Transmembrane peptides influence the affinity of sterols for phospholipid bilayers.
ABSTRACT Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mbetaCD can be measured. Comparison of how cholesterol and CTL partitioning between mbetaCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mbetaCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.
- SourceAvailable from: Terhi Maula[Show abstract] [Hide abstract]
ABSTRACT: To study the role of the interfacial properties of ceramides in their interlipid interactions, we synthesized palmitoylceramide (PCer) analogs in which a methyl group was introduced to the amide-nitrogen or the C3-oxygen of the sphingosine backbone. A differential scanning calorimetry analysis of equimolar mixtures of palmitoylsphingomyelin (PSM) and PCer showed that these sphingolipids formed a complex gel phase that melted between 67°C and 74°C. The PCer analogs also formed gel phases with PSM, but they melted at lower temperatures compared with the system with PCer. In complex bilayers composed of an unsaturated glycerophospholipid, PSM, and cholesterol, the 3O-methylated ceramide formed a cholesterol-poor ordered phase with PSM. However, the 2N-methylated and doubly methylated (2N and 3O) PCer analogs failed to displace sterol from interactions with PSM. Like PCer, the analogs reduced sterol affinity for the complex bilayers, but this effect was most pronounced for the 3O-methylated ceramide. Taken together, our results show that 2N-methylation weakened the ceramide-PSM interactions, whereas the 3O-methylated ceramide behaved more like PCer in interactions with PSM. Our findings are compatible with the view that interlipid interactions between the amide-nitrogen and neighboring lipids are important for the cohesive properties of sphingolipids in membranes, and this also appears to be a valid model for ceramide.Biophysical Journal 12/2011; 101(12):2948-56. · 3.67 Impact Factor
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ABSTRACT: The effects of cholesterol on various membrane proteins are of long-standing interest in membrane biophysics. Here we present systematic molecular dynamics simulations (totaling 1.4 μs) of integral protein phospholamban incorporated in POPC/cholesterol bilayers (containing 0, 11.11, 22.03, 33.33, and 50 mol% of cholesterol). Phospholamban is a key regulator of cardiac contractility and has recently emerged as a potential drug target. In agreement with experiments, our results show that in a cholesterol-free pure POPC bilayer, phospholamban exhibits broad conformational distribution, ranging from the closed T-state to the extended R-state, crucial for its functionality. Increasing cholesterol concentration progressively stabilizes the bent conformers of phospholamban over open structures, and favors extensive interactions of its amphipathic N-terminal helix with the bilayer surface. The interaction energies between the N-terminal helix of PLB and different POPC/cholesterol bilayers quantitatively confirm its stronger interaction with a higher cholesterol-containing membrane. Simulation with 50 mol% of cholesterol further supports the above conclusions, where phospholamban undergoes rapid conformational transition from extended to closed form, which remains stable for the rest of the simulation time and exhibits the strongest interaction with the membrane. Cholesterol participates in hydrogen-bonding and π-stacking interactions with polar and/or aromatic residues and favors membrane association of phospholamban. We observed cholesterol-enrichment in the neighborhood of phospholamban. Moreover, as a modulator of membrane biophysical properties, cholesterol modifies the hydrophobic matching and trans-membrane tilting of phospholamban and also hinders its 2D-lateral mobility. Altogether, our results highlight atomistic details of protein-lipid interplay and provide new insights into the possible effects of cholesterol on conformational dynamics of phospholamban in membrane bilayers.Physical Chemistry Chemical Physics 12/2011; 13(45):20188-98. · 3.83 Impact Factor
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ABSTRACT: The sphingoid bases of sphingolipids, including ceramides, can vary in length from 12 to >20 carbons. To study how such length variation affects the bilayer properties of ceramides, we synthesized ceramides consisting of a C12-, C14-, C16-, C18-, or C20-sphing-4-enin derivative coupled to palmitic acid. The ceramides were studied in mixtures with palmitoyloleoylphosphocholine (POPC) and/or palmitoylsphingomyelin (PSM), and in more complex bilayers also containing cholesterol. The trans-parinaric acid lifetimes showed that 12:1- and 14:1-PCer failed to increase the order of POPC bilayers, whereas 16:1-, 18:1-, and 20:1-PCer induced ordered- or gel-phase formation. Nevertheless, all of the analogs were able to thermally stabilize PSM, and a chain-length-dependent increase in the main phase transition temperature of equimolar PSM/Cer bilayers was revealed by differential scanning calorimetry. Similar thermal stabilization of PSM-rich domains by the ceramides was observed in POPC bilayers with a trans-parinaric acid-quenching assay. A cholestatrienol-quenching assay and sterol partitioning experiments showed that 18:1- and 20:1-PCer formed sterol-excluding gel phases with PSM, reducing the overall bilayer affinity of sterol. The effect of 16:1-PCer on sterol distribution was less dramatic, and no displacement of sterol from the PSM environment was observed with 12:1- and 14:1-PCer. The results are discussed in relation to other structural features that affect the bilayer properties of ceramides.Biophysical Journal 11/2012; 103(9):1870-9. · 3.67 Impact Factor
Transmembrane Peptides Influence the Affinity
of Sterols for Phospholipid Bilayers
Joel H. Nystro ¨m, Max Lo ¨nnfors, and Thomas K. M. Nyholm*
Department of Biochemistry and Pharmacy, A˚bo Akademi University, Turku, Finland
content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient
is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral orga-
nization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral
distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with
bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of
proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between
LUVs and mbCD can be measured. Comparison of how cholesterol and CTL partitioning between mbCD and phospholipid bila-
yers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the
results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL par-
titioning between DMPC bilayers and mbCD was measured. From the results it was clear that WALP23 increased both the order
in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent
way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller
with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and
saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of
KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can
both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distri-
bution within the bilayer and thereby the lateral organization in biomembranes.
Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol
Cholesterol is an essential component in the membranes of
mammalian cells. An interesting property of cholesterol is
that it together with phospholipids can form a so called liquid
ordered phase, by ordering the acyl chains in fluid phospho-
lipids. Thereby, cholesterol can induce liquid-liquid phase
separation in bilayer membranes, i.e., cholesterol can influ-
ence the lateral organization in lipid bilayers.
How the lateral organization of a phospholipid bilayer is
influenced by cholesterol depends on the phospholipid
composition of the bilayer. Cholesterol has been shown to
interact differently with different phospholipid types (1–3),
and to prefere phospholipids with saturated acyl chains
over those with unsaturated chains (see (4) and references
within). Hence, cholesterol can facilitate lateral separation
in complex bilayer membranes by favoring saturated acyl
chains over unsaturated ones. For example, it has been
observed that saturated sphingomyelin together with choles-
terol can form ordered domains that laterally separate from
disordered domains containing unsaturated phosphatidyl-
choline (5,6). As it is known that sphingomyelin and choles-
terol have a similar distribution in cellular membranes it is
thought that they are cosorted, and that sorting is linked to
the formation of domains enriched in sphingolipids and
cholesterol (reviewed in Holthuis et al. (7)).
membrane proteins between Golgi and the plasma membrane
seems to depend, at least partially, on the length of the hydro-
phobic transmembrane segments in the proteins (9–11).
Proteins with longer transmembrane helices are transported
known that cholesterol can increase the bilayer thickness and
that the plasma membranes are thicker than Golgi membranes
may drive the sorting of transmembrane proteins as the
membrane components strive to minimize the hydrophobic
material properties (12). This could mean that proteins with
patches, i.e., cholesterol and sphingolipid enriched domains in
the Golgi, leading to transport to the plasma membrane.
Proteins with shorter transmembrane segments on the other
structure of the surrounding lipids (reviewed in de Planque
Submitted December 9, 2009, and accepted for publication April 22, 2010.
Abbreviations used: CTL, cholestatrienol; DLPC, dilauroylphosphatidcho-
line; DMPC, dimyrisoylphosphatidylcholine; DPH, diphenylhexatriene;
1-palmitoyl-2-oleoyl-phosphatidylcholine; PSM, palmitoylsphingomyelin.
Editor: Amitabha Chattopadhyay.
? 2010 by the Biophysical Society
526Biophysical JournalVolume 99July 2010526–533
in lipid bilayers remains unknown. Membrane proteins could
increase the heterogeneity in cellular membranes e.g., by
having different affinity for different lipids, and in fact it
has been observed that several different membrane proteins
preferentially interact with lipids that have a specific acyl
chain length (16–20). As different proteins seem to have
different acyl chain preferences, deriving from the effective
hydrophobic length of their transmembrane segments,
scopic domain formation in cellular membranes.
If some proteins attract phospholipids with longer acyl
chains, do the same proteins also attract cholesterol? Such
attraction could be a result of the presence of long chain
phospholipids at the surface of the protein or transmembrane
proteins having an ordering effect on the surrounding lipids.
This could result in the formation of nanoscopic domains
around the proteins. Other proteins attracting short chain
phospholipids could have the opposite effect, and expel
cholesterol from the surrounding lipid environment.
The aim of this study was to obtain new information about
how transmembrane proteins can influence how cholesterol
interacts with phospholipid bilayers. A convenient way to
study this is to measure sterol partitioning between LUVs
and mbCD (21–24). For this purpose, we developed and
tested what we believe to be a new fluorescence-based
method. The tests showed that the fluorescent cholesterol
analog, CTL, responded similarly as cholesterol to changes
in the lipid composition, i.e., the probe should also respond
similarly to incorporation of proteins into the LUVs.
Model peptides designed to mimic the transmembrane
helices of proteins were included to the lipid bilayers to
give information about the influence of proteins. Peptide
inclusion increased the affinity of CTL for the phospholipid
bilayer, and the affect was decreased when the phospholipid
acyl chains were shorter or unsaturated. In addition, the
effect of peptides on the affinity of sterols for phospholipid
bilayers was clearly dependent on the peptide structure.
Hence, we conclude that sterols sense proteins in the bilayer
and that based on the results in this study transmembrane
proteins could affect the lateral organization in cellular
membranes in a way that may be important for the sorting
of lipids and proteins within the cell.
All phospholipids werepurchasedfromAvanti PolarLipids(Alabaster,AL).
PSM was purified from egg yolk sphingomyelin by reverse-phase high
performance liquid chromatography (Supelco Discovery C18 column,
dimensions 250 ? 21.2 mm, 5 mm particle size) using methanol/water
(95:5, volume ratio) as the eluent. DPH was obtained from Molecular Probes
(Eugene, OR). CTL (cholesta-5,7,9(11)-trien-3-b-ol) was synthesized as
described by Fischer et al. (25) and purified by reverse-phase high perfor-
mance liquid chromatography with acetonitrile/methanol (7:3, vol/vol) as
the eluent. The concentrations of the fluorophore stock solutions were deter-
mined based on their extinction coefficients (DPH: 88,000 cm?1M?1at
The solutions were stored at ?20?C and warmed to ambient temperature
followed by passage through a Millipore UF Plus water purification system
Partitioning of CTL between mbCD
and phospholipid vesicles
Lipid vesicles for the partitioning studies were prepared by mixing phospho-
lipids, CTL (2 mol %), and peptides in organic solvent. The solvent was then
evaporatedunder a constant streamof nitrogen,after which the resultingfilm
was rehydrated above the gel-to-fluid transition of the phospholipids in the
samples. The rehydrated samples were then vortexed, and briefly bath-soni-
cated to form multilamellar vesicles. To create LUVs the multilamellar vesi-
cles were then extruded through a membrane with 200 nm pores. The quality
of the resulting LUVs was checked by measuring the vesicle size on a Mal-
vern Zetasizer (Worcestershire, UK). For the partition assay 100 nmol lipids
(LUVs) were portioned into 10 glass tubes. mbCD was added to nine of the
tubes, after which the solutions were diluted with milli-Q water to a final
phospholipid concentration of 40 mM. The final concentration of mbCD
in the tubes was 0, 0.04,0.08, 0.15,0.25, 0.35, 0.50,0.60, 0.80,and 1.0 mM.
The samples were then incubated 2 h at 37?C or overnight at 25?C, after
which the steady-state anisotropy of CTL was measured (at 37?C or 25?C)
on a PTI Quantamaster 1 (Photon Technology International, NJ) spectroflu-
orimeter operating in the T-format, with both the excitation and emission
slits set to 5 nm. The samples were excited at 324 nm and the emission
was measured at 390 nm.
The molar concentration of CTL CLUV
calculated from the measured anisotropies according to
CTLin the LUVs in each sample was
where CCTLis the total concentration of CTL in the samples, rLUVis the
anisotropy of CTL in the specific phospholipid bilayer, riis the CTL anisot-
ropy in the sample, and rCDis the anisotropy of CTL in the CTL-mbCD
complex. The anisotropy of the CTL-mbCD complex was measured for
a range of the CTL-mbCD ratios and was determined to 0.175 at 25?C
and 0.170 at 37?C.
The molar fraction partition coefficient KXwas calculated as described by
Tsamaloukas et al. (22) based on the equation
?CL þ CLUV
where CLis the phospholipid concentration, CCDis the cyclodextrin concen-
the concentration of cholesterol in complex with mbCD. The partition coef-
ficients were calculated by plotting the calculated molar concentrations of
CTL in the phospholipid bilayers against the mbCD concentration and
fitting the obtained curves with the following equation
CHOLis the cholesterol concentration in lipid bilayers, and CCD
1 þ 4
Biophysical Journal 99(2) 526–533
Cholesterol-Phospholipid Interactions 527
Determination of the DPH anisotropy
in phospholipid bilayers
Samples were prepared by mixing phospholipids, DPH (0.5 mol %) and
peptides (0–7 mol %) in organic solvent, after which the solvent was evap-
orated under a constant stream of nitrogen. The dry films were rehydrated in
milli-Q water at a temperature above the gel-to-fluid transition of the phos-
pholipids in the sample. The hydrated samples were then vortexed and bath
sonicated briefly to obtain multilamellar vesicles, with a final lipid concen-
tration of 50 mM. The anisotropy of DPH was measured at 25?C and 37?C
using the same instrument as in CTL anisotropy measurements, with the slits
at 5 nm. All samples were excited at 358 nm and the fluorescence emission
was measured at 430 nm.
CTL partitioning between mbCD and phospholipid
The affinity of sterols for lipid bilayers has been measured
successfully previously by measuring how sterols partition
between cyclodextrins and lipid bilayers (21–24,26). In the
majority of these studies,3H-cholesterol was used as a probe,
and in two studies cholesterol partitioning was measured
using isothermal titration calorimetry. A draw back in all
the methods used previously is that rather large quantities
of material is needed, and that relatively high cholesterol
concentrations were used to keep the phospholipid concen-
trations lower. In this study, we wanted to minimize material
use, and especially lower the sterol concentration below
domain forming concentrations. Therefore we developed
what we believe to be a new fluorescence-based method to
determine the affinity of sterols for lipid bilayers with
different composition. CTL was chosen as the probe,
because it has been shown to be a good analog of cholesterol
(27). To avoid formation of sterol rich domains that could
complicate the interpretation of the results the amount of
sterol in the bilayers were set to 2 mol %.
To calculate partition coefficients the fraction of CTL in
LUVs and in complex with mbCD needed to be determined.
If the CTL anisotropy in lipid bilayers is different from that
of CTL in the cyclodextrin complexes this parameter could
be used for this purpose. Therefore, we first measured the
anisotropy in CTL mbCD complexes. Complexes were
prepared by rehydrating dry CTL films in 0.04, 0.25, 0.5,
or 1.0 mM mbCD solutions, with a final CTL concentration
of 0.8 mM. The anisotropy of CTL in these complexes was
measured and as seen in the results shown in Fig. 1 A the
anisotropy was ~0.175 in the complexes, irrespective of
the total CLT-mbCD ratios in the solutions. As the anisot-
ropy of CTL in all studied phospholipid bilayers have been
significantly higher than this, we could distinguish between
CTL in bilayers and in cyclodextrin complexes by measuring
the anisotropy. Next we prepared POPC LUVs containing
2 mol % CTL and prepared a series of samples with
40 mM POPC LUVs and 0–1 mM mbCD. To insure that
equilibrium was reached the samples were incubated over-
night at 25?C before the fluorescence measurements were
carried out. When the anisotropy of CTL in these samples
was measured at 25?C a clear relation between mbCD
concentration and anisotropy could be seen (Fig. 1 A). As
the mbCD concentration increased, the anisotropy decreased
toward the anisotropyobserved in the CTL-mbCD
complexes. This indicated the CTL was being removed
from the lipid bilayers by mbCD.
The recorded anisotropy data could then be used to calcu-
late the fraction of CTL in the LUVs in the presence of
varying mbCD concentrations using Eq. 1. The resulting
data is shown in Fig. 1 B. To obtain the molar fraction parti-
tion coefficients for CTL partitioning between LUVs and
mbCD, and the stoichiometry of the CTL-mbCD complexes
the data in Fig. 1 B was fitted with Eq. 3. As is clear from the
figure, the data fit well with this model for the partitioning.
The stoichiometry of the CTL-mbCD complexes was 2 ac-
cording to the fit, in agreement with what has been shown
previously for cholesterol-mbCD complexes (22). The parti-
tion coefficient was 6.5 mM, which was much lower than has
been observed for cholesterol (22,23), but is in agreement
with the previous observation that CTL is removed much
faster both from pure sterol and phospholipid-sterol mono-
layers by b-cyclodextrin than cholesterol (28).
0.0 0.20.40.60.8 1.01.2
CTL in LUVs (μM)
0.00.20.4 0.6 0.81.0 1.2
POPC and CD
KX = 6.5
n = 2
The anisotropy of CTL in POPC bilayers and in mbCD complexes was
measured as a function of mbCD concentration at 25?C. (B) Using Eq. 1
the concentration of CTL in lipid bilayers can be calculated from the anisot-
ropy data and by fitting the data with Eq. 3 the partition coefficient and the
stoichiometry of the mbCD-CTL complexes are obtained.
Representative data that describes the partition method. (A)
Biophysical Journal 99(2) 526–533
528Nystro ¨m et al.
Lipid effects on CTL partitioning
To further test the method and to learn more about what
determines the affinity of sterols for phospholipid bilayers
we tested how CTL partitioning between LUVs and mbCD
was influenced by the lipid composition in the LUVs.
Because previous studies offered data on cholesterol parti-
tioning between POPC/PSM LUVs and mbCD, we also
studied this system with our method to be able to compare
how CTL and cholesterol interactions with phospholipid
bilayers are affected by changes in the lipid composition.
LUVs of POPC and 0, 33, or 50 mol % PSM were
prepared and CTL partitioning was studied at 25?C and
37?C. The results from measurements at 37?C are shown
in Fig. S2 and Fig. S3 in the Supporting Material. As ex-
pected, the addition of PSM increased the affinity of CTL
for the lipid bilayers both at 25?C and 37?C. Hence, it seems
that CTL responds to changes in bilayer composition simi-
larly as cholesterol.
To further test how the affinity of the sterol is affected by
lipid bilayer composition, we measured CTL partitioning
between mbCD and DLPC, DTPC, and DMPC bilayers at
25?C and 37?C, i.e., in fluid bilayers composed of saturated
acyl chains. The results from the experiments at 37?C are
shown in Fig. S2. The results show that the affinity of
CTL for phospholipid bilayers depend on bilayer thickness
as KXincreased at both temperatures with increasing acyl
chain length in the phospholipids.
How cholesterol interacts with phospholipids has been
shown to correlate well with the chain order in the lipid bila-
yers (23). To see whether CTL partitioning has a similar
correlation on chain order we measured DPH anisotropy in
all different lipid compositions used in the partition studies.
Fig. S3 shows the correlation between KXand the anisotropy
reported by DPH at both 25?C and 37?C, and clearly there is
a fairly good correlation between KXand DPH anisotropy.
A bonus from using CTL in the partition assay is the
recorded anisotropy values for the sterol in different phos-
pholipid environments. Hence, it is possible to evaluate
also how the order of the sterol in different bilayers correlates
with KX. From Fig. S3, it is clear that KXalso correlates with
the anisotropy of the sterol, i.e., the more ordered the bilayer
surrounding the sterol the higher its affinity for the bilayer.
In conclusion, the results from studies of CTL affinity for
different phospholipid bilayers shows that CTL senses phos-
pholipid structure similarly to cholesterol, suggesting that
results obtained with the probe can be used to evaluate
cholesterol interactions with phospholipids.
The effect of peptides on CTL partitioning
To be able to correlate the results from the partitioning assay
with the acyl chain order in the lipid bilayers with and
without peptides we measured chain order in the samples.
The effect of peptides on the acyl chain order in lipid bilayers
was determined by measuring the anisotropy of two probes,
CTL and DPH, in DMPC bilayers containing 0–7 mol %
peptide (Fig. 2). Inclusion of WALP23 led to an increased
order in the bilayers as seen from both the CTL (Fig. 2 A)
and DPH (Fig. 2 B) anisotropy. The effect of KALP23 was
much smaller, but also for this peptide the anisotropy of
both probes was increased slightly with increasing peptide
Having determined the ordering effect of the peptide, the
next step was to determine how this effect influenced sterol
affinity for the DMPC bilayer. This was done using the same
approach as for pure lipid systems, except that 0–7 mol % of
peptide was added to the DMPC bilayers. After the samples
had been incubated 2 h at 37?C, the equilibrium distribution
of CTL between LUVs and mbCD was determined from
CTL anisotropy measurements. First the effect of WALP23
was studied. Representative data are shown in Fig. 3 A,
and as can be seen in the figure an increasing amount of
WALP23 in the bilayers led to less efflux of CTL from the
bilayers. The calculated partition coefficients are shown in
Fig. 3 B. As the figure shows, KXincreased with increasing
WALP23 concentration, reaching a plateau at ~5 mol %
WALP23. This indicates that WALP23 addition increased
the affinity of sterols for the bilayer.
To get further information about how WALP23 influences
the sterol affinity for phospholipid bilayers similar experi-
ments were carried out in DLPC and POPC bilayers. The
yers. The steady-state anisotropy of (A) CTL and (B) DPH was measured in
DMPC bilayers with and without peptides at 37?C.
Effect of peptide inclusion on the acyl chain order in the bila-
Biophysical Journal 99(2) 526–533
Cholesterol-Phospholipid Interactions 529
results from these experiments showed that the influence of
WALP23 (2 or 5 mol %) on KXdepended on the phospho-
lipid composition in the bilayer (Fig. 4). In both DLPC
and POPC bilayers the addition of WALP23 lead to an
increased acyl chain order as seen from DPH and CTL
anisotropy (Fig. 4 and Fig. S4). KXincreased on inclusion
of the peptide, i.e., CTL bound stronger to the bilayer in
the presence of the peptide. However, the effect was much
smaller than with DMPC bilayers. Furthermore, there was
a smaller effect of increased acyl chain order, as probed
with DPH anisotropy, on KXin DLPC and POPC bilayers
than in DMPC bilayers. This suggests that to what degree
proteins affect sterol-phospholipid interactions depends on
The effect of KALP23 on CTL partitioning between
LUVs and mbCD was studied next. This was done in the
same way as with WALP23 and representative data is shown
in Fig. 5 A. The calculated partition coefficients are shown in
Fig. 5 B. As can be seen from the figure, the addition of
KALP23 did not increase the sterol’s affinity for the bilayer.
Instead, KALP23 inclusion led to decreased CTL partition-
ing into phospholipid bilayers. By doing sucrose gradient
centrifugation experiments, as described by Killian et al.
(29), we insured that this was not due to nonhomogenous
incorporation of KALP23 in DMPC bilayers (results not
shown). Hence, we can conclude that addition of peptides,
mimicking the transmembrane helices in proteins, to phos-
pholipid bilayers can have either a positive or a negative
effect on the affinity of sterols for the bilayers.
CTL in LUVs (μM)
bilayers and mbCD at 37?C. (A) Representative data showing the effect of
WALP23 on the partitioning of CTL between DMPC bilayer and mbCD.
(B) Summary of the calculated partition coefficients.
Influence of WALP23 on sterol partitioning between DMPC
acyl chain order in WALP23 containing bilayers. The anisotropy of DPH
and partitioning of CTL was measured in DMPC, DLPC,and POPC bilayers
in the presence of 0, 2, or 5 mol % WALP23 at 37?C.
Relationship between measured partition coefficients and the
CTL in LUVs (μM)
bilayers and mbCD at 37?C. (A) Representative data showing the effect of
KALP23 on the partitioning of CTL between DMPC bilayer and mbCD.
(B) Summary of the calculated partition coefficients.
Influence of KALP23 on sterol partitioning between DMPC
Biophysical Journal 99(2) 526–533
530Nystro ¨m et al.
Cholesterol interactions with phospholipid
CTL has been shown to be the fluorescent cholesterol analog
that mimics cholesterol best (27). For example it was re-
ported that CTL orders the surrounding lipid acyl chains
and interacts with phospholipids similarly as cholesterol.
Therefore, CTL has been used in studies of cholesterol en-
riched domains (30,31). In this study, we have further char-
acterized CTL-phospholipid interactions by measuring the
equilibrium portioning of the probe between phospholipid
vesicles and mbCD. The first apparent difference between
cholesterol and CTL that we observed was that CTL parti-
tioned more into mbCD than cholesterol. For example, KX
at 37?C with pure POPC bilayers was 6.5 mM for CTL
whereas it has been reported to be 30–47 mM for cholesterol
(21–23). This finding was not surprising because it has been
shown that rate of CTL efflux from monolayers is markedly
higher than that of cholesterol (28). We think that this effect
is mostly due to differences in sterol-mbCD interactions, and
not so much dependent on sterol-phospholipid interactions.
To compare how CTL and cholesterol interacts with phos-
pholipids one must compare how the sterol partitioning
behavior is affected by changes in phospholipid composi-
tion. For this purpose, we studied how CTL partitioning
was affected by inclusion of PSM into POPC bilayers. It
has been reported previously that the addition of 50 mol %
PSM to POPC bilayers increases KXfor cholesterol three
to four times at 37?C (21,24). For CTL the KXwas increased
approximately six times by PSM addition at the same
temperature. The higher relative partition coefficient could
mean that CTL has a higher affinity for PSM containing bila-
yers than cholesterol, but we think that the increase was due
to a markedly lower sterol concentration (2 mol %) in the
bilayers than in the cholesterol studies (15–30 mol %).
This presumption is based on observations by Tsamaloukas
and co-workers regarding sterol content and partitioning
behavior (24). Addition of 33 mol % PSM to POPC bilayers
increased KXfor CTL 4 times. This is closely matching what
previously has been observed for cholesterol in the same
systems (23). Further, we confirmed that CTL, like choles-
terol, has an increased affinity for phospholipid bilayers
with increasing acyl chain order. In conclusion, we consider
CTL to mimic the membrane properties of cholesterol well
enough that CTL data can be used to make predictions
regarding cholesterol partition between lipid bilayers and
Effect of transmembrane peptides on sterol
It is well known that cholesterol likes lipids that form
ordered bilayers. We showed recently that the affinity of
cholesterol for phospholipid bilayers is more or less directly
dependent on the acyl chain order in lipid bilayers (23). In
this study, we showed that CTL affinity for bilayers also
depends on the acyl chain order as seen from DPH anisot-
ropy (Fig. S2). Similar results were also obtained in a recent
molecular dynamics study of cholesterol interactions with
phospholipid bilayers (32). In this study, the area per lipid
correlates fairly well with the free energy barriers for choles-
Model peptide studies have shown that on positive hydro-
phobic mismatch, i.e., when the hydrophobic length of trans-
membrane helices exceed the hydrophobic thickness of
phospholipid bilayers, the acyl chains in lipids next to the
peptides are stretched to minimize the hydrophobic
mismatch (33). This indicates that membrane proteins can
influence the chain order in lipid bilayers, and perhaps
thereby influence the cholesterol distribution within the
bilayer. In this study, we studied how inclusion of two trans-
membrane peptides, WALP23 and KALP23, influenced the
affinity of CTL for the lipid bilayers. Both WALP23 and
KALP23 affected the chain order (as seen from CTL and
DPH anisotropy) as could be expected based on previous
studies (34). WALP23 clearly increased the chain order in
the bilayers whereas KALP23 had only a small ordering
effect on the acyl chain order (Fig. 2).
Addition of WALP23 to DMPC bilayers clearly increased
the affinity of CTL for the bilayers (Fig. 3). The affinity
increased with peptide concentration and reached a plateau
at ~5 mol % WALP23. As with the peptide free bilayers,
there was a clear correlation between the chain order in the
bilayers and the sterol affinity for the bilayer up to 5 mol %
WALP23. At higher WALP23 concentrations the partition
coefficients did not increase although the order in the bilayer
cording to both CTL and DPH anisotropy. It has been esti-
mated that the first shell of phospholipids surrounding
a WALP23 peptide is ~10 lipids/monolayer (33). Therefore,
all phospholipids in the bilayer are likely to be in contact
with WALP23 with 5 mol % of the peptide in the bilayers.
Hence, it seems that the maximum effect of WALP23 on
the CTL partitioning is reached at a concentration at which
all DMPC molecules are in contact with the peptide i.e.,
when there is at most a single shell of lipids between the
peptide transmembrane segments.
In POPC and DLPC bilayers the addition of WALP23 also
increased the acyl chain order and the affinity of CTL for the
bilayers (Fig. 4). In both POPC and DLPC bilayers the effect
of WALP23 on the chain order (as seen from DPH anisot-
ropy) was similar to that in DMPC however the effect on
KXwas small compared to that in DMPC bilayers (Fig. 4).
We conclude that if the chains are too short or unsaturated
the CTL (and cholesterol) will not interact more much favor-
ably with the lipids although WALP23 orders and stretches
the chains. This is in agreement with the general view that
cholesterol interacts favorably with phospholipids that have
long saturated acyl chains.
Biophysical Journal 99(2) 526–533
Addition of KALP23 to DMPC bilayers decreased the
affinity of CTL for the bilayers although the peptide at least
at higher concentrations had a small ordering effect on the
bilayers (Fig. 5). Although KALP23 is built up of the
same number of amino acids as WALP23, it has been shown
that it has a shorter effective hydrophobic length than
WALP23 (35). It was estimated that KALP23 has an effec-
tive hydrophobic length in between a WALP16 and
a WALP19, i.e., a 6–10 A˚shorter than WALP23. The hydro-
phobic length of KALP23 is estimated to be a close match
with the hydrophobic thickness of fluid DMPC bilayers
(36). KALP23 has also been shown induced cubic phase in
di-16:1-PE that forms bilayers that are slightly thicker than
DMPC (35). This indicates that there is negative hydro-
phobic mismatch between KALP23 and di-16:1-PE. As the
lysine side chains are thought to prefer a position no deeper
in the bilayer than the lipid phosphate region (34) it is
possible that there may be a slight negative hydrophobic
mismatch between KALP23 and fluid DMPC bilayers.
Because there should be a positive hydrophobic mismatch
between WALP23 and fluid DMPC bilayer we think that
the fact that WALP23 and KALP23 had the opposite effect
on KXis related to the difference in effective hydrophobic
length of the peptides.
The sorting of transmembrane proteins within cells have
been indicated to be dependent on the cholesterol gradient
in cellular membranes (8). It is believed that the membrane
thickening effect of cholesterol can be an important factor
that guides proteins to their destination, and cholesterol is
also known to form so called membrane rafts together with
sphingolipids. These rafts have been indicated to have
a role in lipid trafficking in cells (37).We think colocalization
of proteins and cholesterol in cell membranes could be an
important step in membrane trafficking. The fact that
peptides mimicking proteins transmembrane segments can
increase sterol affinity for a bilayer, or decrease it, indicates
that protein can attract or expel cholesterol from the phos-
pholipid environment in which they are embedded. An
importantfactor seems to be how proteins influence the chain
order, and thereby the thickness of the surrounding lipid
bilayer. As proteins that are destined for the plasma
membrane on average have longer transmembrane helices
than those that reside in the inner membrane compartments
(8) we predict that these proteins may have an ordering effect
on the Golgi membranes, and thereby may attract choles-
terol. It has been predicted that proteins with larger cross-
sectional size, i.e., several transmembrane helices, have
a larger effect on the surrounding lipid bilayer (38). Hence,
such proteins may also affect sterol affinity for bilayers
even more than single-spanning helices. The results in this
study also showed that proteins modulate the affinity of
sterols the most for phospholipids with longer saturated
acyl chains. Therefore, it seems that proteins interactions
with phospholipids can favor formation of lateral domains
enriched in saturated lipids, like sphingomyelin, and choles-
terol. This could lead to a segregation of lipids and protein in
the bilayer plane that can be an initial step in the sorting of
the membrane components.
In this study, we developed what we believe to be a new
method that can be used to measure sterol affinity for phos-
pholipid bilayers. The method uses the fluorescent choles-
terol analog CTL and we showed that the probe interacted
similarly with phospholipids as cholesterol. This method
was then used to study how peptides mimicking the trans-
membrane helices of proteins affect the affinity of sterols
for phospholipid bilayers. We showed that peptides could
both increase and reduce sterol affinity for phospholipid bila-
yers, and that the degree to which peptides affected sterol
affinity also depended on the lipid composing in the bilayers.
The fact that the sterol affinity for the bilayers was altered by
peptides suggests that proteins can affect the lateral organiza-
tion of cholesterol in cell membrane. It is likely that this
influence of proteins on lipid organization can play a role
in the trafficking of lipids and proteins within cellular
The authors thank J. Peter Slotte and Bodil Westerlund for fruitful discus-
sion and J. Peter Slotte for access to excellent laboratory facilities.
This study was supported by the Academy of Finland, the Sigrid Juselius
Foundation, Medicinska Understo ¨dsfo ¨reningen Liv och Ha ¨lsa R.F., the
Magnus Ehrnrooth Foundation, and the Otto Malm Foundation.
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