moiety. The hydrophilic oligosaccharide headgroup protrudes out into the solvent phase. They occur ubiquitously in vertebrate cell membranes. They are particularly abundant in the nervous system and are predominantly localized on the outer layer of plasma membrane. Gangliosides have been implicated in a variety of cellular phenomena.1-5 Changes in ganglioside composition have been observed during both normal6 and tumor cell4 differentiation. The ability of gangliosides to act as cell surface receptors is exploited by certain bacteria for initial recognition and infection of the host cell.7-9 The organization, structure, and dynamics of gangliosides in model membranes have been studied extensively using differential scanning calorimetry,10 31P NMR spectroscopy and conductance and fluorescence measurements,11 fluorescence recovery after photobleaching,12 fluorescence,13,14 X-ray diffraction analysis,15,16 2H NMR spectroscopy,17 timeresolved small-angle X-ray scattering,18 and small-angle neutron scattering combined with small-angle X-ray scattering and dynamic light scattering19 and molecular dynamics (MD) simulations.20,21 Electron paramagnetic resonance (EPR) studies using 5-nitroxystearic acid as a probe on the fluidity and surface dynamics of ganglioside containing egg yolk phosphatidylcholine (EPC) small, unilamellar vesicles (SUVs) showed that gangliosides reduce the mobility of the hydrocarbon chains around the probe in a concentration-dependent (4-22 mol % Galβ3GalNAcβ4(NeuAcR3)Galβ4Glc1Cer (GM1)) manner: the value of the order parameter S increased from 0.587 (no GM1) to 0.629 (22 * Author for correspondence. Phone: +91-22-25 76 77 78; fax: +9122-25 72 34 80; e-mail: [email protected].

mol % GM1).22 This was attributed to strong interactions at the bilayer surface among gangliosides and between ganglioside and phosphatidylcholine (PC). Steady-state fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the membrane probe showed that incorporation of 30 mol % GM1 increases membrane order of 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3phosphatidylcholine (DMPC) membranes in multilamellar liposomes.23 Studies on the effect of bovine brain gangliosides on membrane dynamics of intact cells with the use of fluorescence probes DPH, N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenylammonium p-toluenesulfonate (TMA-DPH), and 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan) showed that the incorporation of gangliosides into the cell membrane substantially enhances the disorder and hydration of the lipid bilayer region near the exoplasmic surface.24 MD simulations of GM1 in DMPC lipid bilayer showed that GM1 induces local disorder in the arrangement of the acyl chain as well as in DMPC headgroups.20 MD simulations of a single GM1 molecule in DPPC bilayer showed that GM1 significantly disturbs the orientation of PfN vector and decreases the order parameter of hydrocarbon chains of neighboring DPPC molecules.21 Oriented multibilayers and unoriented suspensions of multiwalled vesicles of EPC with varying amounts of GM1 (9:1 to 7:3 EPC:GM1) were studied by X-ray diffraction analyses at 20 °C; the samples were subjected to osmotic stress by incubating the dry lipids in a solution of the neutral polymer poly(vinylpyrrolidone); the samples gave diffraction bands characteristic of liquid-crystalline phase and showed that incorporation of up to 30 mol % GM1 has little effect on bilayer organization.15 Furthermore, on the basis of model calculations with electron density strip models, the oligosaccharide headgroup was suggested to extend at least 1.2 nm beyond the EPC headgroup into the fluid space. X-ray diffraction studies of a

10.1021/jp075975l CCC: $40.75 © 2008 American Chemical Society Published on Web 02/26/2008

Lipid Bilayers Composed of Ganglioside

J. Phys. Chem. B, Vol. 112, No. 11, 2008 3347

Figure 1. Schematics showing residue and torsion angle numbering for the oligosaccharide headgroup of GM1 (upper panel), atom nomenclature for ceramide (middle panel), and atom and dihedral nomenclature of DPPC (lower panel). Initial conformations around the C-C bonds of ceramide chain are trans except as indicated and these are from ref 63.

binary mixture of GM1 at 5.7 mol % and DPPC showed that the system is in a gel phase with hexagonally packed hydrocarbon chains and that the addition of 5.7 mol % GM1 to DPPC bilayer has little effect on the preexisting DPPC bilayer structure in this phase.25 Mixed monolayers of 0-100 mol % of GM1 and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) were studied in solid phase at 23 °C by synchrotron gazing incidence X-ray diffraction and reflectivity studies; the GM1 headgroup at the air-water interface was found to extend from the monolayer surface, and the glycolipid did not affect the packing of DPPE hydrocarbon tails.16 Taken together, the above studies disagree with each other with respect to the effect of GM1 on the bilayer: whereas the EPR22 and steady-state fluorescence studies23 suggest that GM1 increases order, fluorescence probes24 and MD simulations20,21 suggest that GM1 decreases order, and X-ray crystallographic studies15,25 suggest that GM1 leaves the bilayer unchanged. In the present study, MD simulations of a DPPC bilayer containing GM1 at five different concentrations have been performed to gain atomic level insights into the structural perturbations caused by GM1 and to resolve these discrepancies. Biological membranes contain GM1 only on the exoplasmic leaflet. However, vesicles prepared in the laboratory contain GM1 in both the leaflets albeit unequally (see, for example, ref 19). Hence, simulations were performed with GM1 present in only one leaflet (asymmetric bilayers) or in both the leaflets (symmetric bilayers) of the bilayer. The data from the simulations of a pure DPPC bilayer (49 molecules in each leaflet) published elsewhere21 have been used here merely to facilitate comparison. Methods Conventions and Definition. The residues of the oligosaccharide headgroup are numbered sequentially starting from the reducing end of the oligosaccharide (Figure 1). The intersaccharide linkages are given the same number as the nonreducing

saccharide of that linkage. The intersaccharide torsion angle φ is defined as H1-C1-O-CX′ except in NeuAc3-R2,3-Gal2 where it is taken as C1-C2-O-C3′; angle ψ is defined as C1-O-CX′-HX′. The conformation of the saccharideceramide linkage Glc1-β1,1-Cer is described by the angles φ1, ψ1, and θ1, which are defined as Glc1:H1-Glc1:C1-Glc1: O1-Cer:C1, Glc1:C1-Glc1:O1-Cer:C1-Cer:C2, and Glc1: O1-Cer:C1-Cer:C2-Cer:C3, respectively. Generation of Initial Configuration. The initial conformation of the ceramide-saccharide and various intersaccharide linkages are as follows: (φ1, ψ1, θ1) ) (60, -60, 180), (φ2, ψ2) ) (60, 0), (φ3, ψ3) ) (180, 0), (φ4, ψ4) ) (30, -60), and (φ5, ψ5) ) (30, 0); these are the same as those used in an earlier study (simulation G in ref 21). The DPPC bilayer configuration obtained at the end of the previously reported simulation ABI26 was taken without the water molecules as the initial configuration for the present study. DPPC molecules to be replaced by GM1 molecules were chosen randomly but in such a way that the ceramides are not in immediate vicinity of each other. The replacement was done such that the X- and Y-coordinates of the C2 atoms of GM1 and DPPC to be replaced (Figure 1) are the same. The Z-coordinate of GM1:O1′ is the same as the average of the Z-coordinates of DPPC:O22/O32 atoms in that leaflet. The resulting system was energy minimized, was enclosed in a box of size 4.9 × 6.1 × 11 nm, and was solvated with SPC water molecules. One water molecule was replaced by a Na+ using the genion module of GROMACS for every sialic acid moiety present in the system. Water molecules in the hydrophobic core of the lipid molecules were removed. Simulation Details. The united atom GROMOS force field with the modifications suggested by Spieser et al.27 was used for carbohydrates. This force field has been used to simulate GM3 bilayer28 and GM1 in DPPC bilayer.21 The RyckaertBellemans potential as used for phospholipid hydrocarbon chains29 was used for the ceramide hydrocarbon tail. The

3348 J. Phys. Chem. B, Vol. 112, No. 11, 2008

Patel and Balaji

Figure 2. Snapshots of the 10 bilayer systems (Table 1) just prior to the equilibration run; the system identifier is at the bottom of each snapshot. All the snapshots are viewed perpendicular to the XZ-plane as can be inferred from the frame of reference shown at the bottom left. DPPC and ganglioside molecules are rendered as wire-frame and space-fill, respectively, using Pymol.64 Water molecules have been omitted from the display for clarity. Depth cueing was set on.

nonbonding parameters between the atoms of DPPC and the atoms of GM1 were taken as suggested for the lipid force field (lipid.itp; http://moose.bio.ucalgary.ca/ index.php?page)Downloads). The nonbonding interaction between NeuAc3:O11/O12 and GalNAc4:HO6/Gal2:HO2 was excluded to prevent the NeuAc3-R2,3-Gal linkage from accessing disallowed conformations. The bond, angle, and dihedral bond parameter around the double bond of ceramide were the same as that used for the simulation of sphingomyelin bilayer.30 The charges of DPPC are taken from Chiu et al.,31 and the bonding parameters of DPPC are from Berger et al.29 Simulations were performed using GROMACS 3.1.432,33 run in parallel on Intel Xeon 2.4/2.8/3.2 GHz dual processor systems under Red Hat Linux 8.0. Simulations were performed in NPT ensemble with anisotropic pressure coupling.26 Pressure was coupled using Berendsen’s algorithm;34 reference pressure was set to 1 bar, coupling constant was set to 1 ps, and isothermal compressibility was set to 4.5 × 10-5 bar-1. The temperature was kept constant at 325 K through an external thermostat34 with coupling constant of 1 ps. The simulation temperature was chosen such that it is higher than the main transition temperature (viz., 315 K) for DPPC.35 van der Waals interactions were cut off at 1.0 nm. Particle mesh Ewald summation method was used for long-range electrostatic interactions since it was found to be better than simple cutoffs;36,37 the cutoff was set to 1.0 nm for real space calculation. The bond lengths were constrained using SHAKE. Periodic boundary conditions were applied in all the three directions. The equations of motion were integrated with a time step of 2 fs, and the trajectories were saved at 4 ps intervals. Systems Simulated. Two types of bilayer systems were simulated: symmetric and asymmetric. Symmetric bilayers have GM1 in both leaflets of the bilayer whereas asymmetric bilayers have GM1 in only one of the two leaflets (Figures 2 and 3). Within each type, systems with varying number of GM1 were simulated (Table 1). The simulation system was first energy minimized, was simulated for 10 ps by restraining the solute

atoms, and was simulated for another 10 ps by restraining the atoms of DPPC alone. This was followed by 1 ns of equilibration and 20 ns of productive run. Analysis. The trajectories were analyzed using the standard GROMACS tools as well as in-house programs. The average area per DPPC and Cer was calculated using Voronoi tessellation and subsequent measurement of area of Voronoi polygons after sorting the vertices of polygons clockwise. For reasons discussed by Pandit et al.,38a the three atoms C2, C21, and C1of DPPC and C2, C1′, and C3 of Cer were used to calculate the area. The area was calculated for each leaflet separately, and the average area of molecules indicates the average over both leaflets. Results Area per Lipid. The most accepted area of DPPC molecule is 0.64 nm2.38b In the absence of GM1, the area per DPPC molecules is 0.654 nm2 (Table 2).21 Increasing the number of GM1 molecules is accompanied by a decrease in the size of the simulation box along the X- and/or Y-directions (Table 2). As the number of GM1 per bilayer increases, the area of DPPC decreases, especially in symmetric bilayer, whereas that of the ceramide moiety increases (Table 2). This is partly because of the replacement of larger area DPPC molecules by smaller area Cer and partly because of the condensing effect of GM1 on DPPC. Such a condensation of DPPC bilayer has been observed in previous MD simulation studies in the presence of 25% cholesterol (by 0.08 nm2, ref 39, or by 0.18 nm2, ref 40), 25% DPPE (by 0.06 nm2, ref 41), or ∼16% 1,2-dipalmitoyl-snglycero-3-phosphatidylserine (DPPS) + ∼0.19 M NaCl (by 0.02 nm2, ref 42). A decrease in the area of DPPC (by 0.08 nm2, compared to pure hydrated DPPC bilayer) in the presence of ∼25% GM1 (simulation system Sy78:20, Table 1) observed in the present study is comparable to the condensation induced by cholesterol (0.08 nm2) or DPPE (0.06 nm2). In the case of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)



Figure 3. Snapshots depicting the configurations of simulated systems at 10 ns. For each block, the snapshots show the systems through the Y-axis (XZ-plane; left panel) and the Z-axis (XY-plane; right panel). GM1 and DPPC are rendered as purple and orange spheres, respectively. In the left panel of each block, DPPC is rendered as yellow transparent spheres to view GM1 molecules. In the right panel, eight periodic frames (X, -X, Y, and -Y) are shown in addition to the actual box. The actual box is shown with black lines. Snapshots are prepared using VMD.65

TABLE 1: Composition of the Simulated Systems no. of molecules of systema

DPPC

GM1

water

GM1 mol %

Sy94:4 Sy90:8 Sy86:12 Sy82:16 Sy78:20 Asy96:2 Asy94:4 Asy92:6 Asy90:8 Asy88:10

94 90 86 82 78 96 94 92 90 88

4 8 12 16 20 2 4 6 8 10

6614 6466 6328 6160 6037 6673 6642 6567 6484 6428

4.25 8.88 13.95 19.51 25.64 2.08 4.25 6.52 8.88 11.36

a The prefixes Sy and Asy denote symmetric and asymmetric bilayers, respectively. The numbers x:y indicate the total number of molecules of DPPC (x) and GM1 (y). Equal number of GM1 molecules is present in the two bilayers in the case of symmetric bilayers. The total number of molecules (DPPC + GM1) is 49 in each bilayer.

bilayers, it was observed that ceramide has a lesser condensing effect than cholesterol at 33% concentration; the effect is more or less the same at lower concentrations (10, 20, or 25%).43 Fluorescence polarization23 and electron paramagnetic reso-

nance22 studies have also observed that the presence of GM1 in DPPC bilayer increases membrane order. The decrease observed in the area of DPPC molecules because of the presence of GM1 is more in symmetric bilayers than in asymmetric bilayers. For example, the area of DPPC is 0.629 nm2 in Sy94:4 but is 0.643 nm2 in Asy96:2 even though only two GM1 molecules are present in both cases. The difference is that in Sy94:4 the second leaflet of the bilayer also has two GM1 molecules whereas in Asy94:2 the second leaflet consists of only DPPC. Such a difference in the areas of DPPC is found in other cases also, namely, Sy90:8 compared to Asy94:4, Sy86: 12 to Asy92:6, Sy82:16 to Asy90:8, and Sy78:20 to Asy88:10 (Table 2). A decrease in the overall system size is observed for asymmetric systems also but not as much as in symmetric systems. The area of the ceramide moiety of GM1 is less than that of DPPC, and it varied between 0.433 ( 0.040 and 0.492 ( 0.033 nm2 for symmetric systems and between 0.406 ( 0.034 and 0.503 ( 0.012 nm2 for asymmetric systems (Table 2). The area of the ceramide moiety in a lipid bilayer has not been reported in literature so far. However, the area of ceramide in a ceramide

98:0c

Sy94:4

Sy90:8

Sy86:12

Sy82:16 (nm2) a

Sy78:20

Asy96:2

Asy94:4

Asy92:6

Asy90:8

Asy88:10

0.630 ( 0.006 0.620 ( 0.000 0.492 ( 0.067 0.622

0.640 ( 0 0.624 ( 0.000 0.503 ( 0.012 0.626

0.627 ( 0.001 0.604 ( 0.000 0.472 ( 0.01 0.605

0.622 ( 0.003 0.596 ( 0.000 0.486 ( 0.015 0.598

0.629 ( 0.002

0.606 ( 0.004

0.599 ( 0.004

Area 0.577 ( 0.006

0.573 ( 0.007

0.656

0.433 ( 0.040 0.623

0.466 ( 0.056 0.598

0.492 ( 0.033 0.591

0.446 ( 0.034 0.559

0.476 ( 0.030 0.557

0.643 ( 0.001 0.633 ( 0.000 0.406 ( 0.034 0.636

5.44 ( 0.17 5.91 ( 0.20

4.98 ( 0.11 6.13 ( 0.14

4.71 ( 0.11 6.22 ( 0.14

4.98 ( 0.05 5.81 ( 0.09

4.75 ( 0.07 5.77 ( 0.11

4.49 ( 0.09 6.08 ( 0.07

5.02 ( 0.14 6.20 ( 0.15

4.86 ( 0.08 6.27 ( 0.15

4.76 ( 0.18 6.45 ( 0.19

4.77 ( 0.10 6.22 ( 0.11

5.20 ( 0.06 5.64 ( 0.10

2.88

3.01 3.97 3.76 0.00 ( 0.15 0.21 ( 0.15 0.48 ( 0.08 0.11 ( 0.15 0.21 ( 0.15

3.27 4.06 4.17 -0.45 ( 0.00 -0.06 ( 0.08 0.39 ( 0.08 -0.17 ( 0.24 0.17 ( 0.40

Peak-to-Peak Distance (nm)e 3.30 3.59 3.66 4.78 5.15 5.00 4.21 4.43 4.26 -0.40 ( 0.24 -0.36 ( 0.00 -0.12 ( 0.17 0.00 ( 0.16 0.06 ( 0.08 0.30 ( 0.09 0.46 ( 0.16 0.48 ( 0.00 0.67 ( 0.09 0.06 ( 0.24 0.18 ( 0.08 0.37 ( 0.17 0.28 ( 0.08 0.42 ( 0.08 0.55 ( 0.43

3.01

3.02

3.19

3.19

3.18

3.87 -0.43 -0.11 0.21 0.00 0.32

4.14 -0.45 -0.11 0.22 -0.11 0.56

3.96 -0.44 0.00 0.44 0.00 0.11

3.99 -0.23 0.11 0.46 0.11 0.11

3.98 -0.11 0.23 0.68 0.23 0.68

C1:Gal2 f C5:NeuAc 3 C1:Gal2 f C4:Gal5 Hydrationf

40 ( 11 80 ( 3 32 ( 3

38 ( 7 71 ( 13 30 ( 3

Orientation of Sugar Headgroup (Degrees) 31 ( 5 28 ( 5 36 ( 2 60 ( 0 61 ( 0 65 ( 9 31 ( 3 32 ( 3 34 ( 3

45 ( 11 52 ( 11 28 ( 4

41 ( 27 61 ( 26 31 ( 4

40 ( 24 67 ( 14 30 ( 4

41 ( 18 75 ( 20 33 ( 2

32 ( 11 75 ( 23 34 ( 3

GM1‚‚‚G M1 GM1‚‚‚DPP C GM1‚‚‚water

4.7 ( 0.8 8.8 ( 1.2 23.4 ( 2.4

5.3 ( 0.6 8.6 ( 1.1 22.6 ( 2.3

5.7 ( 0.6 7.8 ( 1.0 23.1 ( 1.8

5.1 ( 1.2 9.6 ( 1.7 20.6 ( 3.2

5.7 ( 0.9 8.0 ( 1.5 22.9 ( 3.3

5.8 ( 0.7 8.2 ( 1.3 22.9 ( 2.9

5.6 ( 0.6 8.0 ( 0.7 24.4 ( 1.7

6.5 ( 0.6 6.8 ( 0.7 24.6 ( 2.2

DPPC in leaflet with GM1 DPPC in leaflet without GM1 ceramide area per lipid calculated from box sizeb X-box size (nm) Y-box size (nm)

0.654 0.654

DHH (DPPC)e DHH (GM1)d P-P P-Glc1 P-Gal2 P-NeuAc3 P-GalNac4 P-Gal5

3.60

No. of Hydrogen Bonds 6.5 ( 0.4 6.0 ( 0.4 7.2 ( 0.9 6.7 ( 0.7 23.5 ( 1.9 25.2 ( 2.1


TABLE 2: Structural Properties of Systems Simulated in the Present Study

a The values of the areas given for symmetric bilayers are the average values of the areas of the two apposing leaflets of the bilayer. b The overall system size was calculated as X-box × Y-box/total number of lipid molecules (i.e., DPPC + GM1) in a monolayer. c Data from ref 21. d DHH of GM1 was calculated as the peak-to-peak distance from the number density profile of GM1. e Negative values indicate that the saccharide is more toward the hydrocarbon core relative to phosphorus, whereas positive values indicate that the saccharide is more toward the solvent phase. f Number of water molecules in 0.35 nm of oxygen of GM1.

Patel and Balaji



Figure 4. Hydrophobic thickness calculated from the number density profiles of carbon atoms of hydrophobic tail of DPPC/GM1 molecules together for each leaflet of bilayer (upper panel). The interfacial width calculated as distance where the number density of water reduced from 90% to 10% of bulk water (lower panel). The left and right panels show the data of symmetric and asymmetric systems, respectively. The series labeled as “with GM1” and “without GM1” refer to the monolayer with GM1 + DPPC and monolayer with DPPC, respectively, for asymmetric bilayer; for symmetric bilayer, both the layers have the same number of GM1. For the DPPC bilayer system 98:0, the data is from an earlier publication,21 and this has been included in all the figures merely to facilitate comparison with other data.

bilayer has been obtained by two different MD simulations studies: 0.551 ( 0.007 nm2 in a system comprising 100 ceramide molecules per leaflet plus 50 water molecules per lipid at 368 K44 and 0.388 ( 0.005 nm2 in a system consisting of 64 ceramide molecules per leaflet plus 40 water molecules per lipid at 363 K.45 The area of sphingomyelin (18:0) in a pure sphingomyelin bilayer was found to be 0.53,30 0.55,46 and 0.520.56 nm2.47 The area of PSM in a system of 1,-2-dioleoyl-snglycero-3-phosphatidylcholine (DOPC):palmitoylsphingomyelin (PSM):cholesterol, 12:1:1, was calculated to be 0.495 ( 0.004 nm2.38a Interfacial Width and Bilayer Thickness. The interfacial width, as calculated from the distance where the number density of water decreases from 90% to 10% of bulk water, shows broadening of interface as a function of GM1 concentration for both leaflets of symmetric systems (Figure 4). In the density profiles of systems with higher concentrations of GM1, namely, Sy82:16 and Sy78:20, the interface region has two overlapping peaks instead of a single peak observed in other systems (Figure 5). The peak-to-peak distance calculated from the density profiles of DPPC, GM1, and phosphorus atoms increases with increase in GM1 concentration indicating bilayer swelling (Table 2). In asymmetric systems, the lipid water interface broadens only for the GM1-containing leaflet in asymmetric systems, and the density profile shows a shift with the lowest density point not coinciding with the zero of Z-axis (Figure 5). The number density profiles of carbon atoms of hydrocarbon tails of DPPC and Cer together were plotted, and the minimum and the maximum along the Z-box were used to calculate the hydrophobic thickness. For symmetric systems, for each leaflet of bilayer, an increase in the hydrophobic thickness is observed as a function of ganglioside concentration (Figure 4). In contrast, an increase in hydrophobic thickness is observed only for the leaflet containing GM1 for asymmetric systems. The leaflet

without GM1 does not show concentration-dependent pattern in hydrophobic thickness. Protrusion of Saccharide Headgroups. Phosphorus to saccharide peak-to-peak distances were calculated to analyze the distribution of the saccharide headgroup with respect to lipid phosphorus atoms (Figure 6). The peak-to-peak distances tend to increase as a function of GM1 concentration. This is probably due to an increase in GM1-GM1 interactions as a function of GM1 concentration. A somewhat similar trend is observed for asymmetric bilayers also. It appears as if Gal5 is not protruded out of the bilayer in systems Asy92:6 and Asy90:8; however, in these systems, the distribution of Gal5 is quite large along the bilayer normal and the number density profile has two overlapping peaks instead of a single peak. The presence of GM1 in EPC multibilayers/multiwalled vesicles under osmotic stress conditions did not alter the peakto-peak distance between the phosphorus atoms in a PC bilayer.15 A similar observation was made from the X-ray diffraction studies of 5.7 mol % GM1 and DPPC in gel phase.25 In the present study, an increase in the peak-to-peak distance is observed (Table 2), but the system is fully hydrated and is simulated at a temperature above the phase-transition temperature of DPPC. The headgroup of GM1 is rich in functional groups that can participate in hydrogen-bonding interactions. GM1 headgroups tend to interact more strongly with the surrounding DPPC molecules than with each other (Table 2). This may also be responsible for ordering the hydrophobic core of lipid bilayer, consequently leading to an increase in bilayer thickness. MD simulations of mixed bilayer of DPPE and DPPC have shown that increasing the concentration of DPPE in DPPC bilayer leads to an increase in the P-P distance; this has been attributed to hydrogen bonding and smaller headgroup of DPPE compared to DPPC.41 The bilayer swelling observed in our simulations can also be due to the difference in the chain length


Patel and Balaji

Figure 6. The distances from the phosphorus peak to saccharide peaks calculated from number density profiles of symmetric (upper panel) and asymmetric (lower panel) bilayers. The error bars show the standard deviation between the phosphorus to saccharide distances of the two monolayers.

Figure 5. Number density profiles of various groups as a function of Z-box. The simulation box along the Z-axis (i.e., along the bilayer normal) is divided into 100 equal bins, and the density of the solute atoms/atoms of the indicated group was averaged for the entire 20 ns trajectory data. The upper three panels show the profiles of symmetric bilayers whereas the lower three panels show the profile of the asymmetric bilayers.

of N-linked fatty acid of GM1 (18:0) and fatty acid of DPPC molecules (16:0). For asymmetric systems, the increase in P-P and DHH is small compared to P-P and DHH of pure DPPC bilayer. P-P and DHH are very nearly the same for systems Asy92:6, Asy90:8, and Asy88:10. Deuterium Order Parameter. Motional freedom of carbon atoms of hydrocarbon tail can be quantified using deuterium order parameter. The order parameter profiles of DPPC and GM1 as a function of ganglioside concentration show that the deuterium order parameter of hydrocarbon chains increases with increasing concentration of GM1 (Figure 7) implying that the acyl chains become more ordered for symmetric systems. The increase in deuterium order parameter of hydrocarbon chains of DPPC and GM1 in asymmetric bilayer is not as steep as the increase in the order parameter of symmetric lipid bilayers. The presence of GM1 in only one bilayer increases the order parameter of DPPC of both the leaflets compared to that of pure DPPC bilayer; however, the leaflet with GM1 is somewhat less ordered compared to the leaflet without GM1 (Figure 7). Orientation of PC and Saccharide Headgroups. The orientations of PfN vectors of DPPC with respect to bilayer normal follow a more or less normal distribution for systems

Sy94:4, Sy90:8, Sy86:12, Asy96:2, and Asy92:6 whereas the distribution deviates somewhat from a Gaussian distribution for other systems (Figure 8). An earlier MD simulation study of a single GM1 molecule in a DPPC bilayer has shown that GM1 disturbs the orientation of PfN vector of neighboring DPPC molecules; however, when the orientation of PfN vector for the entire bilayer is plotted, it shows Gaussian distribution.21 The orientations of the vectors C1:Gal2 f C5:NeuAc3 and C1:Gal2 f C4:Gal5 with respect to bilayer normal have been used to determine the orientation of the GM1 headgroup (Figure 9; Table 2). In symmetric bilayers, C1:Gal2 f C5:NeuAc3 prefers an orientation parallel to the bilayer normal at higher GM1 concentrations, and this can be attributed to lateral interactions of GM1. The Gal2-GalNAc3-Gal5 branch prefers a surface-parallel orientation. In asymmetric bilayers Asy94:4, Asy92:6, and Asy90:8, the distribution of the angles subtended by the vector C1:Gal2 f C5:NeuAc3 shows bimodal distribution. The major peak is at ∼30° and the minor peak is at ∼90°. For systems Asy96:2 and Asy88:10, a single peak is observed at ∼55° and 25°, respectively. This shows that NeuAc3 becomes surface parallel with increasing concentrations of GM1. Hydration and Hydrogen Bonding. The number of GM1‚ ‚‚water hydrogen bonds does not show linear dependence on GM1 concentration. However, the number of GM1-GM1 hydrogen bonds increases and GM1-DPPC hydrogen bonds decreases with increase in concentration for both symmetric and asymmetric systems (Table 2). As its concentration increases, GM1 is more likely to be in the neighborhood of another GM1 than a DPPC. The variation in the number of water molecules found within 0.35 nm of the oxygen atoms of GM1 (Table 2) is similar to that observed for a single GM1 in a DPPC bilayer.21 A differential scanning calorimetry (DSC) study carried out to determine the effect of various lipids on the lowering of the



Figure 7. The average deuterium order parameter of carbon numbers 5-9 of fatty acid chain of DPPC (left panel) and carbon numbers 8-12 of base and 5-9 of fatty acid chain of GM1 (right panel). The average deuterium order parameters calculated over carbon atoms C5-C9 of DPPC by 2H NMR studies are shown in the left panel by the symbols “+” [at 321 K; ref 66] and “/” [at 314 K; refs 67, 68].

Figure 8. Distribution of the angles subtended by the PfN vectors with the bilayer normal (viz., the Z-axis of the simulation box) for symmetric (left panel) and asymmetric (middle and right panels) systems.

Figure 9. Distribution of the angles subtended by the vectors Gal2:C1fNeuAc3:C5 (left panel) and Gal2:C1fGal5:C4 (right panel) with the bilayer normal (viz., the Z-axis of the simulation box) for symmetric (upper panel) and asymmetric lipid bilayers (lower panel). Value close to zero indicates an orientation parallel to bilayer normal and value close to 90 indicates an orientation parallel to the bilayer surface.

melting point of water found 22-30 molecules of water per molecule of GM1 to be apparently unfreezable.10 On average, about 30 molecules of water were found to be tightly bound to the headgroup of GM1 at 200:1 2H2O/ganglioside molar ratio by 2H NMR spectroscopic study.17 From time-resolved smallangle X-ray scattering measurements, about 50 water molecules per molecule of GM1 was found to be occluded in the micelle at ∼50 °C.18 Electrostatic Potential. The electrostatic potential profiles of the symmetric bilayer systems across the Z-axis are more or less similar to each other and are thus independent of GM1 concentration (Figure 10). The bulk water has a net negative

electrostatic potential, while the interface has a positive electrostatic potential compared to the hydrophobic core. As the numbers of GM1 and DPPC in both the apposing leaflets are equal, no significant difference in electrostatic potential is observed as a function of GM1 concentration. The shifting and broadening of peaks (indicating the interfacial regions) with respect to the Z-axis of the system is in accordance with the swelling observed as a function of GM1 concentration. For asymmetric lipid bilayers, the electrostatic potential of leaflet with GM1 is more on the negative side compared to electrostatic potential of leaflet without GM1 (Figure 10). The difference in the electrostatic potential of apposing leaflet


Figure 10. The electrostatic potential along the Z-direction of the simulation box for symmetric (upper panel) and asymmetric systems (lower panel). Electrostatic potential was calculated by dividing the simulation box into 100 equal-sized bins. The electrostatic potential of the bilayer center has been taken as 0. For asymmetric systems, the calculation proceeded as monolayer containing GM1, water phase, and monolayer without GM1.

increases with increasing concentrations of GM1. The number density profiles of counterions (data not shown) show that they are present even facing the leaflet without GM1. This causes the electrostatic potential to be more positive on the leaflet without GM1 (Figure 10). Discussion Presently, there is a growing interest in the characterization of asymmetric48-50 and mixed symmetric41,42,51-57 lipid bilayers by MD simulations. Simulations of the asymmetric model membranes have phosphatidylserine (PS) in one of the leaflets because of its presence in the inner leaflet of biological membranes. Generally, an asymmetric bilayer implies differences in the nature of phospholipids making up the two monolayers; however, in this study, the phospholipid in both the monolayers is DPPC and the asymmetry arises from the presence of GM1 in only one of the monolayers. The set of simulations performed in this study shows that increasing the concentration of GM1 in symmetric bilayers results in a decrease in the surface area of DPPC (Table 2) and an increase in the deuterium order parameter (Figure 7) and the peak-to-peak distance of DPPC (Table 2). The decrease in the surface area of DPPC (0.08 nm2 at 25% GM1 compared to pure DPPC bilayer, Table 2), that is, bilayer condensation caused by GM1, is as much as that caused by cholesterol (0.08 nm2; ref 39) and DPPE (0.06 nm2; ref 41) at similar concentrations. A decrease in surface area (Table 2) and an increase in deuterium order parameter (Figure 7) of hydrocarbon chains of DPPC were observed in asymmetric systems also. However, the decrease in bilayer area and the increase in bilayer thickness are not as much as in the symmetric bilayer. The ordering observed in the present study can be attributed to differences in the nature of the headgroup/hydrocarbon tail of GM1 and DPPC. GM1 headgroup is rich in hydrogen bond donors and acceptors. At higher concentrations, GM1 is more

Patel and Balaji likely to interact with GM1 compared to DPPC as evident from the number of hydrogen bonds between GM1‚‚‚GM1 and GM1‚ ‚‚DPPC (Table 2). The increase in lateral interactions between GM1 molecules might be responsible for ordering of bilayer at higher concentrations. The ordering effect of GM1 on bilayer has also been observed previously.22,23 The ordering of GM1 observed on EPC SUVs was attributed to strong interactions at the bilayer surface among gangliosides and between ganglioside and PC.22 However, some studies have also suggested that GM1 either decreases the order of20,21,24 or has no effect on15,25 the bilayer in which it is embedded. The different conclusions reached by these studies can be traced to the differences in the nature of the system studied, conditions under which the study was carried out, and the technique used for the study; for example, the gel phase was investigated in one of the X-ray diffraction studies,25 whereas in the other the samples were subjected to osmotic stress by incubating the dry lipids in a solution of the neutral polymer poly(vinylpyrrolidone).15 One of the MD simulations considered only one GM1 embedded in a bilayer of 97 DPPC molecules,21 whereas the other MD simulation study had used single GM1 in a bilayer composed of 15 DMPC molecules.20 The fluorescence studies used bovine brain gangliosides (variable composition) to study membrane dynamics of intact cells.24 An asymmetric bilayer containing 96 DPPC + 48 DPPS in one leaflet and 120 DPPC alone in another monolayer was simulated.48 It was found that the properties of DPPC molecules in the leaflet without DPPS are as in a pure DPPC bilayer; however, in the leaflet containing both DPPS and DPPC, the properties of DPPC are strongly affected by the presence of DPPS. Even in the simulations of the asymmetric bilayer wherein one leaflet had 42 sphingomyelin (SM) + 22 cholesterol and the other had 42 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (SOPS) + 22 cholesterol, it was found that the properties of one leaflet are not perturbed by the composition of the other leaflet.50 In the asymmetric systems simulated in the present study, the surface area decreases (Table 2) and the hydrocarbon chains become more ordered (Figure 7) in the leaflet without GM1 compared to that in a pure DPPC bilayer. These results indicate that the presence of GM1 in DPPC does not only influence the monolayer in which it is present but also influences the apposing leaflet. Charge distribution at the lipid bilayer surface is known to influence the activity of several proteins.58 Bilayer membranes containing GM1 have been used as models for investigating the electrostatic potentials adjacent to biological membranes.11 Even though counterions are present in the system to neutralize the charge of GM1, in asymmetric systems the monolayer without GM1 has a positive electrostatic potential compared to that of the monolayer with GM1. In the simulations of an asymmetric bilayer consisting of 137 POPC, 91 POPE, and 46 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), counterions were found to be more frequently associated with the POPE + POPS leaflet than with the POPC leaflet.49 Even in the asymmetric bilayers simulated in the present study, sodium ions are found more frequently on the side of the leaflet which contains GM1 (data not shown). It has been shown that the electrostatic potential of the PC + GM1 bilayer becomes more negative when the GM1 content increases.11 An interesting observation made in this study is that the protrusion of the saccharide moieties of GM1 increases with increasing concentrations of GM1. This might be due to two structural changes brought about by increasing concentrations of GM1: (1) the bilayer becomes less fluid and somewhat tightly

Lipid Bilayers Composed of Ganglioside packed with increasing concentration of GM1 (Figure 7) and (2) the increase in GM1-GM1 interactions (Table 2). It has earlier been shown that gangliosides tend to self-associate and form clusters.59-62 Since the presence of five GM1 molecules in proximity is required for binding of cholera toxin, it is tempting to suggest that self-association and formation of clusters may facilitate the presentation of GM1 to its macromolecular ligands. The present study provides useful insights into the nature of changes in the properties of DPPC bilayers brought about by increasing concentrations of GM1. Even though the systems simulated in this study are model bilayers, the distribution of GM1 in asymmetric bilayers mimics that in a biological membrane whereas the distribution in symmetric bilayers mimics that in vesicles prepared in vitro for various experimental studies. Further, this study assists in the systematic reconstruction and analysis of physiological membranes containing gangliosides and other proteins. Abbreviations. Galβ3GalNAcβ4Galβ4Glc1Cer; Cer, ceramide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (14:0); DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (18:1, cis-9); DPH, 1,6-diphenyl-1,3,5-hexatriene; DPPC, 1,2dipalmitoyl-sn-glycero-3-phosphatidylcholine (16:0); DPPE, 1,2dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (16:0); DPPS, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylserine (16:0); DSC, differential scanning calorimetry; EPC, egg yolk phosphatidylcholine; EPR, electron paramagnetic resonance spectroscopy; Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine; Glc, D-glucose; GM1, Galβ3GalNAcβ4(NeuAc3)Galβ4Glc1Cer; Laurdan, 6-lauroyl-2-(dimethylamino)naphthalene; MD, molecular dynamics; NeuAc, N-acetyl-D-neuraminic acid; PC, phosphatidylcholine; PS, phosphatidylserine; PSM, palmitoyl sphingomyelin; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (16:0-18:1); SM, sphingomyelin; SUV, small, unilamellar vesicle; TMA-DPH, N,N,N-trimethyl-4-(6-phenyl1,3,5-hexatrien-1-yl)phenylammonium p-toluenesulfonate. Acknowledgment. R.Y.P. thanks the Indian Institute of Technology Bombay for a research fellowship. Part of the hardware used for this work was supported by a grant from the Council of Scientific and Industrial Research, India, to P.V.B. under the NMITLI scheme (5/258/10/2002-NMITLI). The authors are grateful to the anonymous referee for his useful and critical comments, which among other things, helped improve the manuscript. References and Notes (1) Allende, M. L.; Proia, R. L. Curr. Opin. Struct. Biol. 2002, 12, 587-92. (2) Hakomori, S. I. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 225-232. (3) Miljan, E. A.; Bremer, E. G. Science’s STKE 2002, 2002, RE15. (4) Birkle, S.; Zeng, G.; Gao, L.; Yu, R. K.; Aubry, J. Biochimie 2003, 85, 455-63. (5) Schnaar, R. L. Arch. Biochem. Biophys. 2004, 426, 163-72. (6) Yu, R. K.; Bieberich, E.; Xia, T.; Zeng, G. J. Lipid Res. 2004, 45, 783-793. (7) Van Heyningen, S. Science 1974, 183, 656-657. (8) Lindberg, A. A.; Brown, J. E.; Strmberg, N.; Westling-Ryd, M.; Schultz, J. E.; Karlsson, K. A. J. Biol. Chem. 1987, 262, 1779-1785. (9) Fukuta, S.; Magnani, J. L.; Twiddy, E. M.; Holmes, R. K.; Ginsburg, V. Infect. Immun. 1988, 56, 1748-1753. (10) Bach, D.; Sela, B.; Miller, I. R. Chem. Phys. Lipids 1982, 31, 381394. (11) McDaniel, R. V.; McLaughlin, A.; Winiski, A. P.; Eisenberg, M.; McLaughlin, S. Biochemistry 1984, 23, 4618-4624. (12) Goins, B.; Masserini, M.; Barisas, B. G.; Freire, E. Biophys. J. 1986, 49, 849-856.

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Characterization of Symmetric and Asymmetric Lipid Bilayers

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