Phase-Separation and Domain-Formation in Cholesterol-Sphingomyelin
Mixture: Pulse-EPR Oxygen Probing
Laxman Mainali,†Marija Raguz,†‡and Witold K. Subczynski†*
†Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin; and‡Department of Medical Physics and Biophysics,
School of Medicine, University of Split, Split, Croatia
recovery electron paramagnetic resonance spin-labeling methods, in which bimolecular collisions of relaxation agents (oxygen
or nickel ethylenediamine diacetic acid) with spin labels are measured. Liquid-disordered (ld) and liquid-ordered (lo) phases, and
cholesterol bilayer domains (CBDs) were discriminated and characterized by profiles of the oxygen transport parameter (OTP).
In the ldphase, coexisting with the lophase, the OTP profile is bell-shaped and lies above that in the pure ESM membrane.
Changes in the OTP profile across the lophase are complex. When the lophase coexists with the ldphase, the OTP profile
is similar to that across the pure ESM membrane but with a steeper bell shape. With an increase in cholesterol concentration
(up to the cholesterol-solubility threshold), the profile becomes rectangular, with low OTP values from the membrane surface
to the depth of C9, and high values in the membrane center. This approximately threefold increase in the OTP occurs at the
depth at which the rigid ring structure of cholesterol is immersed. Further addition of cholesterol and the formation of the
CBD does not affect the OTP profile across the lophase. OTP values in the CBD are significantly lower than in the lophase.
Membranes made of Chol/ESM (cholesterol/egg sphingomyelin) mixtures were investigated using saturation-
Our motivation for undertaking studies on membranes made
from cholesterol/egg sphingomyelin (Chol/ESM) mixtures
is twofold. First, we are interested in the role of cholesterol
in forming membrane domains, especially raft domains
(1–5). Second, we are interested in the properties and orga-
nization of lipids in fiber cell plasma membranes of the eye
lens (6–10). In our investigations, we extensively used elec-
tron paramagnetic resonance (EPR) spin-labeling methods
to study the organization and dynamics of phospholipid
membranes with different cholesterol contents. Here,
cholesterol not only saturates the phospholipid bilayer but
also leads to the formation of immiscible cholesterol bilayer
domains (CBDs) (3,5,7–9,11,12). These methods provide
a unique opportunity to determine the lateral organization
of membranes, including the investigation of coexisting
membrane phases and domains. Furthermore, these methods
present additional opportunities to determine several impor-
tant membrane properties as a function of bilayer depth,
including alkyl chain order (13), membrane fluidity (12),
hydrophobicity (14), and the oxygen diffusion-concentra-
tion product (called the oxygen transport parameter
(OTP)) (15,16). In some cases, these properties can be
obtained in coexisting membrane phases and domains
Rafts are considered to be representative of dynamic
domains in the cell membrane requiring both cholesterol
and sphingolipids. The operational definition of lipid rafts
(which is closely related to our research) states that ‘‘deple-
tion of either cholesterol or sphingolipids from cell
membranes leads to the disappearance of the detergent-
resistant fraction and the loss (or modulation) of specific
membrane functions (signaling events) connected with
rafts’’ (18). EPR spin-labeling provides information on the
organization and dynamics of raft molecules, as well as
the organization and dynamics of the raft itself in the
membrane (1–3). Our results support the definition of rafts
formulated by Pike (19), which states that: ‘‘Membrane rafts
are small (10–200 nm), heterogeneous, highly dynamic,
sterol- and sphingolipid-enriched domains that compart-
mentalize cellular processes. Small rafts can sometimes be
stabilized to form larger platforms through protein-protein
and protein-lipid interactions.’’ It is believed that raft
domains are in the liquid-ordered (lo) phase (20–22).
Thus, raft researchers can gain some insight into the struc-
ture of and molecular interactions in raft domains by under-
standing lophases and liquid-liquid phase separations in
binary or ternary mixtures of lipids, including cholesterol
and saturated-chain lipids. Ternary lipid mixtures contain-
ing cholesterol, saturated, and unsaturated phospholipids
(PLs) were investigated to confirm the existence of the lo
phase and to describe regions of phase diagrams where
this phase coexists with the liquid-disordered (ld) phase
(23–26). These results clearly show that in the investigated
systems the lophase domain is formed and coexists with the
ldphase domain when the saturated PL is sphingomyelin
(23,24) or distearoylphosphatidylcholine (25). These do-
mains are much smaller than the optical resolution limit
and cannot be discriminated by fluorescence microscopy
(27). Estimated sizes vary from ~2–8 nm (25) to
45–70 nm (24). These works also discuss whether choles-
terol/PL (Chol/PL) interactions are better described as loand
Submitted March 3, 2011, and accepted for publication July 13, 2011.
Editor: David D. Thomas.
? 2011 by the Biophysical Society
Biophysical Journal Volume 101 August 2011 837–846837
ldcoexisting phases or as condensed complexes of PL and
Using the unique abilities of EPR spin-labeling methods,
we found (3) that the commonly accepted statement that
properties of the lophase lie between those for the ldand
solid-ordered (so) phases (29) is true for membranes formed
at a low Chol/PL mixing ratio where the lophase coexists
with the ld or so phase. However, at higher cholesterol
contents, the OTP in the lophase is similar to that in the
sophase from the membrane surface to the depth of C9
and to that in the ldphase at depths deeper than C9, showing
that the cholesterol-based lophase is ordered only near the
membrane surface and still retains a high level of disorder
in the bilayer center. This property may facilitate lateral
mobility in lophases. Thus, investigation of the molecular
dynamics and structures in the direction of the depth in
the coexisting or separated phases and domains is especially
Our earlier research was carried out to characterize
the physical properties of the lophase when it coexists
with either the ldor sophase. We employed one of the
most straightforward model membranes containing the lo
phase: a binary mixture of dimyristoylphosphatidylcholine
(DMPC) and cholesterol (3). We also made preliminary
measurements on Chol/ESM binary mixtures. The effects
of cholesterol on membrane order and the OTP were moni-
tored at the depth of C5 in fluid- and gel-phase ESM
Recently, our attention became focused on the unique
composition of the fiber cell plasma membranes of the eye
lens, which have the highest cholesterol content of any
known biological membrane. In human lenses, the Chol/PL
molar ratio varies from 1:1 to 2:1 in the cortex to as high as
3:1–4:1 in the nucleus (30). The PL composition of the fiber
cell plasma membrane changes drastically with age, with
the preferential depletion of glycerol-PLs and the conse-
quent enrichment of sphingolipids (31). Sphingolipids
(mainly, sphingomyelin (SM) and dihydro-SM) account
for 66% or more of the total PLs in the eye lens of the adult
human (32). The high, saturating level of cholesterol, affects
the organization of lipids in fiber cell plasma membranes,
including the formation of the lophase and immiscible,
pure CBDs. The effects of cholesterol should be strongly
modulated by the presence of sphingolipids. The alkyl
chains of these PLs are highly saturated. Additionally, cho-
lesterol solubility in sphingolipid bilayers is extremely high.
The cholesterol solubility threshold (CST) reported for
these membranes is at a Chol/SM molar ratio of 2:1 (33),
compared with CSTs in phosphatidylcholine (PC), phospha-
tidylethanolamine, and phosphatidylserine membranes at
Chol/PL molar ratios of 1:1 (34), 1:1 (35), and 1:2 (36),
In our present research, we discriminated phases and
domains in membranes made of Chol/ESM mixing ratios
from 0 to 3. Both cholesterol (cholestane spin label (CSL)
and androstane spin label (ASL)) and PL analog spin labels
(n-PC and n-SASL) were used (see Fig. S1 in the Supporting
Material). These spin labels have molecular structures
similar to parent cholesterol or PLs and, therefore, are ex-
pected to behave and similarly distribute across different
membrane domains. Experiments carried out with probe
molecules necessitate due caution in interpreting results;
the labeled molecules cannot be expected to mimic all of
the properties of cholesterol or PLs. Nevertheless, the inter-
action of CSL (or ASL) with PL and/or cholesterol should,
to a certain degree, approximate cholesterol-PL and choles-
terol-cholesterol interactions in the membrane domain
because of the overall similarity in their molecular struc-
tures and phase behaviors in PC-cholesterol membranes
(37). Similarly, phase boundaries drawn for Chol/DMPC
membranes contain points obtained with n-PCs (38).
Furthermore, points obtained with n-SASLs (5) fit perfectly
to these boundaries. Phase boundaries obtained with
5-SASL for Chol/ESM (4) overlap appropriate boundaries
in the phase diagram for Chol/palmitoyl-SM (PSM) pre-
sented by Almeida et al. (39).
We also characterized three-dimensional dynamic struc-
tures of these phases and domains at cholesterol contents
where they coexisted or occupied the entire membrane.
This was possible through the use of a very small probe
(i.e., molecular oxygen). With this approach, a variety of
phospholipid spin labels were incorporated into the
membrane to probe specific depths and domains (see
Fig. S1). The rate of collision between molecular oxygen
and the nitroxide moiety attached to a specific location in
the lipid was measured using the saturation-recovery (SR)
EPR method. The oxygen collision rate (a product of the
local concentration and the local diffusion coefficients of
molecular oxygen within the membrane) is a very sensitive
monitor of membrane fluidity that reports on translational
diffusion of molecular oxygen (15). Detailed profiles of
oxygen collision rates, or the OTP, were obtained for
Chol/ESM mixing ratios, shown as solid dots in the phase
diagram of Fig. 1. The temperature at which experiments
were performed (40?C) is indicated by a broken line.
Fig. 1 also contains a schematic drawing of the organization
of lipid molecules in phases and/or domains that should
exist at certain cholesterol contents. Fig. 1 should be used
as a guideline for the presentation and interpretation of
MATERIALS AND METHODS
ESM, cholesterol, and phospholipid spin labels (1-palmitoyl-2-(n-doxyl-
stearoyl)phosphatidylcholine (n-PC, where n ¼ 5, 7, 10, 12, 14, or 16) or
tempocholine-1-palmitoyl-2-oleoylphosphatidic acid ester (T-PC) were ob-
tained from Avanti Polar Lipids (Alabaster, AL). Nine-doxylstearic acid
spin labels (9-SASL), CSL, and ASL were purchased from Molecular
Biophysical Journal 101(4) 837–846
838Mainali et al.
Probes (Eugene, OR). Other chemicals (of at least reagent grade) were
purchased from Sigma-Aldrich (St. Louis, MO).
Preparation of Chol/ESM membranes
The membranes used in this study were multilamellar dispersions of ESM
and cholesterol containing 1 mol % spin label. The membranes were
prepared using the film deposition method as described in (5). Chloroform
solutions of ESM, cholesterol, and spin label were mixed to attain a desired
mixing ratio. Chloroform was evaporated with a stream of nitrogen and
with the test tube in constant rotation so as to deposit a uniform film of lipid
over the bottom of the tube. The lipid film was thoroughly dried under
reduced pressure (0.1 mm Hg) for 12 h. A buffer solution (0.2 ml of
10 mM PIPES and 150 mM NaCl, pH 7.0) was added to the dried lipids
at 50?C and vigorously mixed. The buffer used for samples with 9-SASL
was 0.1 M borate at pH 9.5. A rather high pH was chosen in this case to
ensure that all 9-SASL carboxyl groups were ionized in the ESM
membranes (40). The properties of ESM membranes should not be sensitive
to a pH range of ~5–~11 because ionization of ESM polar PC headgroups
does not change in this pH range (41).
To avoid traces of solvent (chloroform) in our samples (and due to the
uncertainty of chloroform removal efficiency (42)), we did not use the rapid
solvent exchange method (35) in our preparations. Instead, we used the film
deposition method, which is widely used (39,43,44). A significant advan-
tage of the film deposition method in the application of EPR spin labeling
to discriminate and characterize the CBD (which was demonstrated and
described in detail in our previous work (9)) is that the EPR signal of
ASL and CSL from cholesterol crystals is so broad that it cannot be seen
in conditions where the signal from CBD is recorded.
The membranes were centrifuged briefly, and the loose pellet was used for
EPR measurements. The samplewas placed in a 0.6 mm i.d. capillary made
of a gas-permeable methylpentene polymer called TPX (45). Conventional
EPR spectra were routinely recorded for all samples with a Bruker EMX
spectrometer equipped with temperature control accessories. Samples
were thoroughly deoxygenated, yielding correct EPR line-shapes. The
z-component of the hyperfine interaction tensor, AZ, for ASL and CSL in
the membrane was determined directly from EPR spectra for samples
frozen at about –165?C and recorded with a modulation amplitude of
2.0 G and an incident microwave power of 2.0 mW (14).
equipped with a loop-gap resonator (46). Spin-lattice relaxation times (T1s)
were determined by analyzing the SR signal of the central line obtained in
short pulse experiments (16,46). For measurements of the OTP, the sample
was equilibrated with the same gas that was used for temperature control
(i.e., a controlled mixture of nitrogen and dry air adjusted with flowmeters
(Matheson Gas Products, Montgomery, PA, model 7631H-604)) (15,47). A
relatively low level of observing power (8 mW, with the loop-gap resonator
deliveringanH1fieldof3:6 ? 10?5G)wasusedforallexperimentstoavoid
microwave power saturation (which induces artificial shortening of the
apparent T1). Typically, 105? 106decays were acquired with 2048 data
points on each decay. Sampling intervals 2, 2.5, 4, 5, 10, or 20 ns were
used for measurements. The total accumulation timewas typically 2–5 min.
SR signals were fitted by single- or double-exponential functions. When
a single exponential fit was satisfactory, the decay time constant was evalu-
ated with a standard deviation smaller than 53% from the mean value for
independent experiments (for samples prepared totally independently).
When a double-exponential fit was necessary, and satisfactory, the decay
times were usually evaluated with standard deviations < 55% and 510%
short T1s (due to the presence of molecular oxygen) in the current setting of
the instrument. Itisalso possiblethattheavailablepumppowercannotsatu-
rate the signal when the T1is very short.
The outline of theory for evaluating the OTP and nickel ethylenediamine
diacetic acid (NiEDDA) accessibility parameter is given in the Supporting
RESULTS AND DISCUSSION
Discrimination of membrane phases and domains
using PL-analog spin labels
Fig. 1 is the guideline for our experiments. All measure-
ments were performed in fluid-phase membranes. We chose
40?C to ensure that measurements were performed well
above the phase transition temperature of ESM membranes
(~35?C), which is somewhat broad because of the natural
source of this PL (4,39,43,48). Six Chol/ESM mixing ratios
(indicated in Fig. 1 as black dots) represent cholesterol
contents at which phases and/or domains either exist as
single structures or coexist.
Fig. 2 contains representative SR signals for 7-PC in ESM
membranes with different cholesterol contents. We chose
7-PC because we expected it to present pronounced changes
in T1in the presence of oxygen. In deoxygenated samples,
and solid circles indicate the temperature and Chol/ESM mixing ratios at
which measurements were performed. Schematic drawings of membrane
structures (including phases and domains) at different Chol/ESM mixing
ratios ((a) 0; (b) 1:4; (c) 1:2; (d) 1:1; (e) 2:1; (f) 3:1) are presented.
Phase diagram of the Chol/ESM membrane. The broken line
Biophysical Journal 101(4) 837–846
SR signals for 7-PC were fitted successfully to a single-
exponential function, indicating that the spin label alone
cannot discriminate purported domains. Similarly, single-
exponential curves were good fits for other PL spin labels
in deoxygenated samples. SR signals from samples equili-
brated with 50% air were also single exponential. Only for
samples at a Chol/ESM mixing ratio of 1:4 (Fig. 2 b) could
the SR signal for 7-PC be fitted successfully to the double-
exponential curve, indicating the presence of two phases
(compare the residual for single- and double-exponential
fits). Other PL spin labels also showed the presence of
two phases at that cholesterol mixing ratio. Results are
consistent with the phase diagram shown in Fig. 1. Coexist-
ing ldand lophases are expected at the Chol/ESM mixing
ratio of 1:4. Furthermore, the lo-phase domain and CBD
are expected to coexist at a mixing ratio of 3:1. However,
the CBD cannot be discriminated using PL-analog spin
labels, which do not partition into the pure cholesterol
domain. In double-exponential fits in the presence of
oxygen, shorter T1 values for all PL-analog spin labels
were assigned to the ldphase, and longer values to the lo
phase. These assignments were confirmed by T1measure-
ments. T1values measured at a Chol/ESM mixing ratio of
1:2 were nearly the same as longer T1values measured at
a Chol/ESM mixing ratio of 1:4. T1values in the presence
and absence of molecular oxygen were used to calculate
values of the OTP (see Eq. S1) and to obtain profiles of
the OTP across single or coexisting membrane phases
Discrimination of membrane phases and domains
using cholesterol-analog spin labels
Because the CBD is a pure cholesterol domain (Fig. 1 f), it
can only be discriminated with cholesterol-analog spin
labels. For this purpose, we used CSL (in which the nitro-
xide moiety replaces the –OH group of cholesterol) and
ASL (which is similar to CSL with the isooctyl chain re-
placed by the –OH group (see Fig. S1)). Both CSL and
ASL should be anchored at the membrane surface by their
–OH groups. However, ASL possesses two polar ends,
and, in principle, both can be located in a polar headgroup
region. The –OH group is more polar than the nitroxide
moiety and is expected to be located in the headgroup
region. However, the opposite orientation is possible, espe-
cially when the lipid composition of the membrane changes.
This presents the question of whether the expected double-
exponential SR signals of ASL in the presence of oxygen
indicate 1), formation of coexisting domains (as shown in
Fig. 1, b and f) or 2), the ‘‘upside-down’’ orientation of
ASL. To clarify this problem, we measured local hydropho-
bicity around the nitroxide moiety of ASL in Chol/ESM
membranes (with a mixing ratio between 0 and 3) and
compared it to local hydrophobicity around the nitroxide
moiety of CSL (Fig. 3). Hydrophobicity measured with
curves andresiduals (theexperimental signalminus
the fitted curve) of 7-PC obtained at different
Chol/ESM mixing ratios (indicated in Fig. 1).
Membrane specimens were equilibrated with
nitrogen or a mixture of 50% air and 50% nitrogen.
nentialfunction in the absence ofmolecular oxygen
with time constants of (a) 3.56 5 0.01 ms, (b)
3.78 5 0.01 ms, (c) 4.12 5 0.01 ms, (d) 4.59 5
0.01 ms, (e) 4.62 5 0.01 ms, and (f) 4.66 5
0.01 ms. SR signals in the presence of molecular
oxygen were fitted either to single exponentials
with time constants of (a) 0.73 5 0.01 ms, (c)
1.25 5 0.01 ms, (d) 1.94 5 0.01 ms, (e) 2.00 5
0.01 ms, (f) 1.86 5 0.01 ms, or double exponentials
with time constants of (b) 1.19 5 0.05 ms and
and the lower residuals for double-exponential fits).
Representative SR signals with fitted
in membranes made of Chol/ESMmembranes plotted as a functionof Chol/
ESM mixing ratio.
Hydrophobicity around the nitroxide moiety of ASL and CSL
Biophysical Journal 101(4) 837–846
840Mainali et al.
ASL does not change with an increase in cholesterol content
(with the exception of a small increase after the addition of
25–30 mol % cholesterol). In contrast, hydrophobicity
measured with CSL decreases with an increase in choles-
terol content. Cholesterol molecules separate larger PL
headgroups and increase water penetration to the polar
headgroup region where the nitroxide moiety of CSL is
located (14). In the CBD, the nitroxide moiety of CSL is
fully exposed to the water phase (Fig. 1 f). The difference
between 2AZvalues detected by ASL (~64.5 G) and CSL
(~70.0 G) is so great that the orientation of ASL with the ni-
troxide moiety in the polar headgroup region (like CSL)
should be detected at a high cholesterol content. The results
allow us to infer that the nitroxide moiety of ASL is always
located in the hydrophobic membrane center, independently
of cholesterol content, and that ASL unambiguously detects
two coexisting domains, not the distribution of ASL orien-
tations (see (9) for more detail).
Fig. 4 shows representative SR signals of ASL (A) and
CSL (B) in Chol/ESM membranes with a mixing ratio of
3:1 in the presence and absence of oxygen. SR signals
were fitted using single and double exponentials and
compared. The single-exponential fit was satisfactory for
both ASL and CSL in deoxygenated membranes. For ASL
in the presence of oxygen, the single-exponential fit was
not satisfactory, whereas the double-exponential fit was
excellent (compare the residual for single- and double-expo-
nential fits). T1values from double-exponential curves were
assigned to the lo-phase domain (the shorter time constant)
and to the CBD (the longer time constant) (see (7–9) for
more detail). However, all SR signals obtained with CSL
for membranes in the presence of oxygen were single expo-
nentials (Fig. 4 B). Because CSL should be distributed
between the lo-phase domain and the CBD similar to choles-
terol, we conclude that the collision rate between oxygen
and the nitroxide moiety of CSL is the same in both domains
(see also the discussion in (7).). Thus, the existence of the
CBD in ESM membranes can be confirmed using ASL
and oxygen, but not CSL.
affects the T1values of spin labels with the nitroxide moiety
at the membrane-water interface (like CSL). Fig. 4 C shows
representative SR signals for CSL in the presence and
absence of NiEDDA. In the presence of NiEDDA, a single-
exponential fit was not satisfactory, whereas the double-
exponential fit was excellent (compare the residual for
single- and double-exponential fits). T1s were assigned to
the lo-phase domain (the longer time constant) and to the
CBD (the shorter time constant). Thus, the existence of the
CBD can also be confirmed with CSL and NiEDDA as
a relaxation agent. All SR signals obtained with ASL for
membranes in the presence of NiEDDA were single expo-
nentials, with the time constant nearly the same as in the
absence of NiEDDA (data not shown). We conclude that
NiEDDA does not penetrate to the depth at which the nitro-
xide moiety of ASL is located both in the lo-phase domain
and the CBD. These results are in agreement with hydropho-
bicity measurements for ASL presented in Fig. 3.
Final results for discriminating membrane domains with
ASL and CSL are presented in Fig. 5. As expected, we de-
tected two domains when the Chol/ESM mixing ratio was
greater than the CST in the ESM bilayer. This display also
confirmed our assignments of SR results to the lo-phase
domain and the CBD. Results presented in Fig. 5 A indicate
that the OTP in the center of the CBD is about three to six
times smaller than that in the center of the lo-phase domain.
Although CSL data show a single value for the OTP for all
cholesterol contents, this does not mean that CSL detects
a single homogeneous domain. Data indicate that the colli-
sion rate between the nitroxide moiety of CSL and oxygen
of 3. Signals were recorded for samples equilibrated with (A–C) 100% nitrogen, (A–B) with a gas mixture of 50% air and 50% nitrogen, and (C) in the pres-
ence of NiEDDA. Fordeoxygenated samples, SR signals were satisfactorily fit to a single-exponentialfunction with time constants of (A) 3.00 5 0.01ms, (B)
3.41 5 0.01 ms, and (C) 3.41 5 0.01 ms (upper residuals are for single-exponential fit). The SR signal in the presence of molecular oxygen can be fitted
satisfactorily with a single exponential function only for CSL with a time constant of (B) 2.12 5 0.01 ms (lower residual is for single-exponential fit)
and with double exponential curves for ASL with time constants of (A) 1.72 5 0.07 ms and 0.48 5 0.01 ms (the middle residual is for single- and the lower
residual for double-exponential fits). The SR signal for CSL in the presence of NiEDDA can be fitted satisfactorily only with double-exponential curves with
time constants of (C) 1.57 5 0.04 ms and 0.74 5 0.01 ms (the middle residual is for single- and the lower residual for double-exponential fits).
Representative SR signals with fitted curves and residuals for (A) ASL and (B and C) CSL in an ESM membranewith a Chol/ESM mixing ratio
Biophysical Journal 101(4) 837–846
Cholesterol-Sphingomyelin Membranes 841
in the lo-phase domain and the CBD is the same. Thus, the
value of the OTP obtained with CSL can be used for both
domains to create profiles of the OTP. This hypothesis
was confirmed by measurements with NiEDDA that show
CSL is located in the lo-phase domain and the CBD, and
can discriminate these domains (Fig. 5 B). The nitroxide
moiety of CSL is more exposed to collisions with NiEDDA
when CSL is located in the CBD and the moiety is not pro-
tected by the umbrella effect of phospholipid headgroups (as
in the lo-phase domain). These results are in agreement with
the hydrophobicity measurements presented in Fig. 3, which
show that the polarity around the nitroxide moiety of CSL
increases when the cholesterol content increases beyond
Interestingly, the dual-probe method using ASL and
oxygen has also allowed us to discriminate coexisting ldand
lo phases. Double-exponential signals were measured,
and twovalues of the OTP were calculated for the Chol/ESM
mixing ratio of 1:4 (Fig. 5 A). Furthermore, the use of CSL
with NiEDDA has allowed us to discriminate coexisting ld
Profile of the OTP across the ldphase at different
regions in the phase diagram (Fig. 1) 1), the ldphase in the
with cholesterol that coexists with the lophase (~7.5 mol%
cholesterol in the ldphase for a Chol/ESM mixing ratio of
1:4 at 40?C; Fig. 6 b). OTP values obtained with ASL and
CSL are also included in the profiles.
The profile for the ldphase without cholesterol is a broad
bell shape, with all values of the OTP greater than the OTP
in water. OTP values in the membrane center are about two
times larger than those close to the membrane surface.
Comparison of this profile (Fig. 6 a) with that in the ldphase
containing ~7.5 mol % cholesterol (upper profile in Fig. 6 b)
reveals that the OTP is much greater in the ldphase contain-
ing the very small (~7.5 mol %) concentration of choles-
terol. This is surprising not only because it is contrary to
the general view that cholesterol increases alkyl chain order
and suppresses molecular motion, but also because such
a small concentration of cholesterol induced such large
increases in the OTP. However, a similar increase in the
OTP was induced by ~5 mol % cholesterol in the ldphase
of the DMPC membrane (3). Furthermore, we have previ-
ously shown that small amounts of cholesterol (< 5 mol %)
increase alkyl-chain mobility (gauche-trans isomerization)
in the ldphase (see Fig. 3 in (49)). In that study, a strong
rigidifying effect was observed for cholesterol in concentra-
tions above 5 mol %. This enhanced mobility of the phos-
pholipid spin label and OTP in the presence of very small
amounts of cholesterol may be due to an impurity effect
of cholesterol: it destroys cooperative, dynamic characteris-
tics by forming the membrane of a single molecular species,
which is particularly apparent in the middle of the bilayer
where cholesterol induces vacant space.
Profile of the OTP across the lophase at different
Detailed OTP profiles in the lophasewere obtained with PL-
analog spin labels in the five distinct regions indicated in the
phase diagram of Fig. 1: 1), the lophase that coexists with
the ldphase (~30 mol % cholesterol in the domain for
a Chol/ESM mixing ratio of 1:4; Fig. 6 b); 2), the lophase
that constitutes thewhole membrane and contains the lowest
cholesterol concentration of 33 mol % (Fig. 6 c); 3), the lo
phase that constitutes the whole membrane with 50 mol %
cholesterol (Fig. 6 d); 4), the lophase that is saturated
with cholesterol (at 66 mol % cholesterol; Fig. 6 e); and
5), the lo-phase domain that coexists with the CBD
(~66 mol % cholesterol in the domain for a Chol/ESM mix-
ing ratio of 3:1; Fig. 6 f). OTP values obtained with ASL and
CSL are also included in the profiles.
Profiles for the lophase containing the lowest cholesterol
concentration (Fig. 6, b and c) are not much different than
those for the pure ESM membrane (Fig. 6 a). All profiles
have a bell shape, with a gradual increase in the OTP toward
the membrane center. The major difference is that in the lo
phase the OTP is 50–20% lower and closer to the membrane
surface (T-, 5-, 7-, 9-PC positions). At deeper positions, the
(B) the NiEDDA accessibility parameter for CSL
in Chol/ESM membranes plotted as a function of
the Chol/ESM mixing ratio.
(A) The OTP for ASL and CSL and
Biophysical Journal 101(4) 837–846
842Mainali et al.
OTP values are very similar. Further increase in the overall
cholesterol content—up to the CST—induced a dramatic
change in the OTP profile (Fig. 6, d and e), decreasing the
OTP close to the membrane surface and increasing it in
the membrane center. As a result, the OTP profile became
almost rectangular, with a sharp 3–4-fold change in its value
between the C9 and C10 positions. Formembranes saturated
with cholesterol, overall change in the OTP across the
membrane was as large as a factor of ~9. OTP values
from depths close to the membrane surface were as low as
those observed in gel-phase membranes, and values from
the membrane center were as high (or even higher) as those
observed for the pure ESM membrane. The abrupt increase
in the OTP occurs at the depth at which the rigid tetracyclic
cholesterol structure is immersed. Increase in cholesterol
content beyond the CST does not cause any further change
in the OTP profile in the lo-phase domain (Fig. 6 f), indi-
cating that properties of the lo-phase domain are minimally
affected by the CBD.
Profile of the OTP across the CBD
Because only ASL and CSL can be located in the CBD, an
approximate profile of the OTP can be drawn based on OTP
membranes obtained at Chol/ESM mixing ratios
of (a) 0, (b) 1:4, (c) 1:2, (d) 1:1, (e) 2:1, and (f)
3:1 (schemes for membrane structures are shown
in Fig. 1). Dotted lines indicate the OTP in the
Profiles of the OTP for Chol/ESM
Biophysical Journal 101(4) 837–846
valuesobtainedwith thesespin labels. TwoOTPvalueswere
obtained with ASL. One fits perfectly into the profile across
the coexisting lo-phase domain, and the other indicates the
OTP in the CBD. As was explained earlier, OTP values ob-
tained with CSL are the same in the lo-phase domain and
the CBD. Profiles of the OTP across the CBD are included
in Fig. 6, e and f. Profiles in Fig. 6 f were obtained at
a Chol/ESM mixing ratio of 3:1, at which the CBD coexists
with the lo-phase domain. (The CBD is the pure cholesterol
bilayer, and the surrounding lo-phase domain is the ESM
bilayer saturated with cholesterol (66 mol %)). Under these
conditions, 66% of cholesterol molecules should saturate
the ESM bilayer, and 33% should form the CBD. Values of
the OTP in the center ofthe CBD are about three times lower
than in the water phase and seven times lower than in the
center of the lo-phase domain. These OTP values are similar
to values previously obtained by us for the CBD formed in
lens lipid membranes (7,8) and in binary mixtures of choles-
terol with PLs (9). Our conclusion that the CBD can form
a barrier to oxygen transport is supported.
The CBD was also unexpectedly detected at a Chol/ESM
mixing ratio of 2:1, which reaches the CST in ESM
membranes. The OTP profile across the CBD is presented in
Fig. 6 e based on these measurements. It differs significantly
greater in the CBD center. Profiles in Fig. 6 e were drawn
based on measurements for cholesterol contents close to the
CST. Thus, only a small amount of cholesterol is involved
in the formation of the CBD. With this condition, the CBD
is small. The high exchange rate of lipid molecules between
the small CBD and surrounding lo-phase domain and/or
high frequency of formation and dissipation of the CBD
those of the lo-phase domain. This is in agreement with our
earlier statement that CBDs are highly dynamic domains (9).
After reviewing the literature (20–22,50) and weighting our
results, it was concluded that rafts can be considered
domains of a lophase. On the basis of this concept, we
believe that efforts to characterize lophases and domains
extensively are important. Because the membrane (and,
thus, the lophase and domain) is not really a two-dimen-
sional structure, knowledge of the molecular dynamics
and structures in the direction of the depth in the membrane
is especially important.
as a functionofcholesterol content from0 to50 mol %using
only one spin label (4). The nitroxide moiety was located at
the C5 position where we expected the strongest effects of
cholesterol. We distinguished the lophase from the ldphase
(and the sophase) and obtained values of the OTP for these
phases at the C5 position. We were also able to confirm in
Chol/ESM membranes positions of the boundaries between
regions with coexisting ldand lophases or soand lophases
and the region with a single lophase. In our present research,
which completes the studies presented in our previous work
(4), we obtained detailed profiles of the OTP across single
or coexisting phases in ESM membranes for different
Chol/ESM mixing ratios from 0 to 3. At cholesterol contents
and ldphases using both PL- and cholesterol-analog spin
labels. However, at cholesterol contents equal to or
>33 mol % (Fig. 1, c–e), both types of spin labels indicated
tained for Chol/ESM andChol/PSMmembranes using solid-
content was equal to or exceeded the CST in the ESM
membrane (Fig. 1, e and f), wewere also able to discriminate
and characterize the CBD (see, however, the explanation in
us to make the hypothetical extension of the phase diagrams
for Chol/ESM presented earlier (4,39,51) to the area of the
membrane overloaded with cholesterol, where cholesterol
not only saturates the PL bilayer but also forms the immis-
Our data confirmed that the properties of different
membrane phases and domains are strongly affected by
cholesterol concentration, and that this effect is different at
different depths in the lipid bilayer. The most pronounced
changes were observed for the lo phase in which the
OTP profile changed from a bell shape to a rectangular
shape. The fluidity of the lophase saturated with cholesterol
from the membrane surface to the depth of C9 is suppressed
to the level of fluidity in the gel-phase membrane, whereas
in the membrane center, fluidity is comparable to that in
the center of the fluid-phase membrane without cholesterol.
It should be noted that the fluidity measured here is deter-
the membrane. This work complements our previous work
(3) in which we characterize the binary mixture of DMPC
and cholesterol. Data obtained in these works should be
used as a reference point to compare the properties of lipid
phases and domains in model and biological membranes.
Cholesterol-mediated lipid interactions have important
effects on the lateral organization of PLs in the membrane.
They increase the molecular order of PL alkyl chains, in-
crease membrane thickness, and modulate the packing of
is much stronger for saturated PLs than for unsaturated PLs.
These differences are clearly seen in profiles of the OTP.
(POPC) (6), DMPC (3), and ESM membranes saturated with
cholesterol (the work presented here) show that the most
tightly packed lipids are in the ESM membrane. Values of
the OTP obtained at 40?C for 5-PC in Chol/POPC, Chol/
DMPC, and Chol/ESM membranes are ~0.9, ~0.65, and
~0.4 ms?1, respectively. However, in the membrane center,
2H NMR spectroscopy (43). When the cholesterol
Biophysical Journal 101(4) 837–846
844Mainali et al.
saturating amounts of cholesterol do not change the OTP (as
in POPC and DMPC membranes) or even increase it (as in
our observations and the work of Bunge et al. (24) who
showed, using cholesterol analog spin labels with the nitro-
xide moiety attached to the end of the isooctyl chain, which
in the membrane center cholesterol also induces a greater
order in PSM than in POPC. The order parameter (the static
membrane parameter) cannot differentiate the effects of
cholesterol at different depths, whereas the dynamic param-
the membrane region where the cholesterol ring structure is
located and the deeper region where the isooctyl chain of
cholesterol is located (see (3,46) for more discussion).
These data also contribute to our research on lens lipid
membranes. PL compositions of these membranes change
significantly between species, with age, and with location
in the lens (cortex versus nucleus). For example, the total
PL extract from the lenses of animals with a short life
span (i.e., a mouse or rat) contains ~45% PC and only
~15% SM. From human lenses, the total PL extract contains
~11% PC and 66% SM (32). The ratio of PC/SM in two-
year-old cow lens cortex and nucleus is 0.66–2.0 and
0.21–0.5, respectively (8). The cholesterol content in all
lens lipid membranes is close to the CST or higher (8,30).
We previously obtained detailed profiles of membrane prop-
erties across POPC membranes with cholesterol contents
close to or exceeding the CST, which occurs at a Chol/
POPC mixing ratio of 1 (6). Here, we obtained detailed
profiles of the OTP across ESM membranes at a cholesterol
mixing ratio from 0 to 3. This allowed us to investigate ESM
membranes in conditions similar to those in lens lipid mem-
branes, keeping in mind that the CST in ESM membranes is
at a Chol/ESM mixing ratio of 2 (34). We were able to
discriminate the CBD at a Chol/ESM mixing ratio of 3,
even as well at a mixing ratio of 2. In the latter case,
CBDs were small and/or lipid exchange between them and
a surrounding membrane was fast. In the former case, the
properties of CBDs were similar to those reported earlier
(9). OTP profiles across the surrounding ESM bilayer satu-
rated with cholesterol were rectangular in shape, with an
abrupt increase in OTP value between C9 and C10, and
did not change when the cholesterol mixing ratio was
increased from 2 to 3 and the CBD was formed. Profiles
were practically identical to those obtained for lens lipid
membranes (6–8) and across POPC membranes saturated
with cholesterol or obtained in the presence of the CBD (6).
One figure and additional text are available at http://www.biophysj.org/
This work was supported by grants EY015526, TW008052, EB002052, and
EB001980 of the National Institutes of Health.
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846Mainali et al.