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Phase Transition of Glycolipid Membranes Studied by CoarseGrained Simulations


Raisa Kociurzynski, Martina Pannuzzo, and Rainer A. Bockmann*
Computational Biology, Department of Biology, Friedrich-Alexander University Erlangen-Nurnberg, 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 dierent partitioning and
interaction pattern of glycolipids in the membrane as
compared to those of phospholipids are likely due to their
dierent 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 inuence 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.

INTRODUCTION
Lipid rafts are membrane-ordered microdomains which exhibit
a dierent composition and ordering than the surrounding
environment and can phase separate and oat in an otherwise
disordered membrane. These domains carry out a number of
important physiological functions: they host signal transduction
pathways and inuence 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
dierent phase behavior than pure phospholipid membranes.
2015 American Chemical Society

Lipid bilayers are known to exist in dierent 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 dened as the main phase-transition temperature (Tm). The
phase transition is a highly cooperative, ordereddisordered
transition in which the bilayer undergoes lateral expansion and
a decrease in thickness and density. The hydrocarbon chain
packing changes from an all-trans conguration 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 aects 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
inuenced 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 inuences 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 2040 C as compared to that of
Received: May 3, 2015
Revised: July 17, 2015
Published: August 12, 2015
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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, uorescence spectroscopy and surface pressurearea
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
uid 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 dierential calorimetry studies1317 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 nding is supported by surface
pressure and uorescence 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 uorescence 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 eect 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 eect 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 eect 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, uorescence 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
uorescence 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 1520 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 eect 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
dierently aect the degree of order/disorder of the lipid tails
and inuence the architecture of the membrane. Overall,
important insight into possible functional roles of dierently
sized glycolipid headgroups on the membrane phase and
domain formation is obtained.

SIMULATION METHODS
Coarse-grained simulations were employed to study the phase
transition of mixed DPPC/glycolipid membranes and the
inuence of glycolipids on the membrane structure.
Coarse-Grained Simulations. The framework of the
Martini parametrization2729 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 eld 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 eld,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 eect for the
addition of charged GM1 lipids on the phospholipid phase
transition as compared to experiment. Additionally, GM1
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by sti bonds. The systems were equilibrated for 4 ns at 300 K


to let the lipids relax and the remaining water diuse 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 phasein particular, possible cluster
formationis considerably hampered due to the drastically
reduced diusion, we additionally performed cooling simulations starting from equilibrated uid-phase structures. For
statistics, each simulation was repeated several times by using
slightly diering 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
ParrinelloRahman barostat37 was used with semiisotropic
pressure coupling and a time constant of 4.0 ps, a
compressibility of 4.5 105 bar1, 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 o 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 ve 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, tted using the
Heaviside function. Linear ts 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 leaet. 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 articially enhanced


clustering. In turn, for GCER the phase-transition temperature
was shifted similarly for the standard and for the polarizable
Martini force eld (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 les were obtained
from the Martini Web site (http://cgmartini.nl). Rened force
eld parameters for GM1 were provided by the Martini group,
based on the recently published Martini force eld for
glycolipids.29 The bonded parameters were modied for
increased stability and faithfulness to atomistic force elds.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 Z
2
2

(1)

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.

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.
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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
dierent systems are compared. Although the phase-transition
temperature is well-dened 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 dierent
transition temperatures for the dierent 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 diers 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 eld 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
signicantly decreased the area per lipid for both the gel and
liquid-crystalline states as compared to pure DPPC (Figure 2),
and GM1 aected 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 eect of GCER in the gel phase is coupled
to the small GCER headgroup: the size of the phospholipid
headgroup gives rise to comparably large chainchain
distances. For the mixed DPPC/GCER system, GCER tailmediated interactions between the phospholipid chains lead to
a tightening of the tails. This also aects 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 (t 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

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 signicantly 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 proles (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
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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 628 GM1 molecules were
observed for the DPPC/GM1 system (Figure 5A) in the
separate leaets with 17 GM1 clusters per leaet. (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 ve molecules. Consequently, around 714 GCER
clusters per leaet 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 leaet but also among molecules in opposite
leaets, 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 diering 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 conrmed in a CG simulation at a reduced GM1
concentration of 7% (>1000 lipids in total, 3.2 s 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 dierent membrane components analyzed for the systems DPPC, DPPC + 17% GM1, and DPPC + 17% GCER
for the gel (left) and the liquid-crystalline (right) phase.
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Solvent-Accessible Surface (SAS). The solvent-accessible


surface (SAS) area for the DPPC bilayer is signicantly
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 reects 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 s of simulation. The clusters were
formed in the liquid-crystalline phase. Only the PN 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
reects 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).

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 dierent 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 eect 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.
Dierently, GM1 molecules showed cluster formation in a
DPPC bilayer. The highly dynamic clusters involved a large
fraction of phospholipids as well, forming uctuating 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 (2)
tilt angle (deg)
average cluster size (molecules)

SAS (nm2)

gel
liquid-crystalline
gel
liquid-crystalline
gel
liquid-crystalline
whole bilayer
upper leaet
lower leaet
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)

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).
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Figure 7. Number of glycolipidglycolipid, glycolipidDPPC, and glycolipidwater 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 141168 and 309336 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 specic lipids or sterols
inuences 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 eect 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
eld 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 eld 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 eect on the main phase transition of phospholipid
membranes. The physicochemical properties of both the
glycolipid head and chain regions determine the dierential
interaction pattern with the phospholipids.

Notes

The authors declare no competing nancial interest.

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-Nurnberg.
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 ErlangenNurnberg (RRZE).

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 (0)9131 85-25409. Fax: +49 (0)9131 85-25410.


E-mail: rainer.boeckmann@fau.de.
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