Supplementary material
Cholesterol-phospholipid interactions: new insights from surface X-ray scattering data
Andrey Ivankin, Ivan Kuzmenko, and David Gidalevitz
Analysis of Bragg rod profiles
The Bragg rod profiles for pure DPPC, cholesterol and their mixtures with χCHOL 0.25 and 0.7 are
shown in Fig. 1. Analysis of the Bragg rod profiles was done by approximating the DPPC acyl chains and
cholesterol rigid body as tilted cylinders with constant electron density and length LC [1]. The combined
Bragg rod profile of the (1,1),(1,-1) and (0,2) reflections for DPPC, shown in Fig. 1a, was produced by
integrating scattering intensity through the 1.23 Å-1≤qxy<1.52 Å-1 region of the two peaks. The molecular
tilt of DPPC acyl chains comprises 32 ± 1° from the surface normal in the nearest-neighbor (NN)
direction. The length of the cylinder with constant electron density used to model the Bragg rod profiles
LC was 19.0 ± 1.0 Å. This length is consistent with a calculated length of 19.24 Å for a 16-carbon acyl
chain. A single Bragg rod with the maximum at qz ≈ 0 Å-1 for the cholesterol-DPPC mixtures with χCHOL
0.25-1 (Fig. 1b-d) suggests that in the LO phase molecules are aligned normal to the surface. The LC
value used in fitting the Bragg rods for the mixed and cholesterol monolayers was 10.0 ± 1.0 Å which is
consistent with a calculated size of the cholesterol fused-ring body of 9.5 Å. In mixed monolayers only
the DPPC acyl chains region which is in direct contact with the cholesterol rigid body contributes to the
in-plane scattering diffraction pattern.
Figure 1. Grazing incidence X-ray diffraction data: scattering intensity against scattering vector qZ
measured at the Bragg peak position qxy (symbols). Solid lines are the corresponding fits of the Bragg
rod profiles. (a) DPPC; (b) and (c) mixtures with χCHOL 0.25 and 0.7; (d) cholesterol.
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Biological relevance of the model for cholesterol-phospholipid
Diffraction
patterns
for
the
mixed
monolayer with χCHOL 0.66 at 23 °C and 20
mN/m was compared to that at 37 °C and 32
mN/m are shown in Fig. 2. The contribution
from the thermal diffuse scattering increases
with temperature. Therefore, the intensity of
the diffraction peak of the mixture at 37 °C,
i.e the signal to noise ratio, is lower than that
for the mixture at 23 °C. Nevertheless, the
position of the peak remains the same
signifying that at χCHOL = 0.66 an average
Figure 2. Grazing incidence X-ray diffraction data:
scattering intensity, integrated over qz range, against
scattering vector qxy of cholesterol/DPPC mixture
(60/40 molar ratio) at 23 °C and 20 mN/m (squares)
and at 37 °C and 32 mN/m (inverted triangles).
inter-molecular distance, and, consequently, the molecular composition of the LO phase in the mixed
monolayer at 37 °C and 32 mN/m is identical to that at 23 °C and 20 mN/m.
X-ray reflectivity data analysis
Electron density profiles (z) of the cholesterol-DPPC monolayers were derived by fitting the
measured X-ray reflectivities (XR) (presented in the paper) using the model-dependent slab model
refinement [2-3] and model-independent stochastic fitting [4] routines. Both approaches resulted in very
similar fits, which are in an excellent agreement with the measured reflectivity curves. For convenience,
the electron density profiles are discussed within the framework of the slab model hereafter. Table 1
summarizes the fitting results of slab model refinement, including but not limited to slab thicknesses (L)
and average electron densities ().
Reflectivity curves of both pure DPPC and cholesterol monolayers were fitted with a two slab model.
The top slab is comprised of acyl chains and the bottom corresponds to either the headgroup of DPPC or
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the rigid body of cholesterol. These results agree well with our earlier studies of DPPC [2] and cholesterol
[5] monolayers.
Modeling of XR intensity profiles for the cholesterol-DPPC mixtures was successful using a three slab
model. The vertical relative position of the sterol molecule in the film can be retrieved from the electron
density profile of DPPC and cholesterol as well as from the mean molecular area available from the
surface pressure-molecular area isotherms. For the mixture with cholesterol molar ratio (χCHOL) of 0.130.46 the top slab is composed of the upper part of DPPC acyl chains and flexible acyl chain of
cholesterol. The middle slab was ascribed to the remaining part of DPPC acyl chains and the cholesterol
rigid body. The bottom slab contains the headgroups of both DPPC and cholesterol.
The considerable change in the electron density profile of the mixtures with χCHOL 0.7 and, especially,
0.85 suggests different molecular occupation within the slabs. At χCHOL 0.7, the middle slab contains the
carboxyl groups of DPPC in addition to the phospholipid acyl chains and the rigid body of cholesterol.
The bottom slab was assigned to the choline portion of the DPPC headgroups and the hydroxyl group of
cholesterol. For this mixture, cholesterol molecules are shifted towards the aqueous medium relative to
DPPC, as defined by the reduced thickness of the lowest slab. At higher cholesterol concentrations (χ CHOL
0.85) the sterol rigid body occupies an even lower relative position within the film, in the region of the
DPPC headgroups. According to the analysis of XR data for the mixture with χCHOL 0.85 the top slab
contains only acyl chains of DPPC, the middle slab is occupied by the acyl chains of both cholesterol and
DPPC, and the bottom slab comprises DPPC headgroups and the rigid body of cholesterol.
“Condensing” effect, liquid-liquid immiscibility, and chemical activity behavior phenomena of
cholesterol
The “condensing” effect of cholesterol implies that in cholesterol-phospholipid mixtures the average
area per molecule is smaller than the linear combination of the molecular areas of individual lipids, and
that cannot be explained solely by the change of the molecular tilt in the mixed monolayer. Instead, it is
indicative of an increase in the packing density. The apparent change of the phospholipid acyl chain tilt as
a function of χCHOL cannot explain the cholesterol “condensing” effect on its own. Since cholesterol is
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mainly located within the hydrophobic region of DPPC at χCHOL 0.13-0.46, “condensing” can only mean
that the cross-sectional packing density of cholesterol and DPPC acyl chains is increased. As a result,
cholesterol molecules may seize some space above DPPC headgroups and, therefore, effectively occupy a
smaller area. Our data allow estimation of a decrease in the effective area of cholesterol in the mixtures as
a function of its content.
A smaller average molecular area (A) in mixed cholesterol-DPPC monolayers (Table 1) can be
explained by a simultaneous change in the phospholipid acyl chain tilt and increase in the cross-sectional
packing density. A decrease in the effective area of cholesterol in these mixtures is caused by more
efficient molecular packing and is not due to changes in the molecular tilt. The XR data analysis provides
a value for the average molecular tilt (t) which can be used to derive the cross-sectional molecular area as
ACS = A cost (Table 1). If the packing densities of cholesterol and DPPC in the mixtures were the same
as in the single component monolayers, the average ACS of the mixture would be the linear combination
of ACS of the individual components, as defined by the stoichiometry. For low χCHOL, the experimentally
obtained values of ACS are systematically smaller than the calculated ones, as shown in Table 1. As
estimated, the effective area of cholesterol molecules (not cross-sectional) is approximately 33.5 Å2 for
the mixtures with χCHOL 0.13 and 0.25. This value is consistent with the partial-specific area of cholesterol
in DPPC-cholesterol bilayers, obtained by molecular simulations [6-7]. Therefore, for these mixtures,
DPPC headgroups provide on average 4.5-5 Å2 of screening to each cholesterol molecule.
At χCHOL 0.46, the effective area of cholesterol is larger (~34.6 Å2). DPPC headgroups only screen
~3.6 Å2 of cholesterol, which corresponds to 3.1 Å2 per DPPC headgroup, the maximum screening area
one ultimately tilted phosphocholine group can provide. Since at low χCHOL cholesterol tends to grasp
~4.5-5 Å2 above the phospholipid headgroups, we propose that the screening of cholesterol becomes
incomplete at χCHOL~0.4, which may be responsible for the transition between two regions of liquid-liquid
immiscibility in the cholesterol-phospholipid mixtures at similar χCHOL, as well as a sharp jump in the rate
of cholesterol release to β-cyclodextrin from the mixed monolayers, cholesterol chemical activity, also
occuring at the same χCHOL [8].
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At high cholesterol concentrations (70 and 85 mol%), the molecular areas of DPPC and cholesterol
cannot be easily estimated, due to the shift of cholesterol molecules towards the aqueous medium and
concomitant disordering in the upper part of DPPC acyl chains.
Table 1. X-ray reflectivity data fitting parameters.
Experiment
Slab
Li (Å)
i (e-/Å3)
DPPCAC
15.0
0.321
DPPCHG
9.1
0.460
CHOLAC
6.3
0.260
CHOLRB
9.3
0.414
DPPCAC + CHOLAC
7.9
0.311
DPPCAC + CHOLRB
7.5
0.331
DPPCHG + CHOLHG
8.9
0.424
DPPCAC + CHOLAC
7.4
0.304
DPPC
Cholesterol
 (Å)
L (Å)
t (°)
A (Å2)
Acs (Å2)
3.7±0.5
24.1
38.8
49.8
38.8
2.9±0.1
15.6
NA
38.2
38.2
3.3±0.2
24.3
36.8
47.7
38.2
3.1±0.2
25.2
31.5
44.2
37.7
3.1±0.2
24.5
25.3
40.9
37.0
2.9±0.1
23.6
NA
39.7
NA
3.2±0.3
24.0
37.8
38.9
NA
Cholesterol:DPPC Mixture
13:87
25:75
46:54
70:30
85:15
DPPCAC + CHOLRB
9.1
0.340
DPPCHG + CHOLHG
8.7
0.443
DPPCAC + CHOLAC
7.5
0.281
DPPCAC + CHOLRB
9.9
0.359
DPPCHG + CHOLHG
7.1
0.417
DPPCAC + CHOLAC
7.5
0.281
DPPCAC + CHOLRB
10.3
0.371
DPPCHG + CHOLHG
5.8
0.394
DPPCAC + CHOLAC
9.2
0.056
DPPCAC + CHOLRB
6.0
0.321
DPPCHG + CHOLHG
8.8
0.439
Notations: DPPCAC, DPPCHG – acyl chains and headgroup of DPPC, respectively; CHOLAC, CHOLRB, CHOLHG – acyl chain,
rigid body, and hydroxyl group of cholesterol, respectively; Li and i – thickness and average electron density of the
corresponding slab;  - roughness parameter; L – overall film thickness; t – tilt of DPPC acyl chains; A – average molecular area;
Acs – cross-sectional molecular area
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
J. Als-Nielsen et al., Phys Rep 246, 252 (1994).
F. Neville et al., Biophys J 90, 1275 (2006).
F. Neville et al., Soft Mat 4, 1665 (2008).
S. M. Danauskas et al., J Appl Cryst 41, 1187 (2008).
H. Rapaport et al., Biophys J 81, 2729 (2001).
O. Edholm, and J. F. Nagle, Biophys J 89, 1827 (2005).
S. A. Pandit et al., Langmuir 24, 6858 (2008).
H. Ohvo-Rekila et al., Prog Lipid Res 41, 66 (2002).
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