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Sterols and membrane dynamics

Article in Journal of Chemical Biology · December 2008

DOI: 10.1007/s12154-008-0010-6 · Source: PubMed

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J Chem Biol (2008) 1:63–77
DOI 10.1007/s12154-008-0010-6

REVIEW

Sterols and membrane dynamics

Erick J. Dufourc

Received: 4 May 2008 / Accepted: 7 August 2008 / Published online: 23 September 2008
# Springer-Verlag 2008

Abstract The effect of sterols from mammals, plants, fungi, cell recognition, signalling pathways, energetic, signal
and bacteria on model and natural membrane dynamics are transduction, and cell trafficking. Together with diacylgly-
reviewed, in the frame of ordering–disordering properties of cerols, their collective properties modulate lipid polymor-
membranes. It is shown that all sterols share a common phism, through phase transitions (lamellar, hexagonal,
property: the ability to regulate dynamics in order to cubic, micelles), which are involved in enzyme conforma-
maintain membranes in a microfluid state where it can tional changes, cell division, cell fusion, and apoptosis [1].
convey important biological processes. Depending on the Sterols, the third lipid class, also regulate biological
sterol class, this property is modulated by molecular processes and sustain the domain structure of cell mem-
modifications that have occurred during evolution. The role branes where they are considered as membrane reinforcers
of sterols in rafts, antibiotic complexes, and in protecting [2]. While cholesterol (CHO) is the major sterol of
membranes from the destructive action of amphipathic vertebrates, ergosterol (ERG) plays a key role in fungi.
toxins is also discussed. Plants usually possess more complex sterol compositions.
Stigmasterol (STI) and sitosterol (SIT), two 24-ethyl
Keywords Cholesterol . Alpha-cholesterol . sterols, are major constituents of the sterol profiles of plant
Cholesterol palmitate . Cholesterol sulphate . Cycloartenol . species. They are involved in the embryonic growth of
Sitosterol . Stigmasterol . Ergosterol . Bacteriohopanetetrol . plants [3]. Hopanoids such as bacteriohopanetetrol (BHT)
Bacteriohopaneaminotriol . Model and natural membranes . are sterol surrogates of primitive bacteria (archebacteria,
Membrane rafts . Regulating membrane dynamics . cyanobacteria, etc.) that develop in very extreme conditions
Solid-ordered . Liquid-disordered . Liquid ordered . such as hot springs, very high deep sea pressure, highly
Solid state NMR saline water, and ice-covered lakes. They are considered as
good markers of geological samples containing organic
matter [4].
Introduction: sterols and the dynamic membrane Sterols are critical for the formation of liquid-ordered
(lo) membrane states (lipid “rafts” [5]) that are supposed to
It is widely recognized that lipids play multiple roles that play an important role in fundamental biological processes
either individually or collectively influence cell processes. such as signal transduction, cellular sorting, cytoskeleton
Glycerolipids and sphingolipids through charge and struc- reorganization, asymmetric growth, and infectious diseases.
ture are involved in DNA replication, protein translocation, They have been proposed as key molecules to maintain
membranes in a state of fluidity adequate for function. For
E. J. Dufourc (*) instance, phytosterols have been recently shown to increase
UMR 5248 Chemistry and Biology of Membranes membrane cohesion in order to maintain plant membranes
and Nanoobjects, CNRS–Université Bordeaux 1–ENITAB, in a state of dynamics less sensitive to temperature shocks
CNRS Université Bordeaux,
2 Rue Robert Escarpit,
[6, 7]. In this review, the impact of different classes of
33607 Pessac, France sterols on membrane dynamics from mammals to arche-
e-mail: e.dufourc@iecb.u-bordeaux.fr bacteria will be discussed.
64 J Chem Biol (2008) 1:63–77

Molecular structure of some sterols in mammals, plants, Plant sterols Plants usually possess more complex sterol
fungi, and bacteria compositions. Cholesterol, campesterol, a 24-methyl sterol;
stigmasterol and sitosterol, two 24-ethyl sterols, are major
In general, sterols are synthesized via the mevalonate constituents of the sterol profiles of plant species. Sterol-
pathway of isoprenoid metabolism [3]. C24-methyltransferases are important for plant growth and
development [3] and alkylate the side chain on the D-ring
Mammalian sterols Cholesterol is found in many biological of sterols by two successive methylation steps on C24.
membranes and is the main sterol of animals. It is often Therefore, typical phytosterols like sito- and stigmasterol
equimolar with phospholipids in many membranes. It has a (Fig. 1) possess additional alkyl groups on C24. Stigmas-
tetracyclic structure with the OH group in the equatorial terol has an additional double bond at C22–C23. Both
position on its first A ring (Fig. 1) and possesses a short sterols originate from a precursor, cycloartenol, the first
aliphatic chain, with two ending methyl groups, branched cyclic intermediate in the biosynthesis which features a
on the D pentenic ring. The fused ring system is quasi-rigid. very unusual [9–10] cyclopropyl small ring attached to the
The alpha isomer (OH in axial position) is not encountered B-ring, three additional methyl groups on the fused ring
in nature. Cholesterol sulphate (CHS; Fig. 1), is found in system, and a double bond at the end of the acyl chain. The
the spermatozoa head and in the stratum corneum (the dry absence of double bond in position 6–7 in the B-ring
skin outer layer). It has the same structure as cholesterol confers some flexibility to the molecule [8].
except for a sulphate group substituting the hydroxyl group,
which results to a negative charge. Another class of sterols Fungi sterols The main sterol found in fungi is ERG, which
are ester linked to fatty acids (C16:0, C18:1, and C18:2; structure differs from that of cholesterol by the presence of
Fig. 1). They are found in lipoprotein particles and lipid additional double bonds, on the B-ring and on the acyl
droplets as extra- or intracellular surplus that subsequently chain. A methyl group is also present at C24. As will be
regulate the concentration of free sterols in the membranes. shown later, this sterol has peculiar interacting properties
with antibiotics that act against fungus, such as polyene
antibiotics [9–11].

Bacterial sterols Primitive sterols such as hopanoids are

C D produced by bacteria that develop in very extreme
A B Cycloartenol
(temperature, acidity, pressure, ionic strength) conditions
Cholesterol HO
HO
and are considered as good markers of geological samples
containing organic matter [4]. They possess a pentacyclic
fused ring structure (Fig. 1) with four OH groups
(bacteriohopanetetrol) or three hydroxyls and one NH2
α -Cholesterol Sitosterol group (bacteriohopaneaminotriol, BHAT) on the branched
HO HO
aliphatic chain. There are no OH group on the A ring, as in
cholesterol, conferring to these molecules some kind of
inverted polarity, on comparing to cholesterol, i.e., the
HO
O hydrophilic part of the molecule is no longer on the fused
S
O
O
Stigmasterol
ring system but at the end of the short aliphatic tail.
Cholesterol-sulfate HO

Measuring membrane dynamics using a noninvasive

O
Cholesterol-palmitate approach
O HO
Ergosterol

HO HO
Membrane dynamics is essential for cellular life. A delicate
HO HO
balance between a loose membrane too fluid and akin to
OH
NH 2
OH
OH
great permeability and a rigid membrane forbidding any
transfer across the bilayer has been reached through many
B t i h
Bacteriohopanaminotriol
i ti l B t i h
Bacteriohopanetetrol
t t l
evolutionary steps in nature. As will be shown in the
following, sterols play a central role in this aspect. The
Fig. 1 Molecular structures of mammalian, plant, fungus, and rigidity of a membrane is, however, difficult to define.
bacterial sterols When using macromolecular techniques to probe mem-
J Chem Biol (2008) 1:63–77 65

brane dynamics, one could state that it is the inverse of microsecond to millisecond time scale [12]. For instance,
fluidity, i.e., a macroscopic quantity that unfortunately the narrowing of a solid-state deuterium NMR spectrum
hides in some way the molecular aspects. There are a wide will reflect the presence of motions faster that the
variety of molecular techniques, such as spectroscopic microsecond, due to temperature, hydration, phase changes,
methods, that probe dynamics at several scales, atomic, formation of complexes between molecules, etc. Measuring
single molecule, and molecular assemblies. Liquid-state the spectral width thus represents a simple and powerful
nuclear magnetic resonance (NMR) is a very well-known tool to sample dynamics. The membrane microfluidity can
noninvasive technique used to determine the atomic three- then be accessed and the concept of order parameter, S,
dimensional structure of molecules in solution. It is much which originates from the field of liquid-crystal physics,
less known that NMR may also be very powerful for plays a central role. It is best used in biomembranes to
quantitatively measuring molecular or ensemble dynamics. define the residual ordering (microfluidity) of molecules.
In particular, solid-state NMR is one of the best tools to As shown in Fig. 2b,c, membranes made of molecules that
report in a noninvasive way on several time scales, as have a very low order parameter (near the minimum value
pictured in Fig. 2a. of 0) have little internal cohesion: they are almost as a
Measurement of NMR spin lattice relaxation time (T1) liquid; the solid-state NMR spectrum of a nonoriented
allows sampling the effect of very fast motions, occurring sample will be very narrow. In contrary, a membranous
from the picoseconds to the nanoseconds time scale. molecule that has an elevated molecular order parameter
Spectral lineshapes and transverse relaxation times (T2) (near the maximum value of 1) is almost perfectly oriented
are sensitive to slower motional processes occurring at the with the main axis of motion (usually the bilayer normal);
the solid-state NMR spectrum of a nonoriented sample will
be very wide. As illustrated in Fig. 2c,b, the ordering may
NMR (T2) be described for aliphatic chains, which undergo many
a internal motions, or for the entire molecule, considered as a
NMR (spectra) NMR (T1)
rigid rod, as are most of the fused ring systems of sterol
molecules. Because the membrane ordering is directly
(s) 10+3 1 10-3 10-6 10-9 10-12 10-15
linked to bond or molecule space averages, it can be
translated, using an appropriate theory, to average length of
a molecule in a membrane and hence to membrane
Membrane Molecular Bond thickness (Fig. 2d) [13, 14]. It is shown that increase/
Fusion deformation diffusion rotation, etc decrease in bilayer order means increase/decrease in
membrane thickness.
βm
Molecular Smzz=0 In this review, the effect of sterols on membrane
ZN Order Parameter
dynamics will be reported using solid-state deuterium
b m 1
Szz = 2 3cos2β m−1 NMR. The dynamic information will originate from lipids
or sterols where some hydrogen atoms have been replaced
Smzz=1
by their isotope, deuterium, allowing making use of the
Sizz= 0 quadrupolar interaction that best reports on internal or
βi Intramolecular
ZN molecular order parameters. Of course other spectroscopic
Order Parameter
c techniques could have been used, but many of them present
Sizz=1 Sizz= 12 3cos2β i−1 severe drawbacks such as bulky reporter groups and lead
only to qualitative information. Because we choose to base
our report on deuterium NMR data (the literature is rich of
db such NMR experiments on sterol/lipid systems), our
d S comparison will not suffer from the inherent time scale
differences when using different techniques to report on
dynamics.
Fig. 2 a Motional time scales in biomembranes and NMR windows.
Drawings and microscopy image depict the spatial scale at which
events may occur. Lower panel—order parameter concept. b Molec- Effect of mammalian sterols on model membranes
ular order; c intramolecular (bonds) order; d correlation between order
and membrane thickness, db—left, ordered membrane with little bond
or molecule fluctuations (large db); right, less ordered membrane with
Cholesterol This sterol has been the most studied both in
bond and molecule fast reorientations within the membrane (small db). model and natural membranes. It has a well-described
Adapted from [12] ordering–disordering action, which is best seen on model
66 J Chem Biol (2008) 1:63–77

membranes that undergo in its absence a phase transition

from a fully rigid state at low temperature, the solid-ordered
(so) phase, to a very fluid state at high temperatures, the
liquid-disordered (ld) phase. In the presence of cholesterol,
the high order at low temperatures is slightly reduced and
the low order at high temperatures is markedly increased.
This effect is progressive with concentration and for
elevated amounts of cholesterol in the membrane, the
membrane microfluidity is about the same in the entire
temperature range (Fig. 3a).
The motional processes responsible for maintaining
such a state (molecular rotation, trans-gauche bond
isomerization) occur in the microsecond–nanosecond time
scale and gently increase in speed with increasing
temperature [15]. The lipid bilayer is thus maintained in
a liquid-ordered state (lo), where molecules still undergo
rotational and lateral diffusion in the membrane but to a
lower extent. This state is reached for cholesterol
contents greater than 20 mol% [16]. This behavior has
been reported so far for saturated or unsaturated lipid
chains with variable length and different head groups. In
model membranes, it is noteworthy that cholesterol also
lowers by approximately 30 °C the temperature of the
lamellar-to-hexagonal phase transition of phosphatidyle-
thanolamines (PE), i.e., it stabilizes hexagonal phases of
PE lipids and promotes dehydration of both the lamellar
and hexagonal phases [17]. A minute look at the increase
of internal ordering of the lipid chains at high temper-
atures shows that all aliphatic carbons, from the glycerol
backbone to the methyl terminal, perceive the cholesterol
effect in a similar way (Fig. 3b). Because the membrane
ordering is directly linked to bond or molecule space
averages, it can be translated, as mentioned above, to
average length of a molecule in a membrane and hence to
membrane thickness. For instance, the presence of 30 mol%
cholesterol led to a 7-Å increase in the dimyristoylphos-
phatidylcholine (DMPC; C14 phosphatidylcholine) bilayer
thickness. The same effect is reported using independently
chain-labeled phospholipids or ring-labeled cholesterol.
Using deuterium-labeled cholesterol offers the advantage
Fig. 3 Temperature variation of the first moment (proportional to
of determining the average orientation of the fused ring chain order parameter), M1, of 2H-labeled DMPC spectra in the
system with respect to the bilayer normal [18, 19]. presence of various amounts of cholesterol: pure lipid (filled circles),
Cholesterol sits perpendicular to the membrane surface 10% (empty squares), 20% (empty circles), 30% (empty triangles),
and keeps this orientation independent of temperature [18]. 50% (filled squares). Inserts show typical 2H-NMR powder spectra:
pure lipid at 0 °C (a) and 60 °C (b), lipid plus 50% CHO at 0 °C (d)
The depth of embedment in the membrane has been also and 60 °C (c). b Quadrupolar splitting (proportional to bond order
determined: cholesterol is buried in the bilayer with the OH parameter) from de-Paked spectra (see insert) of [2H27-DMPC] as a
group facing the carbonyl of the fatty acid lipid chains [20]. function of labeled carbon position, at T=50 °C. Same symbols as for
The rotational diffusion of cholesterol has been obtained a with in addition 40 mol% CHO (crosses). Numbers in the de-Paked
spectrum (insert) represent the assignment of labeled carbon positions.
in membranes using NMR relaxation time measurements. Adapted from [42]
The sterol undergoes a fast axial rotation in the bilayer
with a frequency of approximately 109 Hz (correlation
time of 3 ns at 25 °C) [21]. The activation energy of such a
motion is 32 kJ/mol.
J Chem Biol (2008) 1:63–77 67

Alpha cholesterol This isomer of cholesterol, with the OH

group in axial position, does not naturally occur in
membranes. It is produced in the course of the chemical
synthesis and deuterium labeling of cholesterol. Insertion of
comparable amounts of epicholesterol in synthetic mem-
branes produces the order–disorder action as seen for
cholesterol but to a lesser extent. Most interesting, the
fused ring system is tilted (10–20°) with respect to the
bilayer normal, a tilt that tends to reduce when increasing
the temperature. Indeed, at high temperatures, the amplitude
of allowed motions increases, leading to a decrease of the
lateral bilayer pressure; the hydrophobic and hydrophilic
interactions are therefore less demanding, and the α-isomer
will tend to align its axis of inertia toward the perpendicular
to the bilayer surface. Although purely academic, this
shows that a small modification in the structure of
cholesterol (OH axial or equatorial) leads to important
modifications of molecular organization in bilayers: the Fig. 4 Spatial representation of the fused ring system of cholesterol
tilted position of α-cholesterol within the bilayer membrane and cholesteryl palmitate. The arrow indicates the direction of the
could be the reason that this isomer of cholesterol is not rotation axis, n, collinear with the membrane normal. The symbol
present in natural membranes, it would disturb too greatly (empty circle) is the oxygen attached at C3 and deuterium nuclei of
interest are plotted as small open circles. The palmitic chain in
the parallel packing of the lipid chains. cholesteryl palmitate is attached at the oxygen. Molecules are drawn
in the overall motional averaging axis system. The figure does not
Cholesterol esters The effect of cholesteryl palmitate on the account for the ‘wobbling’ of the steroid skeleton (i.e., the drawing is
dynamics of dipalmitoylphosphatidylcholine (DPPC) has made for Sm =1). Adapted from [43]
been investigated. The C16 fatty acid chain folds back into
the bilayer hydrophobic interior [22] with the ester adopting
a “horseshoe” conformation. The ester linkage is located including NMR. As shown in Fig. 5a,b, the sulphate analog
near the bilayer interface. The orientation of the steroid extends the hydrated lamellar phase domain towards high
skeleton of cholesteryl palmitate has been determined using water contents, and substitution of 30 mol% CHO by CHS
deuterium-labeled molecules (Fig. 4). in DMPC lamellae induces the trapping of 30 wt.%
It is markedly tilted away from the position of that of additional water.
2
cholesterol. This reflects the presence of the palmitoyl H-NMR of heavy water allowed to determine that CHS
chain attached to the oxygen at carbon position 3. The itself binds 12 more water molecules at the interface than
palmitoyl chain therefore ‘pulls’ the rigid cholesteryl CHO, accounting for part of the swelling effect observed in
skeleton away from the average orientation found for the presence of the sulfated moiety. Like CHO, CHS
cholesterol. From the orientation of the steroid and the possesses ordering–disordering properties. The disordering
values calculated for the carbon–deuterium bond order effect is much more pronounced than in the presence of
parameter, the molecular order parameter, Sm, for the cholesterol whereas the ordering at high temperatures is
steroid moiety of cholesteryl palmitate and of cholesterol much less important; the same ordering effect is obtained
were found. At 45 °C, the values Sm =0.47 for cholesteryl on the entire bilayer thickness with 30 mol% CHS or
palmitate (5 mol% in the bilayer) and Sm =0.63 for 15 mol% CHO (Fig. 5c,d). The order–disorder transition is
cholesterol (7 mol%) indicate that fluctuations of the rigid completely cancelled and there is almost no variation of
steroid from the axis of motional averaging are greater for order on large temperature ranges. These affects are related
cholesteryl palmitate than for cholesterol. This indicates to the hydrated and negatively charged sulfate group that is
that the ester of cholesterol is less capable of membrane much bulkier than the hydroxyl group. Because of this
ordering than cholesterol, mainly due to the “horseshoe” steric hindrance and/or the electrostatic repulsions between
conformation that diminishes the favorable van der Waals like charges, the DMPC acyl chains are further away from
interactions leading to dense chain packing with cholesterol. the CHS fused ring system than they are from CHO.
Therefore, the attractive van der Waals forces are likely to
Cholesterol sulphate The comparative effect of cholesterol be less intense and, hence, the chains more disordered.
versus cholesterol sulphate on dimyristoylphosphatidylcho- These observations shine light in the behavior of some
line membranes has been investigated by several techniques membranes in which both CHO and CHS are present, e.g.,
68 J Chem Biol (2008) 1:63–77

Fig. 5 Partial phase diagrams of

DMPC–CHO–D2O (a) and
DMPC–CHS–D2O (b) at 25 °C.
Hatched areas represent the la-
mellar phase domain. Composi-
tions are given in weight
percentages or corresponding
D2O-to-DMPC molar ratio, Ri.
Right panel—comparative evo-
lution of the first spectral mo-
ment, M1 (chain order
parameter), and the bond order
parameter, SCD, for DMPC in
the presence and absence of
30 mol% steroid. c Thermal
evolution of M1, Tc is the DMPC
main phase transition tempera-
ture. d SCD as a function of the
labeled carbon position at 25 °C;
DMPC (crosses), DMPC–CHO
(30 mol%; filled circles),
DMPC–CHS (30 mol%; filled
squares). Adapted from [44]

the spermatozoon plasma membrane or the stratum cor- artenol is therefore a key precursor in the synthesis of
neum, where a good regulation of their relative concen- other phytosterols. It has a global shape similar to
trations is essential. This regulation is ensured by cholesterol, but the absence of a double bond in ring B
hydrolysis of CHS to CHO via a sulfatase activity. A confers to the molecule some internal flexibility [8] that is
dysfunction during this step can lead to dramatic changes in not observed with the mammalian sterol. Nonetheless, at
membrane properties. Indeed, an accumulation of CHS in equivalent concentrations, the presence of cycloartenol in
the spermatozoa heads results in the incapacity of sperma- model membranes generates an ordering–disordering effect
tozoa to penetrate ovum. This contraceptive effect can be that is very similar to that of cholesterol [23] (Fig. 6a). This
accounted for by the CHS-driven lamellar stabilization of indicates that the internal flexibility of the fused ring
the sperm membrane (Fig. 5b), which would thus be less system is of little importance and the global shape appears
fusogenic, i.e., less capable to undergo nonlamellar struc- to be sufficient to make cycloartenol a cholesterol equiv-
tures at the fusion point. At the skin level, an increase in the alent as far as regulation of membrane dynamics is
relative concentration of CHS versus CHO leads to a concerned.
thickening of the stratum corneum. This clinical pattern of
scaling results from disorders in desquamation, and again Stigmasterol and sitosterol These two sterols are among the
the swelling and less ordering effect can account for it: the main phytosterol components of plants and are involved in
skin retaining more of its elasticity. the polarized growth of pollen tube and root hair. The
asymmetric growth of plant cells is in general due to the
asymmetric distribution of these membrane components. In
a way similar to cholesterol, stigmasterol and sitosterol
Effect of plant sterols on model membranes obviously have a strong ordering effect on model mem-
branes above their phase transition temperature. At temper-
Cycloartenol As in mammals, plant sterols are synthesized atures below the phase transition, they have a marked
via the mevalonate pathway of isoprenoid metabolism. disordering effect. Remarkably, the decrease of the total
There are, however, particularities of the plant sterol chain ordering in the stigmasterol/sitosterol containing
biosynthetic pathway, plants cyclise 2,3-oxidosqualene into systems has a more sigmoid character compared with
cycloartenol (and not lanosterol like in mammals), whose cholesterol (Fig. 6). From 25 °C to 45 °C, the chain
9b,19-cyclopropane ring is further metabolized. Cyclo- ordering of DPPC with 30% stigmasterol/sitosterol is in
J Chem Biol (2008) 1:63–77 69

is, however, less important than with cholesterol (for

A similar amounts in the membrane), whereas at high
temperatures, the ordering effect is similar to that of
cholesterol. As an overall behavior, one can state that
ergosterol promotes a quasi-linear decrease of model
membrane ordering (DPPC) at the temperature range of
0–60 °C. The order–disorder steep transition is damped
down in the presence of this fungus sterol. A detailed two-
component phase diagram has been built by Hsueh et al.
[24] and shows a very similar shape as that early published
with cholesterol, except that the so+lo and ld+lo phase
coexistence regions are considerably broader.

Effect of hopanoids on model membranes

140
B C 0,9

120 0,8 Hopanoids (Fig. 1) are made anaerobically from squalene.

Unlike mammalian, plant, and fungi main sterols, they are
M 1 (kHz)

100
0,7 |2*<S CD>chain|
pentacyclic triterpenoids and were discovered in the 1970s,
0,6
80
primarily in prokaryotes and in a few plants. Interestingly,
0,5 hydroxyl and amino groups are located at the end of the
60 0,4
, short aliphatic chain, providing some kind of “inverted”
0,3 polarity on comparing to other sterols. They occur almost
40
exclusively in membranes which contain large proton
0 20 40 60 0 20 40 60 gradients [25]. It has been proposed that they have a role
Temperature (˚C)
in inhibiting proton leaks through membranes and would
Fig. 6 a Temperature variation of the first moment (proportional to reinforce membrane cohesion in bacteria as do eukaryotic
chain order parameter), M1, of 2H-labeled DMPC spectra in the sterols [2]. Their dynamical effect on model membranes has
presence of various amounts of cycloartenol: pure lipid (filled circles),
10% (filled triangles), 20% (crosses), 30% (inverted filled triangles). been reported using deuterium NMR [23]. Addition of 10–
Lower panel—first moments of 2H-NMR DPPC spectra with plant/ 20% hopanoid to DMPC leads also to ordering effects on
fungi sterols versus temperature. First moments of pure DPPC-2H62 fluid phases, as seen on Fig. 7a.
spectra (line without symbols) and of DPPC-2H62/CHO (30 mol%) The disordering action observed at low temperature is,
spectra (dashed line without symbols) are shown in all diagrams for
comparison. b DPPC-2 H 62 plus 30 mol% ERG (squares); c however, very weak, if any, the NMR spectra of pure
DPPC-2H62/STI (30 mol%; filled squares); DPPC-2H/SIT (30 mol%; lipids retaining most of its axial symmetry as usually
empty squares). On double y-axis is plotted twice the chain order seen in lo phases. Of interest, some macroscopic
parameter. Adapted from [23] and [6] orienting properties by magnetic fields are seen as shown
by the unusual line shape obtained at 15 °C. Both BHT
and BHAT induce similar effects, which are, however,
between that of the pure membrane and that of the 30% weaker than those observed with cholesterol at similar
cholesterol-containing membrane, indicating that in binary concentrations, as far as ordering of fluid phase is
lipid systems (phospholipid/sterol), both plant sterols order concerned. Inversion of steroid hydrophobicity, as in the
less the membranes than do cholesterol. hopanoid, still leads to membrane ordering as seen with
cholesterol. This suggests that the hopane fused ring
system retains some of the cholesterol properties.
However, there is, at present, no information on the
Effect of ergosterol on model membranes orientation of the molecule in the bilayer. One may
conjecture from hydrophilic/hydrophobic considerations
Ergosterol is the major sterol in fungi. Compared to that the hydroxyl or amino groups that are attached to
cholesterol, it has an additional double bond in ring B, the short aliphatic chain are located at the interface and
which brings some aromatic character and another in the that the fused ring system is embedded in the bilayer
short aliphatic chain. A methyl group is also branched at interior: an inverted situation compared to mammalian,
C24. Its ordering–disordering properties are analogous to plant, and fungi sterols where the polar groups are
those found for cholesterol (Fig. 6). The disordering effect attached to ring A.
70 J Chem Biol (2008) 1:63–77

a 5˚C 15˚C 50˚C

b c

Fig. 7 Hopanes and DMPC membranes. a 2H27-DMPC spectra in the chain order parameter), M1, of 2H-labeled DMPC spectra in the presence
absence (lower row) and presence (upper row) of BHT (10 mol%). of various amounts of hopanes. Pure lipid (filled circles), b BHAT 10%
Lower panel—temperature variation of the first moment (proportional to (filled triangles), 20% (crosses), c BHT 10% (filled triangles)

Sterols and the “raft” concept question about the possible perturbation of nonnatural
probes, we will concentrate in this review on nonperturbing
A relatively new concept has emerged in biology in relation techniques. A general concept, which subtends all the
with the lateral organization of biomembranes: it is above studies, is the rafts rigidity, which would be due to
proposed that lipid microdomains, also denominated rafts, the presence of increased concentrations of cholesterol and
exist in the plane of the membrane and could explain some sphingolipids. This very rigid character, as opposed to the
complex biological activity [26–28]. These lipid micro- rest of the membrane in which rafts diffuse, would play an
domains are rich in cholesterol and saturated chains lipids, important role in the functional properties of proteins that
such as sphingolipids, and could be stabilized by weak are embedded in these regions. As already demonstrated,
forces such as hydrogen bonding and van der Waals NMR is a very potent noninvasive technique to probe this
interactions. The rafts also possess distinct protein compo- aspect.
sition, favoring development of signal transduction or
membrane trafficking because of the formation of confined Cholesterol in rafts Lipid membrane systems that have
regions. Several techniques have been used to evidence been reported to be composed of sphingomyelin (SM)/
theses membrane domains, the most popular being fluores- cholesterol microdomains or “rafts” by Dietrich et al.
cence microscopy where fluorescent molecules, thought to (palmitoyloleoylphosphatidylcholine (POPC)/(SM)/CHO,
be specific of rigid of fluid lipid phases, were added to 1/1/1) [29] and by Schroeder et al. (SCRL: Liver-PC/
model or natural membranes. Because one may always Liverphosphatidylethanolamine/SM/Cerebrosides/CHO, 1/
J Chem Biol (2008) 1:63–77 71

1/1/1/2) [30] were investigated under the form of fully other “rigid” molecules exchange between regions of
hydrated liposomes by the 2H NMR method. Liposomes of different ordering (microfluidity).
binary lipid composition POPC/CHO and SM/CHO were
also studied as boundary/control systems. All systems at Plant sterols in rafts In plants, specialized lipid domains
physiological temperatures were found to be in the liquid- are involved in the polarized growth of pollen tube and root
ordered phase (lo). Use of deuterium-labeled cholesterol hair and the asymmetric growth of plant cells is in general
enabled finding both the position of the sterol axis of due to the asymmetric distribution of membrane compo-
motion and its molecular order parameter. The axis of nents. The effect of sitosterol and stigmasterol, two major
anisotropic rotation of cholesterol is such that the molecule plant sterols, on the structure and dynamics of membranes
is, on average, quasi-perpendicular to the membrane plane, whose composition is representative of domains (rafts) in
in all of the four systems investigated, its molecular area plants was recently documented [6]. Liposomes of phytos-
being minimal with 33 Å2 (Fig. 8a). terols associated with glucosylcerebroside (GC) and with
Cholesterol order parameters greater than 0.8 are deuterium-labeled dipalmitoylphosphatidylcholine (2H-
observed, indicating that the sterol is in a restricted DPPC) were analyzed with deuterium solid-state nuclear
environment in the temperature range 0–60 °C (Fig. 8b). magnetic resonance (2H-NMR). For comparison, membrane
The binary mixtures present “boundary” situations with the systems representative of raft composition in fungi and
lowest values for POPC/CHO and the highest for SM/CHO. mammals were also investigated. Spectra such as that
The SCRL raft mixture has the same ordering as the SM/ shown in Fig. 9, insert, allow detection of the lo phase,
CHO, i.e., the highest order parameter values over the characteristic of a membrane state half-way between solid-
temperature range. It demonstrates that in the SCRL ordered (so) and liquid-disordered (ld) states. The so state,
mixture, cholesterol dynamics is as in the binary system also called “gel”, is found at low temperatures (below 35 °C),
SM/CHO, therefore suggesting that it might be depleted when membranes are essentially composed of SMs or GC
from the rest of the membrane to form complexes as if it (Fig. 9).
were alone with SM. On the other hand, the mixture POPC/ This membrane state allows little biological function
SM/CHO exhibits intermediate ordering situation between because it prevents membrane trafficking due to its very
SM/CHO and POPC/CHO. This strongly suggests that rigid state (order parameter close to 1). In turn, the ld or
cholesterol could be in fast exchange, at the NMR time “fluid” state is found at high temperatures, in the absence of
scale (milli- to microseconds), between two or more SM, GC, and sterols (low order parameter). On the
membrane regions of different dynamics and questions the contrary, such high membrane dynamics may lead to
statement of “rigid domains” made of SM and cholesterol excessive membrane passages. By using 2H-NMR, the
in the model “raft” system POPC/SM/CHO. To summarize, temperature behavior of membrane systems containing GC
rafts systems must be considered in a more cautious way and plant sterols, it was found that the so–ld, order–
and no longer seen as rigid “rocks” floating in fluid lipids. disorder, transition was totally abolished: sitosterol and
A more dynamic view could be adopted where sterols and stigmasterol fluidized the so state and ordered the ld state to

Fig. 8 a Left—representation (stick mode) of cholesterol average area when viewed from above the membrane surface. b Thermal
orientation with respect to its principal axis of motional averaging, n variation of the cholesterol molecular order parameter, Smol, in the four
(membrane normal), when n is parallel to the page plane. Right—view lipid–cholesterol compositions: POPC/CHO-2H5, SM/CHO-2H5,
with n, perpendicular to the page plane, CPK representation is used POPC/SM/CHO-2H5, SCRL/CHO-2H5. Adapted from [45]
here. The dashed line tentatively represents the cholesterol molecular
72 J Chem Biol (2008) 1:63–77

Fig. 9 Regulation of temperature-driven membrane dynamics by liquid-ordered, lo, state. Left panel—schematics of solid-ordered, so
plant sterols. Central panel—first spectral moment (left y-axis) or (gel), and liquid-disordered, ld (fluid), membrane states. Right panel—
order parameter (right y-axis) as a function of temperature; solid line schematics of the lo (raft) membrane state together with the structures
2
H-DPPC with glucosylcerebroside; open circles plus stigmasterol; of cholesterol and sitosterol. Adapted from [6, 7]
filled circles plus sitosterol. Insert 2H-NMR spectrum typical of a

produce the lo state where membrane fluctuations vary tion of smaller membrane domains, may be the evolution
smoothly with temperature (Fig. 9, central panel). This response for plant adaptation to large temperature variations
effect was already documented with CHO in mammals but [7].
on a much narrower temperature range (vide supra). The
case or the fungus system was found in between that of
plants and mammals. In summary, it appears that plant
membranes of “raft” composition are less sensitive to Sterols and toxins in membranes: the protecting effect
temperature variations than those of animals.
This suggests that cell membrane components like Cationic amphipathic α-helical peptides, such as the bee-
sitosterol, stigmasterol, and glucosylcerebrosides, which venom toxin melittin, preferentially disrupt mixed model
are typical of plants, are produced in order to extend the membranes or natural membranes such as those of red
temperature range in which membrane-associated biologi- blood cells [31] potentially causing a release of cell
cal processes can take place. This observation is well in contents. The molecular processes by which this happens
accordance with the fact that plants have to endure higher are multiple and have been summarized in a review by Shai
temperature variations than animals, which usually can [32]. One of them involves the formation of bilayer discs of
either regulate their body temperature or change their 20–40 nm chopped off from the membrane by lateral
location in order to avoid extreme heat or coldness. segregation of α-helical amphipathic peptides [33], see
Compared to cholesterol, the two phytosterols posses inserts in Fig. 10.
additional ethyl groups branched on C-24 (Fig. 9, right). This is translated by the appearance of very sharp and
It has been proposed that the presence of an additional ethyl isotropic lines in solid-state 31P or 2H NMR. This
group may reinforce the attractive van der Waals inter- phenomenon occurs with saturated chain lipids preferen-
actions leading to more membrane cohesion and therefore tially in their gel phase (so), i.e., at temperatures below the
less temperature sensitivity. These results also suggest that order disorder transition [34, 35]. Temperatures above the
domains of smaller size would be promoted in the presence transition lead to the fusion of discs resulting in large
of phytosterols and especially with sitosterol. Such domains vesicles of 200–300 nm (Fig. 10a). In the presence of
may be viewed as dynamic, with sterols laterally exchang- 30 mol% cholesterol, the lo phase is present and leads to a
ing at the microsecond time scale. In plant cells, enzymes total inhibition of the toxin-triggered formation of large
transfer alkyl groups to the C-24 of sterols. If we suppose unilamellar vesicles as seen by the detection or wide NMR
that the relative activities of the different branches of the powder patterns; the appearance of small discs is also
plant sterol biosynthesis are regulated, the concentrations of strongly restricted (decrease of amount of isotropic NMR
major sterols in plants, like sitosterol, stigmasterol, and lines) [36]. Use of deuterium-labeled cholesterol shows that
cholesterol, could be controlled. This shows the importance remaining discs contain small amounts of sterol, phospho-
of equilibrated sterol concentrations for plant growth and lipids being the main component (Fig. 10b,c). Sterols can
development. It thus appears that a fine tuning of the sterol also protect negatively charged membranes from the
structure, i.e., the presence of branched ethyl groups in disruptive effects of other antimicrobial peptides [37]. This
plant sterols increasing membrane cohesion through forma- suggests that bacteria (without sterols) are most susceptible
J Chem Biol (2008) 1:63–77 73

permeability of a number of organisms, thus inducing a

leakage of important cellular constituents and ultimately
lysis and death of the cell. The principal characteristic of
these antibiotics is that they apparently require the presence
of sterols in the cell membrane to promote such an effect.
De Kruijff and coworkers proposed decades ago that
polyene antibiotics and sterols formed molecular complexes
to create channels or solid patches that disrupt the
membrane [9–11]. Amphotericin B has received particular
attention. De Kruijff has proposed that amphotericin B and
cholesterol form an eight–eight molecular complex, span-
ning the membrane vertically and creating a pore. The
dynamic and structural parameters of the interactions
between amphotericin B and sterol-containing model
membranes have been monitored by 2H-NMR of deuteri-
um-labeled lipids and cholesterol. The structural parameters
of deuterium-labeled cholesterol in CHO/DMPC mixtures
did not change upon addition of amphotericin B, i.e., there
is no change in microfluidity and rigid domains are not
detected, at the microsecond time scale, as could be
expected by the presence of a rigid pore where the sterol
molecules are segregated away from the lipids. However,
measurement of NMR relaxation times for labeled choles-
terol showed that the minimum in T1, observed for
cholesterol in DMPC at 32–35 °C, was shifted towards
38–40 °C in the presence of amphotericin B (Fig. 11a).
This minimum allows a direct calculation of the speed of
axial rotation of cholesterol in membranes (50×109 Hz).
The data indicates that the system containing the antibiotic
has to be warmed by approximately 5 °C in order to relax
with the same efficiency as the system without the drug. In
other words, the motions of cholesterol in DMPC are
slowed down by the presence of amphotericin B.
Fig. 10 31P- and 2H-NMR spectra of the melittin/DPPC system in the
presence and absence of cholesterol. a 31P-NMR spectra for selected The interaction of the polyene antibiotic filipin with
temperatures of the cholesterol-free system at lipid to melittin molar membrane sterols is well known and this antibiotic is
ratio of 20. b The corresponding 31P-NMR and c 2H-NMR spectra of nowadays well used as a test to detect the presence of
the system containing 30 mol% of 2H-labeled cholesterol. Inserts sterols in cellular membranes. Although the molecular
show the DPPC/melittin discs at low temperatures and the large
vesicles at high temperatures. In the presence of CHO, remaining discs processes by which this happens are still unclear, filipin
contain small amounts of sterol (insert on right hand side). Adapted has been shown to be particularly efficient in inhibiting
from [36] fungi growth, through interaction with ergosterol. The
effect of filipin on cholesterol containing model membranes
has been followed by 2H-NMR of labeled cholesterol [38].
to the action of such peptides whereas lower eukaryotes At physiological temperatures, there is evidence of filipin-
including fungi (containing ergosterol) exhibit an interme- induced cholesterol immobilization at the membrane. The
2
diate degree of sensitivity, and higher organisms (contain- H NMR spectra of cholesterol show two domains in which
ing cholesterol) are largely resistant to antimicrobial ordering and dynamics are very different. In one of these,
peptides. cholesterol is static on the 2H NMR time scale (Fig. 11b),
whereas in the other, it undergoes rapid axially symmetric
motions similar to those it exhibits in the drug-free
Sterols and polyene antibiotics in membranes membrane. This indicates that the jumping frequency of
cholesterol between the labile and immobilized domains is
Polyene antibiotics such as amphotericin B, filipin, and less than 105 Hz. The distribution of cholesterol between
nystatin are known to mediate changes in the membrane these two sites is temperature dependent: at 0 °C, all sterol
74 J Chem Biol (2008) 1:63–77

for instance, fluorescence and electron spin resonance.

However, most of these techniques, as well as being
invasive, do not directly measure the fluidity of the
membrane. Although NMR is not very sensitive, a limited
number of experiments have nonetheless been reported in
membranes containing all components, i.e., all lipids and
proteins. Human red blood cell membranes have been
investigated some decades ago. The erythrocyte membrane
is known to have approximately 27% of its total lipid
weight as cholesterol, which can be exchanged with that
from sonicated cholesterol/PC vesicles [39]. Figure 12
shows the spectrum of deuterated cholesterol incorporated
in human erythrocytes.
Only one average spectrum is detected, very similar to
that obtained in model membranes at a concentration of
30 mol%. Analysis in terms of orientational order param-
eter has shown that the orientation and the anisotropic
motion of cholesterol are very similar in natural and model
membranes. The other membrane components appear thus
not to affect much the regulating role of cholesterol.
The dynamic properties of whole sea urchin sperm and
purified membranes of subcellular compartments have been
detected by solid-state 2H-NMR spectroscopy [40, 41]. A
deuterium-labeled lipid (POPC) was incorporated into
whole cell membranes using an approach similar to that
developed for labeled cholesterol (vide supra). Mass
spectrometry was used to measure the amount of incorpo-
rated lipid and the time it took for the lipid to statistically
distribute in all the membranes was determined by
recording spectra as a function of time and temperature.
Figure 13 shows the resulting data for whole sea urchin
Fig. 11 a Temperature dependence of the relaxation time, T1z, of sperm cells and nuclear envelope precursor membrane
[6-2H]-CHO in DMPC, in the presence or absence of amphotericin B
(Ampho B). Lower panels—2H-NMR spectra of [2,2,3,4,4,6-2H6] vesicles.
cholesterol in DMPC (30 mol%) in the presence (b) and absence (c) of
filipin, at 25 °C. The antibiotic, when present, is equimolar to
cholesterol. Adapted from [38]

molecules are immobilized, whereas at 60 °C, they are

almost totally in the labile state. To summarize, the effect of
polyene antibiotics may be very diverse on membrane
sterols: from gentle slowing down of axial rotation to
complete immobilization in the membrane, forming “solid”
complexes.

Sterols in complex “live” membranes

Natural membranes contain a number of other molecules

and in particular membrane proteins that may modulate the
sterol behavior as reported above for lipid model mem-
branes. Reporting on the dynamics of entire membranes is a Fig. 12 2H-NMR spectrum of [2,2,3,4,4,6-2H]cholesterol in mem-
difficult task. Several techniques have been used such as, branes of human erythrocytes. Adapted from [39]
J Chem Biol (2008) 1:63–77 75

Membrane evolution, dynamics, and the role of sterols

Sterols have been historically considered as membrane

reinforcers because they induce a molecular order to
membranes. The main motto of this review suggests that
they could better be named as “membrane dynamic
regulators”, by maintaining the membrane in a state of
microfluidity suitable for cell function on large temper-
ature scales (Fig. 14). The so-called “liquid-ordered state”,
which was initially considered as a physicist peculiarity,
seems to be a state present in “rafts” systems that provides
enough “dynamic rigidity” to convey important biological
processes.
It is interesting to remark that each type of cellular
membrane contains a specific sterol as a major compo-
nent: cholesterol in mammals, ergosterol for fungi, sito-/
stigmasterol in plants, and hopanoids in primitive
bacteria. The fused ring system (four or five cycles)
appears to bear the property of increasing the order of
fluid phases. Presence of unsaturations and flexibility of
this fused system does not seem to alter the ordering
effect. On the contrary, the branched chain seems to have
a quite important modulation role. Ethyl groups branched
at C24 bring better ordering over large temperature
scales, a property used by plant sterols to accommodate
thermal shocks. Hydroxyl or amine groups in hopanoids
appear to decrease the dynamic regulating capability: the
so phase is still observed at low temperatures. However,
model membranes in which such sterols are associated with
branched chain lipids (phytanoyl chains) have not been
investigated. Sterols are undoubtedly key molecules in
regulating membrane dynamics but evolution in nature has
Fig. 13 Deuterium solid-state NMR spectra of POPC-2H31 labeled not only led to sterol modification for adaptation but also
sea urchin sperm membranes (a) and precursor egg membrane vesicles
(MV0; b). Sperm and MV0 were labeled with MLVs and SUVs of synthesis of sterols in association with other lipids
POPC respectively for 30 min at 40 °C and the corresponding
deuterium NMR spectra acquired at 10 °C (middle spectra «sperm
Tini», «MV0 Tini»). The systems were equilibrated at 40 °C and
reacquired at 10 °C (bottom spectra «sperm Tfin», «MV0 Tfin»). The
top spectra corresponds to MLVs of POPC and the dashed lines show
the plateau quadrupolar splitting enlargement on sperm and MV0 Solidordered
spectra postequilibrium at 40 °C. Upper images are electron HO
microscopy pictures of sperm and egg membrane vesicles. Adapted
from [40]
HO

HO HO HO
Liquidordered
OH NH2
Spectra postequilibrium are typical of the liquid-ordered
phase as reported in models. It was demonstrated that whole
sperm membranes are more dynamic than nuclear envelope
precursor membranes due to the higher cholesterol levels of Liquiddisordered
the latter. This application is rather new and can be exploited
Fig. 14 Sterols (mammals, fungi, plants, bacteria) as regulators of
as a generic method for monitoring membrane dynamics in
membrane dynamics. Left—the solid-ordered and liquid-disordered
whole cells, various subcellular membrane compartments, states in the absence of sterols. Right—the liquid ordered state with
and membrane domains in subcellular compartments. sterols
76 J Chem Biol (2008) 1:63–77

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