Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy

Article abstract of DOI:10.1021/ja801789t

Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance (²H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state ²H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M₁ and the observed ²H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d₃₁ and POPC-d₃₁ and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.

Full text of DOI:10.1021/ja801789t

Published on Web 10/08/2008
Raftlike Mixtures of Sphingomyelin and Cholesterol
2
Investigated by Solid-State H NMR Spectroscopy
Tim Bartels,† Ravi S. Lankalapalli,‡ Robert Bittman,‡ Klaus Beyer,†,§ and
Michael F. Brown*,§,|,
Laboratory of NeurodegeneratiVe Disease Research, Ludwig-Maximilian-UniVersity,
80336 Munich, Germany, Department of Chemistry and Biochemistry, Queens College of The
City UniVersity of New York, Flushing, New York 11367, and Department of Biochemistry and
Molecular Biophysics, Department of Chemistry, and Department of Physics,
UniVersity of Arizona, Tucson, Arizona 85721
Received March 11, 2008; E-mail: mfbrown@u.arizona.edu
Abstract: Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is
associated with putative microdomains in cellular membranes and undergoes alterations due to aging or
neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and
ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) using deuterium nuclear magnetic resonance (2H NMR) spectroscopy in both macroscopically
aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC
perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state 2H NMR spectra
of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction
and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct
regions of the phase diagram. According to our analysis of the first moments M1 and the observed 2H
NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential
model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural
parameters such as chain extension and thermal expansion coefficient in order to further define the two
coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/
cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to
or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller
mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase
separation phenomena seem to be differences in the effects of cholesterol on the configurational order of
the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin
observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular
membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.
sphingolipids, cholesterol, and certain lipid-anchored proteins.3
Sphingomyelin is abundant in the nervous systems of mammals
Introduction
The concept that biomembranes are two-dimensional fluids
with randomly distributed proteins has been challenged by the
proposal that cellular membranes may contain areas of lateral
segregation known as lipid rafts.1 The presence of these domains
is typically associated with cellular functions such as vesicle
fusion, signal transduction, lipid trafficking, transcytosis, protein
sorting, and virus budding.2 Hence, they constitute an important
target for research in pharmaceutical and physical chemistry as
well as in cellular biology. As currently understood, rafts are
mostly depleted of unsaturated phospholipids and enriched in
and undergoes compositional changes due to aging or neuro-
degeneration. The observation that depletion of cholesterol from
biological membranes induces major changes in the distribution
and function of raft-associated proteins4 indicates that cholesterol
is an essential component of raft domains. However, a thorough
investigation of the physical basis for these observations is
complicated by the intricate lipid compositions of most biologi-
cal membranes. It is therefore desirable to further illuminate
the physical properties of lipids in simple raftlike model systems.
Raftlike domains are believed to occur in lipid systems with
coexisting liquid-disordered (ld) and liquid-ordered (lo) phases.5,6
The lo phase is characterized by a high rate of transverse
† Ludwig-Maximilian-University.
‡ Queens College of The City University of New York.
§ Department of Biochemistry and Molecular Biophysics, University of
Arizona.
(2) (a) Simons, K.; Vaz, W. L. Annu. ReV. Biophys. Biomol. Struct. 2004,
33, 269–295. (b) Riethmüller, J.; Riehle, A.; Grassmé, H.; Gulbins,
E. Biochim. Biophys. Acta 2006, 1758, 2139–2147. (c) Jacobson, K.;
Mouritsen, O. G.; Anderson, R. G. Nat. Cell Biol. 2007, 9, 7–14. (d)
Allen, J. A.; Halverson-Tamboli, R. A.; Rasenick, M. M. Nat. ReV.
Neurosci. 2007, 8, 128–140. (e) Wilflingseder, D.; Stoiber, H. Front.
Biosci. 2007, 12, 2124–2135.
| Department of Chemistry, University of Arizona.
Department of Physics, University of Arizona.
(1) (a) Simons, K.; Ikonen, E. Nature 1997, 387, 569–572. (b) Brown,
D. A.; London, E. Biochem. Biophys. Res. Commun. 1997, 240, 1–7.
(c) Edidin, M. Annu. ReV. Biophys. Biomol. Struct. 2003, 32, 257–
283.
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2008 American Chemical Society
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A R T I C L E S
Bartels et al.
diffusion and positional disorder, as found in the ld phase,
together with relatively ordered acyl chains, as occurs in the
solid-ordered (so) phase.5,7 The ld phase in these systems
typically contains highly unsaturated lipids with a low phase-
transition temperature, whereas the lo phase predominately
consists of a saturated or sphingolipid component with a high
phase-transition temperature and cholesterol. Analyzing the
thermodynamics and structural properties of such lipid mixtures
remains a challenging task.8 Experiments using fluorescence
techniques have led to substantial progress in understanding the
phase behavior and boundaries in ternary mixtures by their
ability to establish binary and ternary phase diagrams for several
lipid systems of current interest.9 Yet these phase diagrams are
often quite fragmentary at physiologically relevant temperatures
and in some cases can lead to questionable conclusions because
of the need to incorporate a fluorescent probe. This last
disadvantage has recently attracted significant attention, as
changes in phase behavior have been discovered upon addition
of even trace amounts of a fluorescent probe.10
abundance of this lipid in the nervous systems of mammals,
where it becomes increasingly enriched as a result of progression
in age,14 but it poses a synthetic challenge. The normal
semisynthetic pathway, namely, removal of the amide-linked
fatty acyl chain followed by reacylation with a deuterated
derivative, is hampered by partial epimerization at the C3 carbon
of the sphingosine backbone. Despite these difficulties, a
chemical synthesis of optically pure and selectively 2H-labeled
sphingomyelin is needed for an investigation of the putative
role of sphingomyelin in the formation of rafts.
In our studies, we used binary mixtures of cholesterol with
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or
N-palmitoylsphingomyelin (PSM), in which the palmitoyl chain
of POPC or PSM was perdeuterated, to study the different
effects of cholesterol on the saturated chains of the lipid.
Additionally, we investigated ternary mixtures containing both
lipids and varying cholesterol content at several temperatures
in order to obtain structural information on the lipids for
representative sections of the phase prism of the ternary mixture.
We observed each 2H-labeled lipid component separately, and
using a structural model, we were able to distinguish the order
parameter profile for each component in its respective phase.
For certain ranges of temperature and cholesterol content, lateral
phase separation due to the preferential interaction of cholesterol
with sphingomyelin was demonstrated. However, our data
suggest that little or no phase separation is detectable at
cholesterol mole fractions (XC) greater than 0.33, indicating that
PSM is in a homogeneous phase with POPC and cholesterol.
These concentrations are lower than those implied by published
phase diagrams9,15 and have important implications for lateral
organization and raft formation in biological membranes in vivo.
2
Solid-state H NMR spectroscopy, on the other hand, is a
versatile technique that lends itself to a noninvasive investigation
of the order and mobility of acyl chains and polar headgroups
in lipid bilayers.11,12 The residual quadrupolar couplings (RQCs)
of chain deuterons located within the lipid bilayer can be
evaluated in terms of segmental order parameters and order
parameter distributions, which may also yield knowledge about
partitioning of the lipids into different bilayer domains. Further
interpretation using structural models can provide information
on the mean interfacial area of a lipid or the average length of
the acyl chain projection, which is directly related to the
membrane thickness.12,13 Combining these measurements with
the overall ordering of the acyl chains, which is directly
accessible by solid-state 2H NMR experiments, enables one to
acquire information about the phase behavior of lipids in the
mixtures. Nonetheless, a limitation of this technique is the
availability of selectively deuterated lipid samples. Deuteration
of sphingomyelin is of particular interest because of the
Methods
Chemicals. Synthetic POPC, egg yolk sphingomyelin (EYSM),
cholesterol, and 1-perdeuteriopalmitoyl-2-oleoyl-sn-glycero-3-phos-
phocholine (POPC-d31) were purchased from Avanti Polar Lipids
(Alabaster, AL). Perdeuterated palmitic acid (palmitic acid-d31) and
deuterium oxide were obtained from Cambridge Isotopes (Promo-
chem GmbH, Wesel, Germany). Labeled N-perdeuteriopalmitoyl-
sphingomyelin (PSM-d31) was synthesized by N-acylation of
D-erythro-sphingosylphosphocholine as described previously.16
Briefly, the p-nitrophenyl ester of palmitic acid-d31 and anhydrous
potassium carbonate were added to lysosphingomyelin (prepared
from EYSM as previously described17) in a mixture of anhydrous
dimethylformamide and dichloromethane under nitrogen, and the
mixture was stirred at ambient temperature for 1 day. The products
were purified by silica gel column chromatography (eluting with
65:35:5 chloroform/methanol/water), and the suspended silica gel
was removed by filtration. Purity was checked by thin-layer
chromatography and liquid chromatography/mass spectrometry.
Lipid Sample Preparation. Oriented lipid multibilayers were
prepared and macroscopically aligned as described elsewhere.18,19
Briefly, 30 mg of the lipid or lipid mixture containing at least 12
mg of deuterated material was dissolved in 2.5 mL of tert-butyl
(3) (a) Brown, D. A.; Rose, J. K. Cell 1992, 68, 533–544. (b) Cinek, T.;
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Anderson, H. A.; Hiltbold, E. M.; Roche, P. A. Nat. Immunol. 2000,
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Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162–172.
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1990, 29, 10670–10675. (c) Mouritsen, O. G.; Zuckermann, M. J.
Lipids 2004, 39, 1101–1113. (d) Veatch, S. L.; Soubias, O.; Keller,
S. L.; Gawrisch, K. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17650–
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604, 129–158. (b) Cutler, R. G.; Mattson, M. P. Mech. Ageing DeV.
2001, 122, 895–908.
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(10) Veatch, S. L.; Leung, S. S.; Hancock, R. E.; Thewalt, J. L. J. Phys.
Chem. B 2007, 111, 502–504.
(15) Veatch, S. L.; Keller, S. L. Phys. ReV. Lett. 2005, 94, 148101-1–
148101-4.
(11) Gawrisch, K.; Eldho, N. V.; Polozov, I. V. Chem. Phys. Lipids 2002,
116, 135–151.
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939–946.
(12) Brown, M. F.; Lope-Piedrafita, S.; Martinez, G. V.; Petrache, H. I. In
Modern Magnetic Resonance; Webb, G. A., Ed.; Springer: Berlin,
2006; pp 245-256.
(17) Bittman, R.; Verbicky, C. A. J. Lipid Res. 2000, 41, 2089–2093.
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2441–2451.
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14522 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Raftlike Mixtures of Sphingomyelin and Cholesterol
A R T I C L E S
alcohol. The solution was spread onto 50 ultrathin glass plates (8.3
× 18.3 × 0.08 mm; Marienfeld Laboratory Glassware, Lauda-
Ko¨nigshofen, Germany) and dried for 20 min under a stream of
warm air and then at room temperature for at least 18 h in vacuo
(20-30 Pa). The glass plates were stacked on top of each other
with gentle pressure and inserted, along with a pair of glass cylinder
segments, into an open glass tube (inner diameter 9.8 mm). Two
small paper strips were soaked in 2H2O and carefully dried in order
to exchange labile hydrogen for deuterium. The strips were attached
to the short sides of the glass stacks, and a few microliters of 2H2O
or a 10:90 (v/v) 2H2O/H2O mixture was applied to the paper surface.
The tube was rapidly sealed with two appropriately machined Teflon
plugs having silicon O-rings. The membranes were annealed for
8 h at 46 °C. The annealing process and the final hydration level
were monitored by observation of the water signal at the offset
omitted because of their inequivalence; the almost parallel alignment
to the membrane surface of the pro-R deuteron and the ∼34° angle
of the pro-S deuteron in the case of 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC) does not allow a comparison with the other
methylene segments. Peak assignments were generally made by
assuming that the order parameters decrease in going from C3 to
the methyl end and that the area under each peak is proportional to
the number of deuterons. A more precise assignment of the 2H
NMR spectra of pure PSM-d31 and PSM-d31/POPC was possible
on the basis of previously published results.16,17
For the oriented samples, the number of water molecules per
lipid headgroup (nw) was obtained by calculating the integral ratio
of the respective 2H signals from 2H2O and the labeled lipids. The
hydration level was adjusted to obtain full hydration23 of the
respective bilayers (25 e nw e 35). The angle θ between the normal
to the glass plates and the magnetic field was varied until the
quadrupolar splittings ∆ν(Qi) reached a maximum, indicating that θ
) 0° (eq 1). In the case of the unoriented samples, the addition of
50 wt % of water assured complete hydration for the duration of
the experiments. For the unoriented samples, the first and second
moments Mn (n ) 1, 2) were calculated according to eq 2:
2
frequency of the H NMR spectrum.19
For unoriented samples, the lyophilized (from tert-butyl alcohol)
lipid mixture containing at least 20 mg of perdeuterated material
2
was weighed in a glass tube, and 50 wt % of H-depleted water
was added. On one side of the tube, a constriction was drawn by
heating the glass with an oxygen burner under Ar. Immediately
thereafter, the top of the constriction was melted and thereby sealed
under vacuum. While the glass was heated, the sample was held at
77 K in a bath of liquid nitrogen. The tube was centrifuged at 5000g
with inversion of the sample several times above the phase-transition
temperature in order to ensure complete hydration. After equilibra-
tion for at least 4 h, both sides of the tube were opened with a
glass cutter, and the sample was transferred to a tubular polycar-
bonate jacket with a diameter of 5 mm by applying gentle pressure
to the nonconstricted end. The jacket was sealed with appropriately
∞ ωnf(ω) dω
∫
0
Mn )
(2)
∞ f(ω) dω
∫
0
where f(ω) denotes the spectral intensity distribution and ω ) 0
corresponds to the Larmor frequency ω0.
Calculation of Structural Parameters via Application of
the First-Order Mean-Torque Model. If it is assumed that the
hydrocarbon chains have an average shape of a prism (cylinder or
cuboid), the distance traveled between carbons i + 1 and i - 1
projected on the bilayer normal can be expressed as
2
machined Teflon plugs. Because of the use of H-depleted water
and the consequent absence of the water signal, the annealing was
monitored by the overall shape of the spectra above the phase-
transition temperature.
2VCH
Solid-State Deuterium NMR Spectroscopy. All of the 2H NMR
spectra were acquired with a Varian VXR-400 spectrometer
operating at 9.4 T (2H frequency 61.4 MHz). Spectra of macro-
scopically aligned samples were obtained using a 10 mm flat-wire
solenoid. A locally constructed goniometer was used for accurate
alignment in the magnetic field at θ ) 0°, where θ denotes the
angle between the normal to the bilayer stack and the field
direction.19 In some cases, it was more practical to acquire spectra
of powder-type samples using a 5 mm flat-wire solenoid, which
were de-Paked using rapid deconvolution by weighted fast Fourier
transformation20 to yield results for the θ ) 0° orientation. The
quadrupolar echo sequence21 was applied for signal excitation, using
composite pulses with a 90° pulse width of 10 µs for the 10 mm
coil and 3 µs for the 5 mm coil. The pulse spacing was 20 µs, and
the recycle delays were chosen to avoid saturation. To increase
the signal-to-noise ratio, the unoriented spectra were symmetrized
by processing only the data points recorded from the real channel.
When samples of several compositions were measured with both
experimental setups, the results were in good agreement, giving
almost identical quadrupolar splittings and 2H NMR spectra. Order
parameters for individual carbon positions in the N-palmitoyl chain
of PSM-d31 or the sn-1 chain of POPC-d31 were obtained from the
resolved quadrupolar splittings ∆ν(Qi) according to eq 1:
2
Di )
(3)
Ai
where Ai is the cross-sectional area per segment and VCH is the
2
volume of a single methylene group, which can be approximated
using the expression VCH (T) ≈ V0CH + RCH (T - 273.15 K),13 in
2
2
2
which the empirical parameters V0CH and RCH have the values 26.5
2
2
Å3 and 0.0325 Å3/K, respectively. The distance Di, which is twice
the travel of one methylene group, can be expressed in terms of
the theoretical maximum projection of the vector connecting the
two neighboring carbon atoms of the segment i onto the bilayer
normal (DM ) 2.54 Å) and the angle ꢁi between the vector and the
bilayer normal: Di ) DM cos ꢁi. The average travel of the carbon
segment is therefore given by eq 4:
Di ) DM cos ꢁi
(4)
This result enables the average projected chain length L/C , which
gives the average distance between the second carbon and the
terminal methyl group, to be calculated by summing up the
projections for each carbon segment:
nC-1
LC/
)
Di
(5)
∑
i)3,5...
3
2
where nC designates the number of carbons per chain. We can also
determine the full chain length projection LC by estimating D2
(i)
CD
|∆ν(Qi)| ) ꢀQ|P2(cos θ)||S
|
(1)
by D3 and adding the extra term Dn -1 to approximate the
C
where ꢀQ denotes the quadrupolar coupling constant (167 kHz for
contribution from the nC ) 16 terminal methyl end:
a C-2H bond), P2(cos θ) is the second Legendre polynomial, and
(i)
1n -1
C
S
is the order parameter for the ith C-2H bond in the hydrocarbon
CD
chain. The quadrupolar splittings of the C2 methylene groups were
LC
)
Di + Dn -1
(6)
∑
C
2
i)2
(20) McCabe, M.; Wassall, S. Solid State Nucl. Magn. Reson. 1997, 10,
53–61.
(21) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P.
Chem. Phys. Lett. 1976, 42, 390–394.
(22) Engel, A. K.; Cowburn, D. FEBS Lett. 1981, 126, 169–171.
(23) Guo, W.; Kurze, V.; Huber, T.; Afdhal, N. H.; Beyer, K.; Hamilton,
J. A. Biophys. J. 2002, 83, 1465–1478.
9
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A R T I C L E S
Bartels et al.
It should be stressed that the hydrophobic thickness and the chain
length are not necessarily the same because of the existence of a
distribution of acyl chain lengths and interdigitation between
opposing bilayers. Although it is possible to determine the average
projected chain length by summing up the projection contributions
for each carbon segment, the hydrocarbon thickness DC can be
calculated by using just the values obtained for the plateau-region
carbon segments, which gives results in good agreement with
available X-ray data.13
If a first-order mean-torque potential is assumed, the following
coupled equations, obtained from integration of eqs 12 and 13 by
cos ꢁ, can be solved numerically:
U1
kBT
U1
cos ꢁ ) coth -
+
(14)
(15)
(
)
kBT
2
kBT
2kBT
U1
cos2 ꢁ ) 1 + 2 -
+
coth -
(
)
(
)
U1
U1
kBT
With the assumption that the average shape of the hydrocarbon
chain constitutes a geometrical prism, the volumetric thickness DC
is then related to A by the expression
An analytical solution for cos ꢁ can then be obtained using the
approximation coth(-U1/kBT) ≈ 1, which for individual segments
gives the relation
2VC
(i)
CD
DC )
(7)
-8S - 1
1
2
A
cos ꢁi
)
1 +
(16)
(
)
ꢀ
3
where VC is the total volume of a single chain. It is well established
that the volume of a methyl group is twice the methylene volume,24
i.e., VCH ≈ 2VCH . Moreover, for the top part of the chain (nearest
It should be noted that in the limit of an all-trans chain with axial
symmetry, cos2 ꢁi ) cos ꢁi ) 1, giving q ) 1. Hence, according
3
2
the aqueous interface), eq 3 can be rewritten as:
to eqs 8 and 9, the limiting area is 2VCH /DM and the limiting
2
volumetric thickness is nCDM/2, suggesting that the model can also
be applied to the gel state at temperatures where rotational diffusion
of the lipids occurs. Although the above formula is applicable only
for order parameters S(CiD) e -1/8, this is sufficient for most plateau
values in 2H NMR of lipid membranes. For the calculation of
cos ꢁi in cases of smaller order parameters, eqs 14 and 15 are
applied. Use of the mean-torque model is justified mainly for large
|SCD| values, but nonetheless, calculating the chain length of highly
mobile methylene groups with small absolute magnitudes of the
order parameter can be a valuable tool, especially for comparing
data for the same acyl chains at different temperatures and in
different mixtures. For a mixture, the area factor q is the weighted
sum and the calculated values of DC and A are then number-
weighted averages over the components, according to the theory
of moments. Lastly, from the values of DC and A , the isobaric
thermal expansion coefficients parallel (R|) and perpendicular (R )
to the membrane normal can be calculated using eqs 17 and 18,
respectively:
2VCH
2
1
A )
(8)
DM cos ꢁ
In the above formula, A represents the mean area per chain at the
aqueous interface and ꢁ corresponds to those segments in the plateau
region of the order profile where SCD ≈ constant (index i is
suppressed). Hence, using eqs 7 and 8, we can express DC as
-1
1
2
1
DC ) nCDM
(9)
cos ꢁ
For calculation of the average interfacial area
A
and the
hydrocarbon thickness DC, the value of the so-called area factor q
) 1/cos ꢁ is needed; it can be approximated by13
q ≈ 3 - 3 cos ꢁ + cos2 ꢁ
(10)
For individual segments (index i), obtaining the value of cos2 ꢁi
from the RQCs is straightforward using eq 11:
(i)
CD
∂DC
1 - 4S
1
cos2 ꢁi
)
(11)
R| )
R )
(17)
(18)
(
)
)
DC ∂T
P
3
Solid-state 2H NMR experiments enable the order parameter |SCD
for the plateau region to be obtained directly from observables using
eq 1.
|
1
A
∂ A
∂T
(
P
Results
However, in the liquid-crystalline state, acquiring the value
of cos ꢁi is more complicated because cos2 ꢁi
* cos ꢁi ,
1/2
Deuterium NMR Spectroscopy of Binary Lipid Mixtures
Containing Cholesterol. Phosphatidylcholines and sphingomy-
elins from cell and organelle membranes are characterized by
distinct physical and spectroscopic properties, regardless of their
which manifests the variance of the length and area distribu-
tions.25 In effect, one needs to know the first and second
moments of the orientational distribution function f(ꢁ). For
statistical treatment of the possible orientational angles ꢁ, the
mean-torque model assumes that the orientational order of the
acyl chain segments relative to the local director frame can be
described by a mean-field orientational potential, denoted by
U(ꢁ), which in a first-order approximation is given by U(ꢁ) ≈
U1 cos ꢁ. The form of the distribution function of ꢁ is given by
the Boltzmann factor, yielding
2
common choline headgroup. Figure 1a,b compares H NMR
spectra of PSM-d31 and POPC-d31 with decreasing temperature.
PSM-d31 shows a sharp liquid-crystalline-to-gel phase transition
in the narrow temperature range 36-38 °C (indicative of high
sample purity), with a remarkably large maximum RQC of 72.3
kHz (for θ ) 0° in eq 1) in the ld state at 40 °C (Figure 1a).
The deviation from the published phase-transition temperature
of PSM (Tm ) 41.5 °C) is a consequence of the perdeuteration
of the acyl chain, which is known to decrease the chain-melting
temperatures.26 In contrast, a spectrum indicating liquid-
crystalline POPC-d31 is obtained over the whole temperature
range of the measurement, with the largest RQC being 54.8
kHz (θ ) 0°) at 15 °C, in good agreement with previous results27
(Figure 1b). The gel phase of PSM-d31 is distinguished by a
1
Z
U(ꢁ)
kBT
f(ꢁ) ) exp -
(12)
(
)
in which the partition function is
π
U(ꢁ)
kBT
Z )
exp -
sin ꢁ dꢁ
(13)
∫
(
)
0
(24) (a) Nagle, J. F.; Wilkinson, D. A. Biophys. J. 1978, 23, 159–175. (b)
Petrache, H. I.; Feller, S. E.; Nagle, J. F. Biophys. J. 1997, 72, 2237–
2242. (c) Armen, R. S.; Uitto, O. D.; Feller, S. E. Biophys. J. 1998,
75, 734–744.
(25) Jansson, M.; Thurmond, R. L.; Barry, J. A.; Brown, M. F. J. Phys.
Chem. 1992, 96, 9532–9544.
(26) Calhoun, W. I.; Shipley, G. G. Biochim. Biophys. Acta 1979, 555,
436–441.
(27) Huber, T.; Rajamoorthi, K.; Kurze, V. F.; Beyer, K.; Brown, M. F.
J. Am. Chem. Soc. 2002, 124, 298–309.
9
14524 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Raftlike Mixtures of Sphingomyelin and Cholesterol
A R T I C L E S
Figure 2. Cholesterol leads to broadening or disappearance of the so phase
2
of PSM, as shown by solid-state H NMR spectra of PSM-d31/cholesterol
with XC ) (a) 0.15, (b) 0.20, (c) 0.25, and (d) 0.33. Oriented samples (θ )
0°) in (a-d) contained 10:90 (v/v) 2H2O/1H2O and a water/lipid molar ratio
of at least 25.
temperatures (see below), implying that substitution of EYSM
for PSM has only a small influence.
Figure 1. Solid-state 2H NMR spectra as a function of temperature
demonstrate phase transitions of pure lipids and lipids in binary mixtures:
(a) PSM-d31; (b) POPC-d31; (c) 1:1 PSM-d31/POPC; and (d) 1:1 EYSM/
POPC-d31. Macroscopically oriented samples (θ ) 0°) were hydrated with
For an assessment of the effect of cholesterol on the palmitoyl
chains of PSM and POPC in ternary mixtures, we first studied
the cholesterol-lipid interaction for each lipid component
separately. Therefore, solid-state 2H NMR spectra were obtained
for PSM-d31 or POPC-d31 bilayers containing various cholesterol
concentrations over a range of temperatures. The spectra of
PSM-d31/cholesterol mixtures (Figure 2) differ substantially from
those of PSM-d31 alone. At the lowest cholesterol mole fraction,
XC ) 0.15, the quadrupolar splitting corresponding to the outer
edges of the spectrum increases by 15 kHz at 55 °C, which can
be attributed to the well-known ordering effect of the rigid
cholesterol ring structure.28-30 This is accompanied by signal
broadening, which to some extent may be the result of rapid
exchange of the lipids between ordered and liquid-disordered
domains. Inhomogeneous line broadening, due, for example,
to a more pronounced mosaic spread in the macroscopically
oriented samples, must be also considered. The entire phase
transition is substantially broadened in this case, i.e., the onset
can be detected at 42 °C, and completion has not been reached
at 20 °C (Figure 2a). When the cholesterol content is increased
to XC ) 0.2, the transition from the ld to the so phase seems to
vanish completely. The significant line broadening and the
2
10:90 (v/v) H2O/1H2O to a final water/lipid molar ratio of 25, as detailed
in Methods. Note the sharp phase transition in (a) at 38 °C and the
emergence of an additional spectral component in (c).
broad doublet with a maximum splitting of 117.0 kHz (for θ )
0°) at 15 °C. This is consistent with axial rotation of the lipids
at temperatures moderately below that of the phase transition,
with little or no biaxiality.
In an equimolar mixture of sphingomyelin and POPC, the
phase transition commences at 25 °C and is not complete at 15
°C. The transition can be directly seen in the mixture containing
PSM-d31 by the emergence of additional gel-phase signals with
a quadrupolar splitting of 105.8 kHz at θ ) 0° (Figure 1c).
The spectrum of PSM-d31 in the liquid-crystalline state is still
superimposed on the gel-state spectrum, indicating that both
2
phases coexist at 15 °C. The observation of distinct H NMR
spectral components implies that the lateral phase segregation
entails a domain size large enough for diffusional exchange of
the deuterated lipids between the domains to be slow on the 2H
NMR time scale. In contrast, there is only one component in
2
the H NMR spectra of POPC-d31 in the same binary mixture
(Figure 1d), suggesting that this phospholipid rapidly exchanges
between ordered and disordered domains. It may be seen that
the POPC-d31 spectrum broadens significantly at 25 °C, i.e.,
close to the phase transition identified in Figure 1c. One should
also note that because of the limited availability of chemically
defined PSM, use of EYSM/POPC-d31 mixtures could yield
slightly different phase behavior compared to PSM-d31/POPC
mixtures. However, the fraction of PSM in EYSM is sufficiently
large (84%), and the major changes in PSM-d31/POPC mem-
branes are reflected in EYSM/POPC-d31 mixtures at the same
(28) (a) Oldfield, E.; Meadows, M.; Rice, D.; Jacobs, R. Biochemistry 1978,
17, 2727–2740. (b) Trouard, T. P.; Alam, T. M.; Zajicek, J.; Brown,
M. F. Chem. Phys. Lett. 1992, 189, 67–75. (c) Martinez, G. V.;
Dykstra, E. M.; Lope-Piedrafita, S.; Job, C.; Brown, M. F. Phys. ReV.
E 2002, 66, 050902-1–050902-4.
(29) Brzustowicz, M. R.; Stillwell, W.; Wassall, S. R. FEBS Lett. 1999,
451, 197–202.
(30) Brzustowicz, M. R.; Cherezov, V.; Caffrey, M.; Stillwell, W.; Wassall,
S. R. Biophys. J. 2002, 82, 285–298.
(31) Henriksen, J.; Rowat, A. C.; Brief, E.; Hsueh, Y. W.; Thewalt, J. L.;
Zuckermann, M. J.; Ipsen, J. H. Biophys. J. 2006, 90, 1639–1649.
9
J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008 14525

A R T I C L E S
Bartels et al.
2
Figure 3. De-Paked H NMR spectra of a POPC-d31/cholesterol mixture
with XC ) 0.33 at various temperatures. The powder-type samples contained
50 wt % 1H2O. Note the similiarity to the PSM-d31 spectrum at low
temperatures (Figure 2d).
increased splittings suggest the coexistence of an ld phase and
a new lo phase instead of the so phase, with the ld phase
prevailing. The spectra at 38 °C shown in Figure 2b display a
shift of the phase equilibrium from the ld phase toward the lo
phase, which becomes the main phase below 35 °C. This trend
is continued at a mole fraction of XC ) 0.25 (Figure 2c), with
the phase transition being less notable and already begun at
45 °C.
(i)
Figure 4. Order parameters |S | vs carbon segment i for chain-deuterated
CD
PSM and POPC alone and in binary lipid/cholesterol mixtures: (a) PSM-
d31 alone; (b) PSM-d31/cholesterol, XC ) 0.20; (c) PSM-d31/cholesterol, XC
) 0.33; (d) POPC-d31 alone; (e) POPC-d31/cholesterol, XC ) 0.20; and (f)
POPC-d31/cholesterol, XC ) 0.33. Note that the largest differences in
orientational order of POPC and PSM occur at intermediate cholesterol
mole fractions.
Finally, at XC ) 0.33, the main phase transition is no longer
detectable, and the 2H NMR spectrum at 55 °C reflects a
homogeneous lo state (Figure 2d). Thus, at XC ) 0.33, the
amount of cholesterol seems to be sufficient to completely
eliminate the ld phase in a binary mixture with sphingomyelin.
In addition, at 40 °C the largest splitting shifts from 65.7 kHz
for PSM-d31 alone to 104.8 kHz (θ ) 0°), corresponding to an
order parameter of SCD ) 0.42, which is indicative of an lo state.
The large quadrupolar splitting is close to the calculated
maximum splitting of 127.5 kHz expected for complete parallel
alignment of the acyl chains with respect to the magnetic field
when in the trans state and rotating about the bilayer normal.
Order Parameter Profiles of Binary Lipid-Cholesterol
Mixtures. Order parameter profiles derived from the quadrupolar
splittings for PSM-d31 and POPC-d31 and for binary mixtures
of these lipids with cholesterol are shown in Figure 4. A general
feature of the profiles is the decrease of the order parameters
with increasing distance from the lipid-water interface. How-
ever, the profiles clearly reveal distinct differences between the
lipids. For example, for POPC-d31 alone at 45 and 60 °C (Figure
4d), the almost identical splittings of the interfacial chain
segments give rise to an order parameter plateau, whereas the
same chain positions for PSM-d31 (Figure 4a) are characterized
by slightly decreasing order parameters.
2
A comparison with the H NMR spectrum of an analogous
The distinction between the sphingo- and glycerophospho-
lipids becomes even more conspicuous in the presence of
cholesterol. For PSM-d31, the profile changes drastically in the
presence of 20 mol % of cholesterol. At 45 and 60 °C, a broad
unresolved doublet now appears, i.e., there is a plateau region
comprising ∼10 methylene segments. The quadrupolar splittings
from the interfacial segments increase at temperatures <40 °C,
yielding an order parameter close to the value of -0.5 that
would be expected for hydrocarbon chains in an all-trans state.
At 33 mol % of cholesterol, the high-temperature order
parameters for all of the PSM-d31 segments increase signifi-
cantly, and the ordering profiles are much less temperature-
sensitive. In sharp contrast, there is no sudden increase in order
parameter with decreasing temperature for POPC-d31 at a
cholesterol concentration of 20 mol %. Moreover, the temper-
ature dependence of the profiles is stronger with increasing
cholesterol content. At 33 mol % cholesterol, the plateau order
parameter values vary from 0.42 to 0.26 on going from 20 to
60 °C, which may be contrasted to the variation from 0.46 to
POPC-d31/cholesterol mixture (XC ) 0.33) reveals further
differences (Figure 3). The splittings at lower temperatures
reflect similar chain ordering for the POPC-d31 and PSM-d31
bilayers. In the former system, however, the splittings decrease
much faster with temperature, reaching 66.5 kHz at 60 °C, which
is more akin to a mixture of ld and lo phases rather than to a
pure lo state. However, compared to those for PSM-d31, the
signals in the POPC-d31 spectra are much better resolved in both
the oriented and powder-type samples, most probably as a
consequence of the fluidizing effect of the adjacent oleoyl chain
on the palmitoyl chain. We thus conclude that cholesterol at
concentrations below a mole fraction of XC ) 0.33 has less
influence on the ordering of the palmitoyl chain of POPC than
on ordering of the sphingomyelin N-palmitoyl chain. A more
2
detailed study of the POPC-d31/cholesterol system using H
NMR techniques has recently been published elsewhere.31,32
(32) Hsueh, Y.-W.; Chen, M.-T.; Patty, P. J.; Code, C.; Cheng, J.; Frisken,
B. J.; Zuckermann, M.; Thewalt, J. Biophys. J. 2007, 92, 1606–1615.
9
14526 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Raftlike Mixtures of Sphingomyelin and Cholesterol
A R T I C L E S
phase with a quadrupolar splitting of 17.7 kHz, while the smaller
fraction pertains to an ld phase characterized by a splitting of
11.0 kHz. A comparison of the ternary PSM-d31 spectra with
those obtained with POPC-d31 (shown in Figure 5b) displays
similarities and differences in the methyl group splittings. As
lower temperatures are reached, the appearance of a second
phase with domains large enough to generate a separately
observable spectrum on the 2H NMR time scale becomes evident
for the mixture containing 1:1 POPC-d31/EYSM with XC ) 0.2.
At 30 °C, the second phase becomes noticeable by a sudden
broadening of the methylene peaks. At 25 °C, a second set of
plateau peaks appears, with a splitting of ∼96 kHz for θ ) 0°,
indicating a partitioning of POPC into the lo phase formed by
PSM and cholesterol. A closer look at the inset in Figure 5b
again shows doublet splittings for the methyl group signal, which
arise from the main peak (8.8 kHz) and lead to a well-resolved
second peak (11.5 kHz), indicating a spectral component due
to the segregated POPC fraction in the lo phase. The integral
ratio of the two peaks, which changes in the temperature range
25-15 °C in favor of the outer methyl peak, further substantiates
this point. Interestingly, the integral ratio is reversed in
comparison with the methyl group splitting seen for 1:1 PSM-
d31/POPC with XC ) 0.2 (Figure 5a). For 1:1 POPC-d31/EYSM
with XC ) 0.2 (Figure 5b), the main fraction of POPC-d31 is in
the ld phase, as the outer peak at 8.8 kHz is larger, and the
smaller part of the lipid fraction partitions into an lo phase with
PSM-d31, as indicated by the smaller peak at 11.5 kHz. When
the cholesterol content is increased to XC ) 0.33 (Figure 5c)
the 2H NMR spectra of PSM-d31 in a ternary mixture with POPC
and cholesterol become simpler, with splittings similar to the
binary mixtures of PSM-d31 with XC ) 0.33. The splittings
correspond to an lo phase over almost the whole temperature
range studied. Nonetheless, the broadening of the peaks at 60
°C again may indicate the emergence of a second liquid-
crystalline phase at higher temperatures. Additionally, the overall
decrease in the splittings of the plateau peaks for the ternary
1:1 PSM-d31/POPC mixture with XC ) 0.33 (95.2 kHz for θ )
0° at 40 °C; Figure 5c) compared with those for the binary
mixture of PSM-d31 with XC ) 0.33 (104.8 kHz at 40 °C; not
shown) imply that significant amounts of POPC are incorporated
in the lo phase. This leads to smaller quadrupolar splittings for
Figure 5. De-Paked solid-state 2H NMR spectra of ternary raftlike mixtures
as a function of temperature show phase separation: (a) 1:1 PSM-d31/POPC,
XC ) 0.2; (b) 1:1 EYSM/POPC-d31, XC ) 0.2; (c) 1:1 PSM-d31/POPC, XC
) 0.33; and (d) 1:1 EYSM/POPC-d31, XC ) 0.33. The insets in (a) and (b)
show expansions of the methyl group signal at 25 °C. Powder-type samples
contained 50 wt % 1H2O. Note that for XC ) 0.2, discrete components due
to POPC-d31 and PSM-d31 phase separation are observed at lower temper-
atures, whereas for XC ) 0.33, the lipids mix almost ideally.
0.37 observed for PSM-d31. The numerical RQCs for the θ )
0° orientation and their assignments to the respective carbon
segments are provided in the Supporting Information.
Deuterium NMR Spectroscopy of Ternary Mixtures and
Moment Analysis. A simple model for elucidating the physical
properties of lateral inhomogeneities in biological membranes,
often called lipid rafts, is a ternary mixture comprising sphin-
2
gomyelin, POPC, and cholesterol. Figure 5 compares the H
NMR spectra acquired for ternary mixtures of PSM, POPC, and
cholesterol at a series of different temperatures. The addition
of cholesterol at a mole fraction of XC ) 0.2 has a major impact
on the spectra of PSM-d31 in ternary mixtures with POPC
(1:1), as shown in Figure 5a. The plateau splitting at higher
temperatures (71.3 kHz at 60 °C for θ ) 0°) falls between the
RQC values obtained for binary mixtures of PSM-d31 and POPC
(56.6 kHz at 55 °C for θ ) 0°; not shown) and those for PSM-
d31/cholesterol with XC ) 0.33 in the lo phase (91 kHz for θ )
0° at 60 °C; not shown). At 35 °C, an extensive line broadening
becomes noticeable and persists to 15 °C (Figure 5a). In contrast,
the POPC-free binary mixture of PSM-d31 and cholesterol with
XC ) 0.20 (Figure 2b) shows the same phase transition, but
with only a small range of phase coexistence near 40 °C that
merges near 35 °C to give an lo phase at lower temperatures.
This difference in phase behavior indicates that the addition of
POPC seems to destabilize the formation of an lo phase,
therefore delaying and prolonging the ld-to-lo phase transition.
Interestingly, in Figure 5a, the methyl group signal consists
of doublet splittings (17.7 and 11.0 kHz for θ ) 0°, respec-
tively), which are attributed to the coexistence of two laterally
separated phases with different lipid compositions.32 When the
system is cooled from 60 °C, the doublets appear at ∼38 °C.
This subset of splittings indicates that the phase domains are
2
PSM-d31 and hence to diminished average order in the H-
labeled palmitoyl chain.
Next, the complementary experiment employed POPC-d31
instead of PSM-d31 in the same ternary system (XC ) 0.33), as
2
shown in Figure 5d. This lipid also exhibits uniform H NMR
spectra with large methyl group splittings (89.9 kHz for θ )
0° at 40 °C for the plateau peak), which are even greater than
those in binary mixtures with cholesterol alone (85.0 kHz at 40
°C; not shown). The presence of sphingomyelin seems to have
an additional ordering effect on the palmitoyl chain in POPC,
which leads to increased quadrupolar splittings in a ternary PSM/
POPC-d31/cholesterol mixture in comparison with a binary
POPC-d31/cholesterol mixture at the same total cholesterol molar
ratio (XC ) 0.33). This observation is in line with almost ideal
mixing of POPC and sphingomyelin in these mixtures. Another
noticeable feature is the remarkably high resolution of individual
resonances with lower quadrupolar splittings for the nonplateau
methylene groups (C11 to C15) in the palmitoyl chain of POPC-
d31 in comparison with PSM-d31. This is a general feature of
the POPC spectra, as already shown in the binary mixtures with
POPC-d31 and cholesterol alone. The unsaturated sn-2 chain of
POPC obviously has a disordering effect on the later methylene
2
large enough to yield discrete spectral components on the H
NMR time scale. According to the integral ratio of the methyl
peaks (Figure 5a), the main fraction of PSM-d31 is in the lo
9
J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008 14527

A R T I C L E S
Bartels et al.
Figure 6. First spectral moments of powder-type samples as a function of
temperature reveal phase separation. Data are shown for mixtures of 1:1
PSM-d31/POPC (9) and 1:1 EYSM/POPC-d31 (3) with cholesterol for (a)
XC ) 0.2 and (b) XC ) 0.33. The overall ordering of the acyl chain in
POPC-d31 is significally different from that in PSM-d31 in ternary mixtures
with XC ) 0.2 but almost the same for XC ) 0.33.
groups in the acyl chain, probably due to the double bond in
the oleoyl chain. Values of the quadrupolar splittings for the θ
) 0° orientation and their segmental assignment are provided
in the Supporting Information.
The first spectral moments M1 of the 2H NMR spectra (eq 2)
are shown in Figure 6 and reflect the average orientational order
in the bilayer membrane as a function of temperature. The plots
of M1 versus temperature again show the clear difference
between POPC-d31 and PSM-d31 with XC ) 0.2, as seen in
Figure 6a. Obviously, PSM is situated in an environment of
higher segmental order on average, while the palmitoyl chain
of POPC is highly mobile. This underscores the coexistence of
the two phases, one formed by an ld mixture of POPC and
cholesterol with a very low PSM content and the other consisting
of lo domains that are rich in PSM and cholesterol and almost
depleted of POPC. At a cholesterol concentration of XC ) 0.33
(Figure 6b), the differences between the two first spectral
moments vanish almost completely, giving values characteristic
of an lo phase for both lipids. There is a divergence of the
moments only at higher temperatures, most likely because POPC
begins to form an ld phase earlier than PSM. This strongly argues
for ideal mixing of the lipids at temperatures below 50 °C with
cholesterol mole fractions of XC ) 0.33.
Figure 7. Segmental projections onto the bilayer normal for PSM-d31/
cholesterol binary mixtures at different temperatures reveal the effect of
cholesterol on the orientational order of PSM-d31 chains. The indexing (nC
- i + 2) starts from the terminal methyl group (i ) nC) and ends at the C2
carbon (i ) 2). Data are shown for pure PSM-d31 (2), PSM-d31 with XC
)
0.2 (b), and PSM-d31 with XC ) 0.33 (9) at temperatures of (a) 20, (b) 30,
(c) 45, and (d) 60 °C. The chain extent of the lo state corresponds to that
of the so phase, where the full chain length of the lo state is reached at XC
) 0.2. The full chain extent corresponds to L/C from eq 5.
(20 and 30 °C), the lo phase is assumed for a sphingomyelin
membrane with XC ) 0.33, whereas the gel phase is adopted
for pure PSM-d31. In both cases, however, the chain-extension
profiles exhibit nearly identical characteristics with regard to
the segmental projections onto the bilayer normal (Figure 7a,b).
Although the narrow line shape of the 2H NMR spectra of PSM-
d31 with XC ) 0.33 (Figure 2d) clearly indicates the existence
of a homogeneous lo phase over the whole temperature range,
the chain extensions shown in Figure 7a,b are almost the same
as in the so gel phase, which is identified by the line shape of
the 2H NMR spectra for pure PSM-d31 at 15 and 30 °C (Figure
1a). At higher temperatures (45 and 60 °C), as shown in Figure
7c,d, the chain-extension profiles clearly show the difference
between pure PSM-d31 and the cholesterol mixtures, yet
surprisingly, almost identical projected chain lengths are
calculated for XC ) 0.2 and XC ) 0.33. It is also interesting
that the largest difference between the two cholesterol mixtures
is observed for the interfacial region (i ) 2-5), despite the near
saturation of the ordering effect for the other chain segments.
This indicates that cholesterol is located directly in the vicinity
of the water interface, as already implied by the recent
literature.33 In this regard, cholesterol can be considered as a
spacer molecule that increases the separation between the lipid
polar headgroups.34
Discussion
Dynamical Structure and Chain Packing of Lipid-Raft
2
Components. Analysis of the H NMR data by means of the
first-order mean-torque model13 is very useful for gaining an
understanding of the forces affecting membrane lipid bilayers.
This approach can be used to determine the average projected
length onto the bilayer normal for each segment of the acyl
chain, thereby allowing calculation of the average carbon
position relative to an arbitrary reference point (here, the
terminal methyl group). Accordingly, we reindex the carbon
atoms i using the alternate index i′ ) nC - i + s, where s is an
arbitrary shift value. For s ) 2, the indexing is reversed, giving
i′ ) 2 for the terminal methyl group and i′ ) 16 for the C2
carbon. This is more convenient for direct comparison of the
headgroup region. The chain-extension profiles obtained in such
a manner facilitate the analysis of the different bilayer structures.
Applying this approach to binary mixtures of PSM-d31 or POPC-
d31 with cholesterol allows a comparison of the specific ordering
effects of cholesterol on these lipids, thus revealing the factors
leading to lipid mixing or phase separation.
However, for POPC-d31, as shown in Figure 8, striking
differences in chain packing upon interaction with cholesterol
in binary mixtures become evident upon comparison with the
results for PSM-d31. The acyl chains of the glycerophospholipid
clearly are not affected as strongly as the sphingolipid by the
rigid sterol backbone at low cholesterol concentrations. In
In Figure 7 we compare the effects of cholesterol on the
cumulative projected chain length at the level of single-carbon
segments of the PSM-d31 palmitoyl chain. At lower temperatures
(33) Cournia, Z.; Ullmann, G. M.; Smith, J. C. J. Phys. Chem. B 2007,
111, 1786–1801.
(34) Brown, M. F.; Seelig, J. Biochemistry 1978, 17, 381–384.
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Raftlike Mixtures of Sphingomyelin and Cholesterol
A R T I C L E S
Figure 9. Semilogarithmic plots of relative variation of (a, b) the average
area per lipid A and (c, d) the hydrophobic thickness DC (as calculated
from the plateau splittings of the respective mixtures) vs temperature. The
effect of cholesterol on the phase transitions of (a, c) PSM-d31 and (b, d)
POPC-d31 in binary mixtures is shown: pure lipid (2), XC ) 0.2 (b), and
XC ) 0.33 (9). Increasing the cholesterol concentration diminishes the phase
transition of PSM-d31 but may promote a phase transition in POPC-d31 at
XC ) 0.33, as indicated by a small curvature of the plots. Note that the
plots are always steeper for POPC-d31 than for PSM-d31 in a one-phase
regime.
Figure 8. Chain-extension profiles (cumulative segmental projections) for
POPC-d31/cholesterol binary mixtures show the influence of cholesterol on
the orientational order of POPC-d31 acyl groups. The indexing (nC - i +
2) begins with the terminal methyl group (i ) nC) and ends at the C2 carbon
(i ) 2). Results are shown for pure POPC-d31 (2), POPC-d31 with XC
)
0.2 (b), and POPC-d31 with XC ) 0.33 (9) at temperatures of (a) 20, (b)
30, (c) 45, and (d) 60 °C. For POPC-d31, the chain length markedly depends
on the cholesterol concentration.
comparable glycerophospholipid. Direct comparison of the chain
length to that of the sn-2 palmitoyl chain in POPC still seems
to be justified, as the difference in chain lengths for the sn-1
and sn-2 chains is minimal (e.g., a 0.15 Å difference for the
two chains in DPPC40).
addition, a more continuous dependence of chain order on
cholesterol concentration becomes observable in the case of
POPC. This is obviously due to the less favorable interaction
of the unsaturated acyl chain of POPC with cholesterol, as has
already been shown for different unsaturated lipids.29,30,35-37
The exact basis for the diminished ordering effect of cholesterol
on POPC at intermediate concentrations could have several
origins. Molecular dynamics simulations have shown that the
hydroxyl group of cholesterol displays a preferential interaction
with the carbonyl oxygen of the ester bond in the oleoyl chain
of POPC,38 where the molecularly rough ꢁ side of cholesterol
would be associated with the oleoyl chain, while the smooth R
side could have a preferential interaction with saturated chains.39
However, at higher cholesterol concentrations (XC ) 0.33), the
amount of the sterol seems to be sufficient to induce the same
order as in a PSM/cholesterol system, as also observed in other
studies.32 It should be noted that the N-palmitoyl chain in PSM
structurally corresponds more to the sn-1 acyl chain of a
In terms of miscibility, comparison of the full chain extents
of the two lipids, which indicate their hydrophobic chain lengths
in the lipid bilayer, leads to further interesting conclusions.
Hydrophobic mismatch (or interfacial-area mismatch) between
sphingomyelin and POPC in binary mixtures or in ternary
mixtures with cholesterol at XC ) 0.20 should lead to detectable
phase separation. Conversely, as the values of the chain extent
are matched much better in mixtures with higher cholesterol
contents, the two lipids should mix almost ideally, at least at
low temperatures. This of course assumes that neither of the
lipids has a strong influence on the acyl chain order of the other,
and (in the case of ternary mixtures) that cholesterol is evenly
distributed between POPC and sphingomyelin, as suggested
previously.41,42 Additionally, POPC has a stronger tendency than
PSM to become disordered at higher temperatures.
The first-order mean-torque model13 provides two important
structural parameters for lipid bilayers: A , the average area
per acyl chain, and DC, the hydrophobic thickness of one bilayer
leaflet. Both quantities are useful for following the expansion
of the acyl chains perpendicular or parallel to the membrane
normal (director). Figure 9 shows the semilogarithmic temper-
ature dependence of A and DC for the two lipids. The phase
transition in PSM-d31 is suppressed by stepwise addition of
cholesterol, as indicated by a sudden increase in area or decrease
in hydrophobic thickness with increasing temperature (Figure
9a,c). In contrast, POPC-d31 shows a small curvature in this
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A R T I C L E S
Bartels et al.
is now at 45 °C, which is evident in Figure 2c. This observation
can be explained easily by assuming that the transition is not
between an ld phase and an so phase but rather marks the end
point of phase separation between ld and lo phases, as already
2
implied by the H NMR spectra shown earlier. The DC values
provide further evidence that sphingomyelin forms an lo phase
and not an so phase at low temperatures and high cholesterol
concentrations. While the so phase in pure PSM-d31 is typified
by a chain length of 19.9 Å in Figure 10, the lo phase of
sphingomyelin in a binary mixture with XC ) 0.33 exhibits a
slightly but distinctly smaller hydrophobic width of 19.5 Å at
20 °C. By addition of such large amounts of cholesterol, the
phase separation seems to be suppressed over a large temper-
ature range. Only at temperatures above 38 °C is there a small
kink in the temperature dependence of DC, suggesting that small
amounts partition into an ld phase coexisting with the much
larger fraction of lo domains.
The effect of cholesterol on the average hydrophobic length
of the palmitoyl chain in a POPC membrane is again distinct
from that in sphingomyelin, as evinced by Figure 10b. The value
for DC at 15 °C increases from 14.8 Å in pure POPC-d31 to
19.2 Å at a mole fraction of XC ) 0.33, indicating an lo phase
for which DC is, however, 0.3 Å smaller than in a comparable
PSM-d31 membrane. At XC ) 0.33, the slope of DC versus
temperature changes between 35 and 40 °C, thereby suggesting
the appearance of POPC-d31 in a more disordered state. Again,
the main difference is that the ordering effect of cholesterol is
less distinct for POPC-d31 than for PSM-d31 at the intermediate
mole fraction of XC ) 0.2. At temperatures of 35-60 °C, the
hydrophobic thickness is 1.7 Å smaller on average for POPC-
d31 than for PSM-d31, which remains in the presence of
cholesterol. Different ordering effects of cholesterol on PSM
and POPC are suggested as the driving force behind the acyl
length mismatch and therefore the phase separation.
Figure 10. Chain thickness DC as a function of temperature and cholesterol
concentration: (a) PSM-d31; (b) POPC-d31; (c) 1:1 PSM-d31/POPC; and (d)
1:1 EYSM/POPC-d31. Application of the mean-torque model shows the
influence of hydrophobic mismatch at intermediate cholesterol mole
fractions, leading to formation of lipid rafts. Phase transitions are seen as
a jump in the temperature dependence of DC. In ternary mixtures with
intermediate cholesterol concentrations, the hydrophobic mismatch between
POPC-d31 and PSM-d31 becomes evident. Note that the hydrophobic
thickness in the ternary mixtures approximately corresponds to the values
in the binary samples. This implies an equal distribution of cholesterol
between the two lipids.
temperature region (Figure 9b,d) at very high cholesterol
concentrations. This implies that at high temperatures, the binary
POPC-d31/cholesterol mixture with XC ) 0.33 undergoes a
change from a pure lo phase to one with greater longitudinal
order. Conversely, for PSM-d31, a cholesterol concentration of
XC ) 0.33 seems to be sufficient to stabilize the lo phase over
the whole temperature range studied. Another observation of
interest is that the slopes of the semilogarithmic plots are steeper
for POPC-d31 than for PSM-d31 in a one-phase region. The more
pronounced temperature dependence of POPC-d31 should further
drive phase separation in ternary mixtures at high temperatures.
A summary of the structural parameter values obtained for the
pure lipids and their binary mixtures with cholesterol by use of
the first-order mean-torque model is provided in the Supporting
Information.
Ideal or Nonideal Mixing of Sphingomyelin, Phosphatidylcho-
line, and Cholesterol. By calculating the hydrophobic thicknesses
of the palmitoyl chain in the two lipids, it is straightforward to
obtain a descriptive and easily interpretable picture of phase
separation in the ternary mixtures. Figure 10 gives a first view
of the temperature dependence of DC in different mixtures of
PSM-d31 and cholesterol and immediately shows the usefulness
of the model. In the so phase close to the phase transition and
in the solid-disordered (sd) phase, the lipids undergo rotational
diffusion about the bilayer normal, so the mean-torque model
can be applied. Although a biaxial contribution is not evident,
a detailed analysis may require a modification of the formalism.
The phase transition of pure PSM-d31 at 38 °C can be readily
observed in Figure 10a by the sudden increase in DC from 15.9
Å, which is characteristic of the ld phase of sphingomyelin, to
19.8 Å, which corresponds to the hydrophobic thickness of a
membrane in the so phase. At a cholesterol mole fraction of XC
) 0.15, the phase-transition temperature is decreased to 34 °C
(Figure 2a), which is the typical effect of adding a solute to a
pure material. In contrast, at a cholesterol mole fraction of XC
) 0.25, the onset of the broadened phase-transition temperature
In ternary mixtures, the mean-torque model can provide us
with additional arguments for answering the question of whether
the lipids are in coexisting phases having different segmental
orientational order. As observed by atomic force microscopy
in comparable systems,43 the microdomains consisting of
sphingomyelin and cholesterol are distinguished by an increased
bilayer thickness compared with that in the surrounding fluid
phase. This should be directly observable by a difference in DC
calculated for the two lipids, one participating in the ld phase
and the other in the lo phase. Another interesting question is
whether the phase behavior of the binary systems allows
predictions for the ternary systems or a greater affinity between
some components leads to different properties. In order to
answer these questions, we first examine the plots of the
temperature dependence of DC for ternary mixtures of 1:1 PSM-
d31/POPC with cholesterol (Figure 10c). In the cholesterol-free
1:1 binary mixture of PSM-d31 and POPC, a phase transition at
∼25 °C results in a sudden increase of the hydrophobic thickness
from 16.2 to 19.1 Å. This phase transition becomes less
pronounced with increasing cholesterol mole fraction and
completely vanishes for XC g 0.33. Only a slight increase in
DC (0.3 Å) was obtained upon increasing the cholesterol mole
fraction to XC ) 0.5, indicating that the chain-ordering effect
is nearly at a maximum when the cholesterol concentration
reaches XC ) 0.33.
(43) (a) Giocondi, M. C.; Boichot, S.; Plenat, T.; Le Grimellec, C. C.
Ultramicroscopy 2004, 100, 135–143. (b) Giocondi, M. C.; Milhiet,
P. E.; Dosset, P.; Le Grimellec, C. Biophys. J. 2004, 86, 861–869.
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Raftlike Mixtures of Sphingomyelin and Cholesterol
A R T I C L E S
However, the corresponding plot for POPC-d31 in mixtures
with sphingomyelin and cholesterol (Figure 10d) shows a
completely different picture. For the 1:1 binary mixture of
EYSM and POPC-d31 in the absence of cholesterol, the acyl
chain of POPC displays a clearly smaller hydrophobic thickness
than does that of sphingomyelin. Whereas sphingomyelin
undergoes a clear phase transition in the binary mixture at 30
°C, the POPC-d31 in the same mixture does not deviate from
its monotonic dependence of order on temperature. This result,
together with the chain-extension profiles displayed in Figure
8a-d, indicates only a small partitioning of sphingomyelin into
the ld phase mainly formed by POPC. Obviously, no ideal
mixing between the two components takes place over a broad
temperature range. However, the difference of almost 1 Å in
the hydrophobic chain lengths at 30 °C does not seem to be
enough to completely exclude sphingomyelin from the much
more disordered POPC phase, as can be seen in Figure 1c. The
addition of cholesterol seems to strengthen this phase separation
at first. The difference in hydrophobic thicknesses then increases
from 1.6 Å at 60 °C to 4.4 Å at 15 °C. It should be noted that
the jump in the temperature dependence for XC ) 0.2 in the
plot for PSM-d31 (Figure 10c) is reflected in the curve for POPC-
d31 in the identical mixture (Figure 10d). For the sphingolipid,
the chain length suddenly increases with decreasing temperature
starting at ∼25 °C, indicating the emergence of an lo phase with
a larger cholesterol mole fraction. However, for POPC-d31, we
see the contrary behavior at 25 °C. Instead of increasing, the
thickness decreases at 25 °C, manifesting a decreased order of
the palmitoyl chain comparable to that in a pure POPC phase.
Although cholesterol is distributed evenly between the two lipids
at higher temperatures, it now participates to a greater extent
in the lo phase of sphingomyelin, thereby increasing its order
and leaving POPC in a phase of low cholesterol content. This
can be explained by a slightly stronger affinity of cholesterol
for the sphingolipid. Thus, the thermal energy of the lipids is
not sufficient at lower temperatures to overcome the binding
enthalpy between cholesterol and sphingomyelin.
cholesterol concentrations, as in the ternary mixture of 1:1 PSM/
POPC with XC ) 0.2 below 25 °C.
Implications for Rafts in Cellular Membranes. Although
glycerophospholipids are sufficient to form lipid bilayers,44 most
eukaryotic cells contain sphingolipids and sterols as additional
classes of lipids, each available only through elaborate biosyn-
thesis. This considerable anabolic expense, as well as the
abundance of these lipid classes in the plasma membrane
(typically 30-40 mol % cholesterol and 10-20 mol %
sphingomyelin45), naturally raises the question of their evolution
and function in the organization of the bilayer. The implication
that the well-known ordering effect of cholesterol for sphingo-
myelin and other saturated glycerophospholipids leads to lateral
phase segregation and microdomains needs further clarification,
as the situation in vivo is far too complex to be determined
directly. However, the structural properties and phase-transition
temperatures of sphingomyelins near body temperature (37 °C)
suggest they may play an important role in the formation of
specialized domains in membranes such as lipid rafts.46 The
data assembled in this study give some interesting insights into
the effect of cholesterol on the lateral organization of such
mixtures. The approach of using a model membrane, comprising
a lipid that forms a fluid bilayer together with a lipid known to
form a more ordered bilayer as well as cholesterol in varying
amounts, is probably most accessible as a mimic of biomem-
branes with a vast number of components.47 Interestingly,
cholesterol has two different functions in our model membrane
system: on the one hand, it further increases the hydrophobic
mismatch of the lipids at low concentrations and thereby
enhances phase separation, and on the other hand, it functions
as a mixing agent at high concentrations. This variation in
function is interesting, insofar as other sterols which appear in
lower organisms, e.g., lanosterol or ergosterol, show acyl chain
ordering in POPC-d31 up to just 25 mol % sterol.32 Consequently
the saturating behavior seems to be a distinct feature of each
lipid in its interaction with a specific sterol. This has already
been shown for 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) membranes, in which, contrary to the case for DPPC
membranes, cholesterol has smaller ordering effects than
ergosterol48 but greater effects than lanosterol.49 Additionally,
cholesterol has been found to have a much higher mobility
between the two leaflets than ergosterol or lanosterol,50 further
indicating a possible role of cholesterol as a mediator of
interactions between the different monolayers.
The incorporation of cholesterol up to a mole fraction of XC
) 0.33 leads to the following interesting effect in ternary
mixtures. Although the addition of cholesterol initially drives
the phase separation by inducing greater lateral order in
sphingomyelin than in POPC, in mixtures with high amounts
of cholesterol, saturation of the ordering effect for PSM seems
to facilitate mixing of the components. This is indicated by the
2
If we now consider the phase separation as being mostly
driven by hydrophobic mismatch of the acyl chains of the
various lipids, it is normally assumed that the thickness
difference is induced by unequal sterol partitioning into the two
phases. However, our study shows that this assumption is not
needed for lateral segregation to be generated, because the
difference in ordering upon addition of cholesterol already seen
similarity of the H NMR spectra obtained for the two lipids
(Figure 5c,d) as well as by their nearly identical DC values at
lower temperatures (19.3 vs 19.6 Å, respectively, at 15 °C). At
a higher temperature of 60 °C, the differences in chain length
increase to 2 Å, which is also reflected in significant broadening
of the respective signals seen in Figure 5c,d. This again shows
the stronger temperature dependence of the POPC acyl chain
order, as already evident in the binary mixtures. As a rule, the
ternary mixture behaves as implied by the results for the binary
mixtures with cholesterol. Moreover, the lipids in the ternary
mixture have quite similar structural parameters, as expected if
cholesterol is almost evenly distributed between them. Phase
separation occurs only if the thermal energy of the molecules
is lower than the enthalpy of the cholesterol-sphingolipid
interaction and the cholesterol concentration is low enough to
enable competition between the two lipids for the sterol. The
stronger affinity of cholesterol for sphingomyelin is then
sufficient to drive the formation of membrane domains. In our
work, this phenomenon was observed only at intermediate
(44) Bloom, M.; Evans, E.; Mouritsen, O. G. Q. ReV. Biophys. 1991, 24,
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A R T I C L E S
Bartels et al.
in binary mixtures is sufficient to drive phase separation.
Additional measurements by us (data not shown) and other
studies42 of perdeuterated cholesterol in PSM/POPC/cholesterol
mixtures display an intermediate ordering between those of
PSM/cholesterol and POPC/cholesterol, in contrast to the results
for the chain-perdeuterated lipids. In line with recent pulse-
field-gradient NMR studies of similar systems,41 this implies
that cholesterol is involved in exchange between the different
membrane regions that is fast on the NMR time scale (mil-
liseconds to microseconds). Nonetheless, it should be noted that
preferential affinity of the sterol for the sphingolipid probably
appears at small cholesterol mole fractions and low temperatures,
as shown here for PSM-d31/POPC with XC ) 0.20 below
25 °C.
are largest and most stable at intermediate cholesterol content.
If our results were to be compared to those for biological
membranes with high cholesterol content (up to 50 mol %), it
would seem unlikely that patches of lo domains would be
floating in a completely segregated ld phase in vivo. More
probably, the plasma membrane would exist mainly in a highly
ordered lo phase that is interrupted by smaller, more disordered
areas of high fluidity. Of course, this does not take into account
the possibility of domain formation due to cytoskeleton-linked
membrane proteins or the asymmetric distribution of the diverse
lipid species throughout the two leaflets of the bilayer. It has
been shown in model membranes52 that formation of such
domains entails Gibbs free energies that are much lower than
those for protein-lipid interactions, which therefore may
indicate a role for proteins in domain formation. Further detailed
information regarding protein-lipid interactions can contribute
to an understanding of the lateral organization of cellular plasma
membranes and how rafts may be implicated in their functional
mechanisms.
An alternative41,42 is to consider a different mechanism for
phase separation, namely, the influence of the configurational
entropy on the partitioning of lipids with disordered acyl chains
in the ld phase. This mechanism assumes that the overall greater
order parameters of sphingomyelin should lead to a smaller
entropy penalty than for POPC upon interaction with cholesterol.
Miscibility of SM/cholesterol mixtures strongly depends on the
degree of unsaturation in similar systems employing phosphati-
dylethanolamine.37 This effect could not be seen in our systems,
however, as the order in the binary POPC-d31/cholesterol
systems is far more sensitive to temperature changes than in
comparable mixtures of PSM-d31/cholesterol. In ternary mix-
tures, this should lead to increased phase separation at high
temperatures, which could be seen only weakly in our work
and in a recent study on very similiar mixtures.51 The largest
domains with distinct detectable subspectra for each phase were
observed in mixtures with XC ) 0.20 at low temperatures,
indicating a strong influence of enthalpy rather than entropy.
Finally, we note that the phase separation seems to occur
mainly in regions with smaller cholesterol mole fractions, with
distinct subspectra observable on the 2H NMR time scale.
Hence, it is probable that domain size strongly depends in the
same way on sterol concentrations. In other words, domains
Acknowledgment. This work was funded by the Deutsche
Forschungsgemeinschaft (Sfb 596), the National Institutes of Health
(EY012049, EY018891, and HL083187), and the Arizona Biomedi-
cal Research Commission. T.B. thanks Prof. Dr. Daniel Huster and
Dr. Alexander Vogel for generously providing the de-Pakeing
program.
Supporting Information Available: Complete lists of the
spectral assignments as well as the structural parameters obtained
using the first-order mean-torque model. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA801789T
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