Báo cáo khoa học: An X-ray diffraction study of model membrane raft structures doc

An X-ray diffraction study of model membrane raft
structures
Peter J. Quinn
1
and Claude Wolf
2
1 Biochemistry Department, King’s College London, UK
2 ER7-Faculte
´
de Me
´
decine-UPMC, APLIPID, Universite Paris 6, France
Introduction
Cell membranes, once regarded as uniform structures,
are now yielding up a complexity that is required to
explain the multiplicity of tasks they are reputed to
perform. One particular function that demands a
highly specific assembly of membrane components is
the receipt and transmission of molecular signals from
one side of the membrane to the other. Current think-
ing favours the, so-called, raft hypothesis, which postu-
lates that the signalling elements are segregated and
assembled in ordered lipid domains in the membrane
[1–6]. This membrane heterogeneity is rationalized on
the basis that, for the efficient operation of a signalling
system, the protein components must be closely associ-
ated and organized in such a way that structural
Keywords
lipid rafts; liquid-ordered phase; membrane
rafts; sphingomyelin; X-ray diffraction
Correspondence

P. J. Quinn, Biochemistry Department,
King’s College London, 150 Stamford
Street, London SE1 9NH, UK
Fax: +442078484500
Tel: +442078484408
E-mail:
(Received 6 July 2010, revised 1 September
2010, accepted 9 September 2010)
doi:10.1111/j.1742-4658.2010.07875.x
Protein sorting and assembly in membrane biogenesis and function involves
the creation of ordered domains of lipids known as membrane rafts. The
rafts are comprised of all the major classes of lipids, including glycero-
phospholipids, sphingolipids and sterol. Cholesterol is known to interact
with sphingomyelin to form a liquid-ordered bilayer phase. Domains
formed by sphingomyelin and cholesterol, however, represent relatively
small proportions of the lipids found in membrane rafts and the properties
of other raft lipids are not well characterized. We examined the structure
of lipid bilayers comprised of aqueous dispersions of ternary mixtures of
phosphatidylcholines and sphingomyelins from tissue extracts and choles-
terol using synchrotron X-ray powder diffraction methods. Analysis of the
Bragg reflections using peak-fitting methods enables the distinction of three
coexisting bilayer structures: (a) a quasicrystalline structure comprised of
equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a
liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phos-
pholipid bilayers. The structures have been assigned on the basis of lamel-
lar repeat spacings, relative scattering intensities and bilayer thickness of
binary and ternary lipid mixtures of varying composition subjected to ther-
mal scans between 20 and 50 °C. The results suggest that the order created
by the quasicrystalline phase may provide an appropriate scaffold for the
organization and assembly of raft proteins on both sides of the membrane.

Co-existing liquid-ordered structures comprised of phospholipid and
cholesterol provides an additional membrane environment for assembly of
different raft proteins.
Abbreviations
brainSM, bovine brain sphimgomyelin; egg-PtdCho, hen egg-yolk phosphatidylcholine; GPI, glycerylphosphorylinsitol; SAXS, small-angle
(1°–14°) X-ray scattering; WAXS, wide-angle (12°–30°) X-ray scattering.
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4685
changes accompanying the generation of a signal are
coupled to the transducing elements responsible for
execution of the response [7].
Critical tests of the hypothesis have largely been
aimed at characterizing the forces that govern the crea-
tion of lipid rafts rather than identifying the way in
which the signalling complexes are assembled within
the structure [8]. The main obstacle to progress has
been the use of unreliable methods to isolate mem-
brane rafts. The conventional protocol, irrespective of
the type of membrane, has been to recover a mem-
brane fraction that survives dissolution by Triton X-
100 treatment at 4 °C. The integrity of this method
has recently been challenged [7] and alternative meth-
ods based on a milder detergent treatment that is more
compatible with physiological conditions have been
developed [9]. The resulting membrane raft fraction
retains properties consistent with an arrangement of
constituents expected of its biological progenitor.
Using such methods, it has been possible to demon-
strate that subpopulations of raft vesicles which con-
tain predominantly one surface antigen or another can
be separated by immunoadsorption. Moreover, analy-

ses of the composition of these subpopulations show
that they contain different proportions of specific polar
lipids [10]. The fatty acid substituents attached to cere-
brosides and sphingomyelins also differ and represent
products of different metabolic pools; they are con-
sequently remodelled via different pathways. One
remarkable feature of the lipid analysis is the relatively
high proportion (20–30%) of monounsaturated polar
lipids. Moreover, the proportion of polyunsaturated
molecular species of phospholipids, particularly phos-
phatidylinositols, increases following the activation of
raft proteins [11]. These findings appear contrary to
the idea that cholesterol preferentially forms liquid-
ordered phases with saturated molecular species of
phospholipid [12]. Taken together, these results sup-
port not only the notion that the rafts are truly
domains present in the parental membrane, but also
that the lipids are distinct in each raft population. The
results also infer that membrane lipids may fulfil more
specific functions in the segregation and assembly of
protein components in the raft domains than hitherto
contemplated.
Many attempts have been made to model membrane
lipid rafts, some of which are focused on gel-phase
separation of lipid mixtures comprised of molecular
species that differ in the temperature of their transition
between gel and liquid–crystal phases [13,14]. The
relevance of these studies was underscored by the fact
that molecular species of sphingomyelin found in
membranes and enriched in membrane raft fractions

exhibited order–disorder transitions poised around
physiological temperatures. Attention switched to cho-
lesterol when it was reported that the condensing effect
of sterols on phospholipids, particularly sphingomye-
lins, created a bilayer phase that has properties inter-
mediate between a gel and a liquid–crystal phase,
referred to as a liquid-ordered phase [15,16]. Choles-
terol is known to be a prominent lipid component of
membrane rafts irrespective of the isolation method
[6,17].
A third type of lipid enriched in membrane rafts are
the glycosphingolipids [18]. Because the molecular
species of sphingolipids are characterized by a high
proportion of long N-acyl fatty acids (C-22 to C-26) it
was suggested that these lipids may act to couple the
two leaflets of the bilayer by interdigitation of the long
chain fatty acid from one side to the other of the
structure [19,20]. Other suggested functions of these
asymmetric lipids have been to stabilize highly curved
membrane domains formed transiently in the process
of membrane budding and fusion during progress
along the secretory pathway [21], or to increase hydro-
carbon packing density to impede the permeability of
small solute molecules [22]. More recent molecular
dynamics simulation studies are more equivocal on this
point and although long hydrocarbon chains are able
to penetrate the opposing monolayers of fluid bilayers,
the terminal region of the chain appears to be localized
in the centre of the bilayer [23]. Other experimental
and thermodynamic arguments have also cast doubt

on the action of long-chain molecular species of lipids
in coupling domains of bilayer structures [24,25]. The
role of these long-chain molecular species has now
been reassessed in the light of the action of these
asymmetric sphingolipids to form stoichiometric com-
plexes with phospholipids that have the properties of a
quasicrystalline phase [26,27].
We have undertaken an examination of the phase
behaviour of ternary mixtures containing representa-
tives of all the lipid classes identified in membrane raft
preparations. Phospholipids of biological extraction
were used so that a range of molecular species of phos-
phatidylcholines and sphingomyelins are present. The
thermotropic phase behaviour was examined in multil-
amellar dispersions at temperatures spanning the phys-
iological range to characterize the miscibility of the
different lipids under conditions in which mammalian
membrane rafts are likely to form. The use of synchro-
tron X-ray powder diffraction methods is able to pro-
vide detailed information on phase coexistence in
complex bilayers as well as on coupling of the two
monolayers of the bilayer, an essential feature in the
formation of a membrane raft.
Membrane raft structure P. J. Quinn and C. Wolf
4686 FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS
Results
Thermotropic phase behaviour of ternary
mixtures
To characterize the thermotropic phase behaviour of
ternary mixtures of egg-phosphatidylcholine (Ptd-

Cho) ⁄ brain sphingomyelin (SM) ⁄ cholesterol, aqueous
dispersions equilibrated at 20 °C were subjected to ini-
tial heating scans to 50 °C and subsequent cooling
scans to 20 °Cat2°Æmin
)1
. The intensity of scattered
X-rays was recorded simultaneously in the small-angle
(SAXS = 1°–14°) and wide-angle (WAXS = 12°–30°)
scattering regions during the scans. The results
obtained from an initial heating scan of a ternary mix-
ture comprised of egg-PtdCho ⁄ brainSM ⁄ cholesterol in
molar proportions 80 : 10 : 10 are presented in
Fig. 1A. Two series of reflections in the SAXS region
can be detected and they are in the order 1 : 1 ⁄ 2:1⁄ 3
(only the first two-orders are shown), indicating that
all structures are lamellar. Within each order of Bragg
reflection more than one lamellar phase is present; this
is particularly evident from the second-order reflections
in which overlapping peaks are obvious. The absence
of a sharp WAXS peak indicates that no gel or crystal
phases are present in the mixture [28]. The scattering
intensity profiles were subject to a peak fitting analysis
to characterize the coexisting lamellar phases. The
SAXS data were best fitted by three Gaussian +
Lorentzian curves as seen in Fig. S1C,D. The fit of
two peaks to the Bragg peak is shown for comparison
in Fig. S1A,B. The relationship between d-spacings of
the three individual peaks and temperature is plotted
in Fig. 1B. The fact that discrete lamellar reflections
can be deconvolved from the scattering bands means

that the two leaflets of each of the respective bilayer
structures are coupled.
An analysis of the scattering intensity profiles
recorded during a subsequent cooling scan (see Figs S2
and S3) indicates that the changes observed in lamellar
d-spacings (Fig. 1B) during the heating scans are
completely reversible with no significant temperature
hysteresis. This is consistent with the absence of any
structural alteration in the bilayer or thickness of the
AB
CD
EF
Fig. 1. Characterization of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10. An
overview of small- and wide-angle X-ray
scattering intensity profiles recorded from
an aqueous dispersion of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol in molar propor-
tions 80 : 10 : 10, recorded during a heating
scan at 2°Æmin
)1
between 20 and 50 °C, is
shown in (A) as the scattering intensity
profiles from the first two orders of lamellar
repeat structures and wide-angle scattering
profiles. (B) Lamellar d-spacings. (C) Scatter-
ing intensities. (D) Peak shape (scattering
amplitude ⁄ full-width at half maximum inten-
sity) of the first-order lamellar structures.
(E) WAXS d-spacings. (F) WAXS scattering

intensities.
P. J. Quinn and C. Wolf Membrane raft structure
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4687
hydration layer characterizing the dimensions of the
lamellar unit cell. The scattering intensities of the three
peaks and an index of the peak sharpness (peak ampli-
tude ⁄ full width at half maximum intensity) are
presented in Fig. 1C,D, respectively. Unlike lamellar
d-spacings, the decrease in scattering intensity observed
during the initial heating scan is not reversed during
the subsequent cooling scan (Fig. S1B,C). Likewise,
the simultaneous broadening of these peaks is not
reversed on cooling. This suggests that the size, but
not the structure as judged by lamellar d-spacing, of
the scattering arrays decreased during heating from the
equilibration temperature to $ 35 °C as a consequence
of the fragmentation of the scattering units into smal-
ler, possibly less well-ordered, arrays. Heating to
higher temperatures appears to have no additional
effect on the arrangement of the scattering units, there-
fore, a reliable indication of the relative amounts of
lamellar structure in the deconvolved peaks contribut-
ing to the overall scattering intensity can be obtained
at 38 °C. It is noteworthy that the parameters of the
peak of greatest d-spacing, which contributes least to
the total scattering intensity, are relatively constant
during the temperature scans. This may indicate that
the arrangement and presentation of the scattering
units in this lamellar structure do not change signifi-
cantly with temperature.

A peak-fitting analysis of the WAXS intensity pro-
files was undertaken and the results are presented in
Fig. 1E,F. There is no evidence of a sharp peak at
$ 0.42 nm to indicate the presence of a gel phase. A
minor peak located at a d-spacing of 0.45 nm can be
deconvolved from the scattering profiles recorded at
temperatures < 30 °C during the initial heating scan,
but this peak becomes indistinguishable from a broad
scattering band at $ 0.463 nm typical of disordered
hydrocarbons at higher temperatures.
A ternary mixture containing higher proportions of
brainSM and cholesterol was then examined and the
results are presented in Fig. 2. The scattering intensity
patterns recorded in the SAXS and WAXS regions
during the initial heating scan from 20 to 50 °C
from the ternary mixture comprised of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol, 60 : 20 : 20, are presented
in Fig. 2A. The SAXS intensity peaks in this mixture
are best fit by only two Gaussian + Lorentzian curves,
in contrast to the mixture shown in Fig. 1. Lamellar
d-spacings, scattering intensity profiles and peak
AB
CD
EF
Fig. 2. Characterization of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol; 60 : 20 : 20. An
overview of small- and wide-angle X-ray
scattering intensity profiles recorded from
an aqueous dispersion of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol in molar propor-

tions 60 : 20 : 20, recorded during a heating
scan at 2°Æmin
)1
between 20 and 50 °C, is
shown in (A) as the scattering intensity pro-
files from the first two orders of lamellar
repeat structures and wide-angle scattering
profiles. (B) Lamellar d-spacings. (C) Scatter-
ing intensities. (D) Peak shape of the
second-order of the lamellar structures.
(E) WAXS d-spacings. (F) WAXS scattering
intensities.
Membrane raft structure P. J. Quinn and C. Wolf
4688 FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS
shapes derived from analysis of the thermal scans are
shown to be distinct in Fig. 2B,C,D, respectively. It
can be seen that the peak of shortest d-spacing
observed in the mixture comprised of 80 : 10 : 10
(Fig. 1B) is absent from this ternary mixture. More-
over, the remaining two lamellar phases have corre-
spondingly greater lamellar d-spacings than those
observed in the mixture shown in Fig. 1. The tempera-
ture-dependent change in scattering intensity is consid-
erably less marked, suggesting that the scattering units
are more stable when the proportions of brainSM and
cholesterol in the mixture are increased relative to egg-
PtdCho. The Bragg peaks also tend to be sharper. The
dominant scattering peak in the WAXS region is
shifted to shorter d-spacings indicating that increased
proportions of brainSM and cholesterol bring about

a closer packing in the hydrocarbon region of the
bilayers.
The effect of increasing only the proportion of
brainSM in the ternary mixture is exemplified by the
behaviour of a mixture comprised of egg-PtdCho ⁄
brainSM ⁄ cholesterol, 10 : 80 : 10 shown in Fig. 3. The
scattering intensity profiles in the SAXS region show
the first two orders of reflection of lamellar phases and
the presence of a relatively sharp WAXS peak at
0.42 nm, indicating that a gel phase forms on equili-
bration at 20 °C. This WAXS peak coexists with a rel-
atively weak scattering band at $ 0.47 nm which can
no longer be distinguished from the main peak at a d-
spacing at 0.44–0.45 nm upon heating above $ 32 °C.
The changes observed in the SAXS ⁄ WAXS profiles are
consistent with a progressive replacement of a gel
phase of brainSM and the disappearance of a small
proportion of a coexisting highly disordered lamellar
phase with a homogeneous liquid-ordered phase of d-
spacing 0.44 nm during heating to 32 °C. At higher
temperatures, a new lamellar phase of greater d-spac-
ing appears but represents only a relatively minor com-
ponent of the overall scattering intensity. It can be
concluded from analysis of the behaviour of this ter-
nary mixture that the properties of the major constitu-
ent of the mixture, namely, long N-acyl chain
molecular species of sphingomyelin, tend to dominate
the temperature-dependent structural parameters of the
bilayers.
Assignment of lamellar structures

The next task was to establish the identity of the
coexisting lamellar phases in ternary mixtures contain-
AB
CD
EF
Fig. 3. Characterization of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol; 10 : 80 : 10. An
overview of small- and wide-angle X-ray
scattering intensity profiles recorded from
an aqueous dispersion of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol in molar propor-
tions 10 : 80 : 10, recorded during a heating
scan at 2°Æmin
)1
between 20 and 50 °C, is
shown in (A) as the scattering intensity pro-
files from the first two orders of lamellar
repeat structures and wide-angle scattering
profiles. (B) Lamellar d-spacings. (C) Scatter-
ing intensities. (D) Peak shape of the
first-order lamellar structures. (E) WAXS
d-spacings. (F) WAXS scattering intensities.
P. J. Quinn and C. Wolf Membrane raft structure
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4689
ing relatively high proportions of the fluid host
phospholipid, egg-PtdCho, which are representative of
the lipid composition of mammalian membrane extra-
cellular leaflet embedding the raft microdomains. Three
possible lamellar structures comprised of varying
proportions of lipids are brainSM ⁄ egg-PtdCho, brain-

SM ⁄ cholesterol or egg-PtdCho⁄ cholesterol; a ternary
complex of the three lipids is excluded as implausible in
this analysis because most ternary mixtures are com-
prised of more than one bilayer component. Figure 4
shows a method of assigning the composition of the dif-
ferent lamellar phases on the basis of the relationship
between lamellar d-spacing and temperature. The result
of a peak-fitting analysis of the SAXS intensity profile
recorded from the ternary mixture comprised of
egg-PtdCho ⁄ brainSM ⁄ cholesterol, 80 : 10 : 10, at 38 °C
is presented in Fig. 4A. The profile can be seen to be
best fit by three Gaussian + Lorentzian peaks which
are shown in Fig. 4B together with the difference
between the observed and calculated fit to the data
(Fig. 4C) (see Fig. S1).
Peak 1 represents $ 10% of the total scattering
from the first-order Bragg reflections. The d-spacings
of this peak coincide closely with d-spacing recorded
from a binary mixture of egg-PtdCho ⁄ brainSM in
equimolar proportions recorded under the same condi-
tions (Fig. 4D). It is known that gel-phase separation
occurs in this binary mixture when equilibrated at
20 °C [29], however, there is no evidence that gel-phase
separation occurs in this ternary mixture (Fig. 1E).
The presence of 10 mole% cholesterol in the ternary
mixture apparently hinders formation of a gel phase
by brainSM in this mixture. Assignment of peak 1 to a
structure of pure brainSM can also be excluded on this
evidence. The fit of peak 1 to brainSM ⁄ cholesterol
mixtures was considered from the respective dimen-

sions of the unit cell (d-spacings) and peak shape
parameter representing the order of the diffracting
units. The effect of varying proportions of cholesterol
in binary mixtures with brainSM is presented in
Fig. 5A. It can be seen that the d-spacing of brainSM
bilayers at 38 °C is 8.3 nm and this is progressively
reduced by increasing the proportions of cholesterol
(Fig. 5C). An equimolar proportion of cholesterol
would be required to reduce the d-spacing to that
observed for peak 1 (6.8 nm) in Fig. 4C. The assign-
ment of peak 1 as comprised of egg-PtdCho and
$ 20 mole% cholesterol (Fig. 5D) cannot be excluded
on this criterion. Other evidence presented below, how-
ever, indicates that cholesterol is not a constituent of
peak 1.
The scattering intensity of peak 2 contributes
$ 30% to the total intensity of the first-order Bragg
peaks of the mixture shown in Fig. 4. The d-spacing of
peak 2 in Fig. 4B coincides closely with bilayers
formed from a binary mixture of egg-PtdCho and pro-
portions of cholesterol of $ 25 mole% (Fig. 5D). The
effect of cholesterol on d-spacings of egg-PtdCho
bilayers is complex. The presence of only 10 mole%
A
B
C
D
Fig. 4. Assignment of lamellar structures. An analysis of the ternary
mixture of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10 recorded
at 38 °C. (A) Fit of scattering intensity from the first-order Bragg

reflection (d) to three Gaussian + Lorentzian area curves (s). (B)
Peak deconvolution from the scattering intensity profile shown in
(A). (C) Difference between observed and calculated fits to the data.
(D) Lamellar d-spacings as a function of temperature recorded dur-
ing heating scans at 2°Æmin
)1
. d, peak 1; s, egg-PtdCho ⁄ brainSM,
50 : 50; j, peak 2; h, egg-PtdCho ⁄ cholesterol, 70 : 30; m, peak 3;
D, egg-PtdCho.
Membrane raft structure P. J. Quinn and C. Wolf
4690 FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS
cholesterol causes a dramatic increase in d-spacing
because of the full extension and vertical orientation of
the hydrocarbon chains of the phospholipid in the
bilayer. Increasing proportions of cholesterol up to
30 mole% result in a progressive decrease in repeat
spacing because of hydration effects at the bilayer–
water interface. An assignment of peak 2 to a binary
mixture of brainSM and cholesterol can be excluded
on the basis of lamellar d-spacings > 7 nm at 38 °C
[30]. Assuming peak 2 is comprised of phospholipid
and 25 mole% cholesterol, the contribution of the
peak to the total scattering from the ternary mixture is
calculated to be 25%. This is close to the observed
proportion of the total scattering from peak 2 in the
ternary mixture.
Assignment of the dominant peak (peak 3) repre-
senting $ 60% of total scattering at 38 °C in the
ternary mixture egg-PtdCho ⁄ brainSM ⁄ cholesterol,
80 : 10 : 10, was made by comparison with bilayers of

pure egg-PtdCho, the most abundant phospholipid in
the mixture. As can be seen from Fig. 4C, the d-spac-
ing of the pure phospholipid is $ 0.5 nm less at equiv-
alent temperatures than observed for peak 3. Because
the d-spacing is less than binary mixtures containing
high proportions of cholesterol in egg-PtdCho, it fol-
lows that the increase in d-spacing of peak 3 must be
caused by the presence of proportions of cholesterol
< 10 mole%. That peak 3 is comprised predominantly
of egg-PtdCho is also evident from the absence of this
peak in ternary mixtures containing lower proportions
of egg-PtdCho, as demonstrated in the ternary mix-
ture consisting of egg-PtdCho ⁄ brainSM ⁄ cholesterol,
60 : 20 : 20 examined in Fig. 2. Thus, assignment of
peak 3, as judged by d-spacing, can be made as egg-
PtdCho containing a relatively small proportion of
cholesterol.
Further evidence consistent with assignment of
peak 1 to a liquid-ordered lamellar phase comprised of
an equimolar proportion of egg-PtdCho and brainSM
was obtained by relating the relative mass of brainSM
in ternary mixtures of egg-PtdCho⁄ brainSM⁄ choles-
terol to the scattering contribution from peak 1 to the
total scattering intensity recorded at 38 °C. A peak of
lamellar repeat of 6.7–6.8 nm could be deconvolved
from first-order Bragg reflections in 12 ternary mix-
tures examined; no peak at this position was observed
in mixtures with proportions of cholesterol exceeding
50 mole%. There was no correlation between the scat-
tering intensity of this peak and the relative mass of

egg-PtdCho, brainSM, cholesterol or any binary com-
binations of the lipids in the mixtures (Fig. S4). How-
ever, if the contribution to the scattering of the peak
was limited to a mass of brainSM equivalent to the
proportion of egg-PtdCho in those ternary mixtures
where the mol% of brainSM exceeds that of egg-Ptd-
Cho, a correlation is obtained. A plot of the relation-
ship between the scattering intensity of the peak and
the mass of equimolar proportions of brainSM ⁄
AB
CD
Fig. 5. Binary mixtures of phospholipid and
cholesterol. Small-angle X-ray scattering
intensity patterns of binary mixtures of (A)
brainSM and (B) egg-PtdCho with the
indicated mol% cholesterol at 37 °C. (C)
and (D) show the respective relationships
between lamellar d-spacing and mol%
cholesterol.
P. J. Quinn and C. Wolf Membrane raft structure
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4691
egg-PtdCho in different ternary mixtures is shown in
Fig. 6.
A third method to investigate the assignments of
composition of peaks 1 and 2 was to compare relative
electron densities through the bilayer repeat structures.
The results of such calculations are summarized in
Fig. 7. It can be seen that relative electron density dis-
tributions across the bilayer repeats calculated at
38 °C for peaks 1 and 2 are different. By contrast, the

thickness of the bilayers and the water layers of peak 1
are almost identical to those calculated for bilayers
consisting of an equimolar mixture of egg-PtdCho and
brainSM. The bilayer thickness of peak 2 is signifi-
cantly greater, and the water layer significantly less,
than calculated for peak 1. Resolution of the electron-
density calculation for peak 2 was relatively low
because only three orders of reflection were detected in
this mixture. Nevertheless, the thickness of the bilayer
and water layer are almost identical to the parameters
calculated from a binary mixture comprised of egg-
PtdCho and 30 mole% cholesterol, a characteristic Lo
phase [31] where three orders of reflection were used in
the calculation.
Discussion
The lipids identified in rafts isolated from biological
membranes differ from those of the parent membrane,
but such results need to be regarded with some cau-
tion at this stage. The reason is that reliable methods
of isolating rafts have not generally been employed.
The size of domains in living cell membranes is
defined as between 10 and 200 nm [32], and vesicles
derived from rafts occupying areas of the parent
membrane of this order would be between 5 and
30 nm in diameter. This is at odds with the size of
vesicles isolated as detergent-resistant membranes.
Estimates of the size of detergent-resistant membrane
vesicles prepared from rat brain indicate a relatively
homogeneous population of unilamellar vesicles of
diameter ranging from 130 to 240 nm [33]. This corre-

sponds to an average domain diameter in the parent
membrane in the order of 600 nm, somewhat larger
than areas envisaged for membrane raft domains.
Subpopulations of these vesicles can be separated by
immunoadsorption methods containing different sur-
face antigens, which argues against the fusion and
amalgamation of domains in the parent membrane.
Electron microscopy examination of these vesicles
indicates that the prion protein (PrP
c
) and thymus-
derived antigen 1 (Thy-1) associated with these raft
preparations are generally clustered together and
occupy only a small fraction of the vesicle membrane
[9]. This suggests that the rafts are not homogeneous
Fig. 6. Relationship between brainSM and scattering intensity. Cor-
relation between relative scattering intensity of peak 1 in Fig. 4
from the first-order Bragg reflection (d-spacing 6.7–6.8 nm) and
mass of brainSM + equimolar egg-PtdCho in different ternary mix-
tures of the two phospholipids and cholesterol recorded at 38 °C.
Mixtures with proportions of brainSM greater than egg-PtdCho
were taken as equimolar to the proportion of egg-PtdCho in the
mixture.
Fig. 7. Electron-density calculations. Relative electron density pro-
files were calculated through the lamellar repeats of peak 1 and
peak 2 recorded at 38 °C from the data in Fig. 1A. Relative electron
densities calculated from binary mixtures of brainSM ⁄ egg-PtdCho
in equimolar proportions and egg-PtdCho ⁄ 30 mole% cholesterol at
38 °C are shown for comparison.
Membrane raft structure P. J. Quinn and C. Wolf

4692 FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS
bilayers of lipids in liquid-ordered phase but that an
organization is imposed on the proteins that causes
their association within the structure.
The question of whether membrane proteins or lip-
ids alone or together play a part to bring about the
segregation of raft components is a moot point. It is
known that successful delivery of plasma membrane
raft proteins from the Golgi in yeast depends on the
biosynthesis of ergosterol and sphingolipids [34].
Genetic screening of defective mutants indicated a lack
of a functional fatty acid elongating system for synthe-
sis of long-chain molecular species of sphingolipids
[35], or a defect of dihydrosphingosine C4 hydroxylase
in the biosynthesis of phytosphingosine [36] may be
responsible. One possible mechanism for organizing
proteins in the liquid-ordered phase is by homotypic
interactions between the proteins themselves or interac-
tions mediated by intermediary proteins. An example
of the latter is the clustering of Pma1p, the plasma
membrane H
+
-ATPase of yeast, in raft lipid domains.
This has been shown to require a peripheral membrane
protein, Ast1, in the endoplasmic reticulum, a process
that is an essential step in the transfer of the raft pro-
tein to the plasma membrane [37]. In the case of the
N
+
⁄ H

+
antiporter in yeast (Nha1p), however, the
sorting signal apparently resides in the hydrophobic
domain of the membrane [38] and sphingolipid is
essential for retention of the protein in the plasma
membrane [39]. There is also a strong possibility that
the lipid anchors that tether proteins to membrane
rafts may interact in a specific way with the lipids
forming the raft.
Clearly, there is scope for different methods of orga-
nizing proteins within membrane rafts, but it is not
easy to envisage how the specificity required to bring
about clustering of one type of receptor protein on
one side of the membrane, and co-localizing this with
specific lipid-anchored proteins on the opposite side of
the membrane, can occur simply within a liquid-
ordered phase of polar lipid and cholesterol. On the
basis of the evidence obtained in this study, such speci-
ficity can be proposed. The structures formed by
binary mixtures of long N-acyl molecular species of
sphingolipids and phospholipids consist of stoichiome-
tric complexes of 1 : 2 phosphatidylethanolamines [27]
and 1 : 1 phosphatidylcholine [26]. A phase with
hydrocarbon chain spacings consistent with a liquid-
ordered quasicrystalline phase, formed from equimolar
proportions of egg-PtdCho and brainSM [29], has
been identified in this study in ternary mixtures with
cholesterol.
We propose that the structure formed by long
N-acyl fatty acid molecular species of sphingolipids

and phospholipids creates a matrix that is coupled
across the raft membrane. According to such a model,
glycosphingolipids based on molecular species of
galactosylceramides with long N-acyl fatty acids and
phosphatidylcholines would reside in the outer mono-
layer in mammalian plasma membrane. These domains
are coupled with glucosylceramides and phosphatidy-
lethanolamines in the cytoplasmic leaflet. These struc-
tures form a matrix into which GPI-anchored receptor
proteins are interpolated on the outer surface and are
coupled with corresponding lipid-anchored effecter
proteins located in the cytoplasmic leaflet. The specific-
ity of these interactions may involve the sugar residues
of the glycosphingolipids and the domains of the pro-
teins, or the configuration of the lipid anchor, or both.
That activity-associated remodelling of lipid anchors is
a recognized process in raft function [40,41] suggests
that the configuration of the raft anchors is a likely
candidate.
The lipid matrix model of the membrane raft struc-
ture can be formulated according to a two-stage pro-
cess of molecular assembly. Cartoons of the structures
comprising the model and their relationship to bilayer
spacings are presented in Fig. 8. Liquid-ordered
domains comprised of more saturated molecular spe-
cies of phospholipid and cholesterol serve to exclude
most membrane proteins and accommodate those
proteins required for raft function. Glycosphingolipids
with long N-acyl fatty acid chains and phospholipids
form a quasicrystalline matrix acting to concentrate

and organize the protein components into a functional
complex in the raft. It is reported that cholesterol is
excluded from such phases [42]. The remodelling of
lipid anchors takes place in the liquid-ordered domain
in a manner that allows them to interpolate into the
matrix component of the raft membrane. The intimate
association between receptors and effectors brought
about by their integration into the matrix is an essen-
tial feature designed to facilitate the transmission of
molecular signals generated on one side of the matrix
to the other.
The model of raft structure we propose fits current
knowledge of the lipid composition of membrane rafts
obtained without detergent treatment. Eighty-three
molecular species of membrane lipid have been identi-
fied and quantified in highly purified raft preparations
from yeast [43]. Glycosphingolipids with almost exclu-
sively long-chain hydroxylated N-acyl substituents
[C26:0(OH)] are present in equimolar proportions with
di-unsaturated molecular species of phosphatidylcho-
line and would be expected to form a quasicrystalline
bilayer structure. The remaining phospholipids are
dominated by phosphatidylinositol with a saturated
P. J. Quinn and C. Wolf Membrane raft structure
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4693
fatty acid acylated to the C-1 position of the glycerol.
This would form a liquid-ordered phase with a compo-
sition comprised of 38 mole% ergosterol. The order of
the lipids in the membrane rafts as measured by
spectroscopic studies using C-Laudan is consistent with

the tight packing of acyl chains in the raft model
membrane.
Materials and methods
Lipids
Egg-yolk phosphatidylcholine (egg-PtdCho, 715 Da),
bovine brain sphingomyelin (brainSM, 788 Da) and choles-
terol (387 Da) were purchased from Sigma (Sigma-Aldrich,
St. Quentin-Fallavier, France). A complete lipid analysis of
each phospholipid was performed by ESI-tandem MS [29]
and the data are presented in Table S1.
Sample preparation
Samples for X-ray diffraction examination (Table S2) were
prepared by dissolving lipids in warm (45 °C) chloro-
form ⁄ methanol (2 : 1, v ⁄ v) and mixing them in the desired
proportions (denoted as molar ratios in binary mixtures).
The organic solvent was subsequently evaporated under a
stream of oxygen-free dry nitrogen at 45 °C and any
remaining traces of solvent were removed by storage under
high vacuum for 2 days at 20 °C. Dry lipids were hydrated
with an equal mass of water. This was sufficient to fully
hydrate egg-PtdCho [44] and brainSM [45], respectively.
The lipids were stirred thoroughly with a thin needle, sealed
under argon, and annealed by 50 thermal cycles between 20
and 65°C, ensuring a complete mixing of phospholipids.
Samples were stored under argon at a temperature not
below 4 °C. X-Ray diffraction examination was performed
after 5 h sample equilibration at 20 °C and after careful stir-
ring before transfer into the sample cell. In order to check
Fig. 8. Cartoons of the molecular composition of the different structures proposed for the lipid matrix model of membrane raft struc-
ture and their relationship to the Bragg spacings of ternary mixtures of egg-PtdCho ⁄ brainSM ⁄ cholesterol. Other evidence is reviewed in

Quinn [6].
Membrane raft structure P. J. Quinn and C. Wolf
4694 FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS
for possible dehydration or demixing of the components,
various control measurements were undertaken such as
checking for reversibility of phase behaviour during
subsequent heating and cooling cycles. The samples were
also checked for the absence of SAXS and WAXS
diffraction peaks from crystals of cholesterol. The mean
transition temperatures of lipids were found in the expected
range documented in the data bank Lipid Data Bank (LDB;
A list of the samples examined in
this study are tabulated in the Supporting information.
Synchrotron X-ray diffraction measurements
X-Ray diffraction measurements were performed on beam-
line 2.1 at the Daresbury Laboratory. The X-ray wave-
length was 0.154 nm with a beam geometry of
$ 0.2 · 0.5 mm in a mica sandwich cell with a surface of
2 · 5 mm and a path length of 0.5 mm. Simultaneous
SAXS and WAXS intensities were recorded so that a corre-
lation could be established between the mesophase repeat
spacings and the packing arrangement of acyl chains. The
SAXS intensity was recorded using a 2D RAPID area
detector and the signal was circularly integrated to give a
1D pattern. The WAXS intensity was recorded with a 1D
HOTWAXS detector. The sample to SAXS detector dis-
tance was 1.5 m and calibration of d-spacings was per-
formed using silver behenate (d = 5.838 nm). Wide-angle
X-ray scattering intensity profiles were calibrated using the
diffraction peaks from high-density polyethylene [46]. The

measurement cell was mounted on a programmable temper-
ature stage (Linkam, Tadworth, UK) and the temperature
was monitored by a thermocouple inserted directly into the
lipid dispersion (Quad Service, Poissy, France). The set-up,
calibration and facilities available on Station 2.1 are
described comprehensively in the website; .
ac.uk/srs/stations/station2.1.htm. Data reduction and analy-
sis were performed using originpro8 software (OriginLab
Corp., Northampton, MA, USA).
Analysis of X-ray diffraction data
The small-angle X-ray scattering-intensity profiles were
analysed using standard procedures [47]. Polarization and
geometric corrections for line-width smearing were assessed
by checking the symmetry of diffraction peaks in the pres-
ent camera configuration using a sample of silver behenate.
The orders of reflection could all be fitted by Gauss-
ian + Lorentz symmetrical (Voigt) functions with fitting
coefficients greater than R
2
= 0.99. Deconvolution is con-
sistent with the sample to detector distance used [48]. It
was noted that scattering intensity from some lamellar
repeat structures decreased significantly during heating sam-
ples equilibrated at 20 °Cupto$ 35 °C and remained con-
stant irrespective of the temperature for the duration of a
heating and cooling cycle. Because the changes in d-spac-
ings were completely reversible, the changes in scattering
intensity are consistent with a decrease in the size and order
within the diffracting units from that achieved during tem-
perature equilibration (see methods of powder diffraction

analysis in the Supporting information).
The scattering intensity data from the first four orders of
the Bragg reflections from the multilamellar liposomes were
used to construct electron density profiles [49]. After correc-
tion of the raw data by subtraction of the background scat-
tering from both water and the sample cell, each of the
Bragg peaks was fitted by a Lorentzian+Gaussian area
(Voigt) distribution by peak fitting performed using
peakfit 4.12 (Systat Software Inc., Bangalore, India).
Details of the peak fitting procedure are described in the
Supporting information.
The square root of integrated peak intensity I(h) is used to
determine the form factor F(h) of each respective reflection:
FðhÞ¼h
ffiffiffiffiffiffiffiffi
IðhÞ
p
ð1Þ
where h = order of peak reflection, I(h) = integrated
intensity of each respective reflection.
The electron density profile is calculated by the Fourier
synthesis:
qðzÞ¼
X
Æ FðhÞcosð2phz=dÞð2Þ
d = d
spacing
= d
pp
+ d

W
(d
pp
: phosphate-phosphate bilayer
thickness, d
W
: hydration layer) at a resolution of
d ⁄ 2h
max
$ 12 nm for four orders.
The phase sign of each diffraction order is either positive
or negative for a centro symmetric electron-density profile
for lamellar phases. The phasing choice was made by
inspection of all possible phase combinations; for all bilay-
ers examined the phase combination ())+ )) of signs
uniquely provides the expected electron density profile with
the minimum density appropriately located at the bilayer
centre, the maxima at the two electron-rich interfaces and
the hydration layer density (0.33e
)
⁄ A
3
) at the intermediate
value on the relative electron density scale. All other phase
combinations result in aberrant distributions.
Deconvolution of the WAXS intensity peaks was also
undertaken using peakfit software. No corrections for
polarization or geometric factors were necessary with the
HOTWAXS detector. Scattering in the WAXS region origi-
nates predominantly from hydrocarbon chains of the phos-

pholipids with a diffuse scattering contribution from the
polar head-groups. The presence of cholesterol in the
bilayer is known to impose an orientation of the chains
normal to the bilayer plane [50].
Acknowledgements
The authors are grateful to Dr Gunter Grossman for
assistance in setting up beamline 2.1 at the Daresbury
P. J. Quinn and C. Wolf Membrane raft structure
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4695
Laboratory. Drs Galya Staneva and Lin Chen are
thanked for providing assistance in preparing samples.
The work was aided by a grant of beamtime
(DL49098) and funds provided by the Human Science
Frontier Program (RGP0016 ⁄ 2005C).
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Supporting information
The following supplementary material is available:
Fig. S1. First-order Bragg scattering intensity peak (•)
recorded from the mixed aqueous dispersion of egg-
PtdCho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10 at 38 °C.
Fig. S2. (A) Lamellar d-spacing, (B) scattering inten-
sity and (C) amp ⁄ FWHM for a dispersion of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10, equilibrated for
5 h at 20 °C during an initial heating and subsequent
cooling scan at 2°Æmin
)1
.
Fig. S3. (A) Lamellar d-spacing, (B) scattering inten-
sity and (C) amp ⁄ FWHM for a dispersion of egg-Ptd-
Cho ⁄ brainSM ⁄ cholesterol; 60 : 20 : 20, equilibrated for
5 h at 20 °C during an initial heating and subsequent
cooling scan at 2°Æmin
)1
.
Fig. S4. Relationship between the per cent scattering
intensity observed in the peak of lamellar d-spacing of
$ 6.7 nm deconvolved from ternary mixtures of
P. J. Quinn and C. Wolf Membrane raft structure
FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4697

egg-PtdCho, egg-SM and cholesterol in varying pro-
portions and (A) mass of egg-PtdCho, (B) mass
brainSM, and (C) mass cholesterol.
Table S1. Fatty acid analysis of egg phosphatidylcho-
line and bovine brain sphingomyelin.
Table S2. Mixtures examined in this study.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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should be addressed to the authors.
Membrane raft structure P. J. Quinn and C. Wolf
4698 FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS