Biophysical Journal Volume 100 June 2011 2633–2641 2633

Sterols Have Higher Affinity for Sphingomyelin than forPhosphatidylcholine Bilayers even at Equal Acyl-Chain Order

Max Lonnfors,† Jacques P. F. Doux,‡ J. Antoinette Killian,‡ Thomas K. M. Nyholm,† and J. Peter Slotte†*†Biochemistry, Department of Biosciences, Abo Akademi University, Turku, Finland; and ‡Biochemistry of Membranes, Bijvoet Center forBiomolecular Research, Utrecht University, Utrecht, The Netherlands

ABSTRACT The interaction between cholesterol and phospholipids in bilayer membranes is important for the formationand maintenance of membrane structure and function. However, cholesterol does not interact favorably with all types of phos-pholipids and, for example, prefers more ordered sphingomyelins (SMs) over phosphatidylcholines (PCs). The reason for thispreference is not clear. Here we have studied whether acyl-chain order could be responsible for the preferred sterol interactionwith SMs. Acyl-chain order was deduced from diphenylhexatriene anisotropy and from the deuterium order parameter obtainedby 2H-NMR on bilayers made from either 14:0/14:0(d27)-PC, or 14:0(d27)-SM. Sterol/phospholipid interaction was determinedfrom sterol bilayer partitioning. Cholestatrienol (CTL) was used as a fluorescence probe for cholesterol, because its relativemembrane partitioning is similar to cholesterol. When CTL was allowed to reach equilibrium partitioning between cyclodextrinsand unilamellar vesicles made from either 14:0/14:0-PC or 14:0-SM, the molar-fraction partitioning coefficient (Kx) was approx-imately twofold higher for SM bilayers than for PC bilayers. This was even the case when the temperature in the SM samples wasraised to achieve equal acyl-chain order, as determined from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy and the deuteriumorder parameter. Although the Kx did increase with acyl-chain order, the higher Kx for SM bilayers was always evident. At equalacyl-chain order parameter (DPH anisotropy), the Kx was also higher for 14:0-SM bilayers than for bilayers made from either14:0/15:0-PC or 15:0-/14:0-PC, suggesting that minor differences in chain length or molecular asymmetry are not responsiblefor the difference in Kx. We conclude that acyl-chain order affects the bilayer affinity of CTL (and thus cholesterol), but that it isnot the cause for the preferred affinity of sterols for SMs over matched PCs. Instead, it is likely that the interfacial properties ofSMs influence and stabilize interactions with sterols in bilayer membranes.

INTRODUCTION

Cholesterol is an important fluidity and permeability modu-lator in cell membranes and also helps to form and maintaina specific lateral organization in such membranes as well asin multicomponent model membrane systems (1–4). Theinteraction of cholesterol with phospholipids plays a majorrole in the regulation of membrane fluidity and permeability(4), and for the creation of lateral structure (5–7). However,cholesterol does not appear to interact with all types ofphospholipids equally. Whereas cholesterol is able tointeract with, e.g., monounsaturated phosphatidylcholines(PCs) which are abundant in cell membranes, when givena choice it prefers to associate with saturated phospholipids(8,9). The most common saturated phospholipids in cellmembranes are the sphingomyelins (SMs). They have thesame headgroup as PCs but in this case, it is attached toa long-chain base (usually 2-amino-4-octadecene-1,3-diol)to which saturated or monounsaturated C16-C24 acyl-chains are N-linked (10).

If cholesterol can choose to interact with either acyl-matched PCs or SMs, cholesterol prefers to interact withSMs (8,11,12). Some studies have suggested that choles-terol/SM interactions could be stabilized by hydrogenbonding (13,14). However, both NMR studies (15) and

Submitted February 24, 2011, and accepted for publicationMarch 25, 2011.

*Correspondence: jpslotte@abo.fi

Editor: Heiko H. Heerklotz.

� 2011 by the Biophysical Society

0006-3495/11/06/2633/9 $2.00

recent molecular dynamics (MD) calculations have sug-gested that hydrogen bonding, although important for stabi-lizing interlipid interactions among sphingolipids, is notresponsible for cholesterol/SM association (16). Alterna-tively, it is possible that cholesterol prefers to interact withSM over an acyl-matched PC due to differences in acyl-chainorder. Cholesterol is known to preferentially interact withordered phospholipids (17,18), and indeed, the affinity ofcholesterol for binary phospholipid bilayers appears to corre-late with acyl-chain order in the bilayer, as measured from1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy (8,19).

Cholesterol’s interaction with palmitoyl SM has oftenbeen compared with its interactions with dipalmitoyl PC(20–22), because the N- and sn-2-linked acyl-chains areidentical, and because the two lipids have almost an iden-tical gel-to-liquid crystalline phase transition temperature(~41�C (23–25)). However, in the fluid phase the deuteriumorder parameter for the palmitoyl chain in the N-linked(SM) or the sn-2-linked (PC) position is very different.According to atomistic MD simulation and deuteriumNMR experiments, the SM acyl-chain is much more orderedthan the corresponding chain in PC (26,27). Because it is notfully established how much the difference in acyl-chainorder can influence the interaction of cholesterol with,e.g., SM or PC, a systematic study to resolve this questionis warranted.

doi: 10.1016/j.bpj.2011.03.066

2634 Lonnfors et al.

Thus, we have compared the affinity of cholestatrienol(cholesta-5,7,9-trien-3-b-ol; CTL) for membrane bilayerswhich are fluid, and contain either myristoyl SM or chain-matched PCs. The myristoyl chain length for SM wasselected to yield bilayers which are fluid at experimentallyfeasible temperatures. Affinity was deduced from theequilibrium-partitioning coefficient (Kx) of CTL, and wasdetermined under conditions where the bilayer acyl-chainorder was as identical as possible. Acyl-chain order wasdeduced from DPH steady-state anisotropy measurements,and from the deuterium order parameters of the N-linkedor the sn-2-linked tetradecanoate(d27) chains. Our resultsshow clearly that CTL has a significantly higher affinity toSM bilayers when compared to PC bilayers at identicalacyl-chain order. These results show that acyl-chain order,per se, does not explain the preferential interaction betweenCTL and SM.

MATERIALS AND METHODS

Highly pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),

sphingosylphosphorylcholine, 1-tetradecanoyl-2-hydroxy-sn-glycero-3-

phosphocholine, and 1-pentadecanoyl-2-hydroxy-sn-glycero-3-phospho-

choline were purchased from Avanti Polar Lipids (Alabaster, AL).

Tetradecanoyl anhydride and tetradecanoyl(d27) acid were from Sigma-

Aldrich (St. Louis, MO). Pentadecanoyl acid was from Larodan Fine

Chemicals (Malmo, Sweden). Sphingosylphosphorylcholine and lyso-

phosphatidylcholines were acylated as described previously (11,28). The

synthesized phospholipids were purified by preparative high-performance

liquid chromatography (RP C18, elution with methanol) and their

identities were verified by electrospray ionization mass spectrometry

(purity >99%).

Stock solutions of lipids were prepared in hexane/2-propanol (3/2, by

vol). All phospholipid solutions were taken to ambient temperature before

use. The concentration of all phospholipid solutions was determined by

phosphate assay subsequent to total digestion by perchloric acid (29). Stock

solutions of the phospholipids were stored in the dark at �20�C. CTL was

synthesized and purified as described previously (30,31). The identity of

CTL was positively verified by mass spectrometry. DPH was purchased

fromMolecular Probes (Leiden, the Netherlands). Probes were stored under

argon at �87�C until dissolved in argon-purged ethanol (CTL) or methanol

(DPH). The concentration of stock solutions of CTL and DPH was deter-

mined by spectrophotometry using their molar absorption coefficients (3)

values: 11,250 M�1 cm�1 at 324 nm for CTL, and 88,000 M�1 cm�1 at

350 nm for DPH. Stock solutions of fluorescent reporter molecules were

stored in the dark at �20�C and used within a week. Deuterium-depleted

water (2–3 ppm deuterium) was bought from Cambridge Isotope Laborato-

ries (Andover, MA).

All other inorganic and organic chemicals used were of the highest purity

available. The solvents used were of spectroscopic grade. Water was puri-

fied by reverse osmosis followed by passage through a UF Plus water puri-

fication system (Millipore, Billerica, MA) having final resistivity of 18.2

MU cm.

Preparation of vesicles

Multilamellar vesicles at a final lipid concentration of ~50 mM were

prepared by mixing the used lipids and probes (DPH or CTL) from organic

solvent, after which the lipids were dried under nitrogen at ~37�C, followedby exposure to high vacuum for 45 min. The dry lipids were hydrated for

20 min above the Tm for the highest melting lipid with repeated shaking.

Biophysical Journal 100(11) 2633–2641

Determination of steady-state fluorescenceanisotropy

The steady-state fluorescence anisotropy of DPH or CTL was measured on

a T-format Quanta-Master spectrofluorometer (Photon Technology Interna-

tional, Birmingham, NJ), essentially following the procedure described in

Jaikishan et al. (32). The multilamellar vesicles had the indicated lipid

compositions, with 1 mol % DPH or 2 mol % CTL. The wavelengths of

excitation and emission were 368/430 nm and 324/390 nm for DPH and

CTL, respectively. The steady-state anisotropy was calculated as described

in Lakowicz (33).

Sterol partitioning into unilamellar vesicles

The distribution of CTL between methyl-b-cyclodextrin (Sigma-Aldrich)

and extruded large unilamellar phospholipid vesicles (200-nm average

pore diameter) was determined as described previously (19,34). The assay

yields the molar fraction partition coefficient Kx, for CTL. The CTL and

thus sterol concentration was 2 mol % in all partitioning experiments. A

high Kx indicates a higher affinity of CTL for the bilayer as compared

with methyl-b-cyclodextrin.

Deuterium NMR measurement of acyl-chain order

Two to three milligrams of lipid powder was hydrated with 50 ml of deute-

rium-depleted water at 40�C for 3 h and subjected to three freeze-thaw

cycles. The samples were then transferred by centrifugation into 4 mm

o.d. ZrO2 NMR-tubes. These were closed using Kel-F (3M Company, St.

Paul, MN) inserts and caps.2H-NMR measurements were carried out on a wide-bore Ultrashield

magnet (Bruker BioSpin, Billerica,MA) of 11.7Twith anAvance III console

(Bruker BioSpin). A single-channel goniometer probe was used, tuned at

76.783 MHz. The pulse sequence was a solid echo p/2-t -p/2-t- acquisition

as described elsewhere (35), using 600-ms recycling delay, 50-ms echo

delay, and 2.5-ms pulse duration. Sweep width was 200 kHz, and acquisition

time was 4 ms. The spectra were collected with TopSpin 3 (Bruker BioSpin)

using a digitization modewhich optimizes the baseline in solid experiments.

The spectra composed of 20 k scans were multiplied with a cosine function

and zero-filled to 8192 points before Fourier transformation. The measure-

ment were done at several temperatures with a precision of 0.1�C and an

accuracy of ~1�C.The 2H spectra were dePaked using the NMR-DePaker software avail-

able from the Launchpad website (https://launchpad.net/). The Peak-Fitting

Module in the Origin 7.5 software (OriginLab, Northampton, MA) was

then used to deconvolute the dePaked spectra and to assign the peaks to

carbon atoms along acyl-chains. The quadrupolar splittings determined

from the dePaked spectra were used to calculate the order parameter,

SCD(i), according to

DyQðiÞ ¼ 3

4

�e2qQ

h

��3cos2 q� 1

�SCDðiÞ;

where e2qQ/h ¼ 167 kHz and q ¼ 0�.

RESULTS

Acyl-chain order versus temperature

The objective of this study was to compare CTL equilibriumpartitioning into fluid unilamellar PC or SM bilayers atequal bilayer acyl-chain order. Because CTL fluorescenceis very temperature-sensitive (water and oxygen-inducedquenching becomes severe above 40�C and signal intensity

FIGURE 1 DPH anisotropy versus temperature in pure phospholipid

bilayers. Steady-state DPH anisotropy was recorded as a temperature func-

tion from bilayers prepared from the indicated phospholipids (see Materials

and Methods). Each value is the average from two-to-three different

measurements (mean 5 SE). The lines shown are polynomial fits to the

data, and are given to guide the eye.

Sterol/Phospholipid Interaction 2635

decreases markedly), we had to limit the phospholipid selec-tion to species with acyl-chains that are fluid between roomtemperature and ~45�C. We chose to study 14:0/14:0-PC(Tm 24�C (36), and our data not shown) and 14:0-SM(Tm ~ 27�C, data not shown). In addition, we prepared14:0/15:0-PC and 15:0/14:0-PC (Tm 32 and 31�C, respec-tively; our data not shown, but see also (37)) to allow forcomparison of the effect of moderate changes in chainlength and molecular asymmetry in PC on CTL partitioning.

Measuring DPH steady-state anisotropy above the transi-tion temperature for vesicles prepared from each of thephospholipids, we obtained information about relativeacyl-chain order in the fluid phase and plotted it againsttemperature (Fig. 1). It can be seen that 14:0-SM was themost ordered phospholipid of the ones tested, whereas14:0/14:0-PC was the most disordered (at equal tempera-ture). 14:0/15:0-PC and 15:0/14:0-PC were intermediate inacyl-chain order. This trend is wholly expected based onthe acyl-chain composition and the respective Tm for each

A B

fully hydrated phospholipid. It is likely that the extensivehydrogen-bonding network present at the SM bilayer inter-face contribute to the stabilization of intermolecular interac-tion, thus leading to more ordered SM bilayers (13,14). PCbilayers do not have similar interlipid hydrogen-bondingnetworks.

To get more-detailed direct information about acyl-chainorder at different membrane depths, we also measured the2H-NMR spectra of perdeuterated 14:0 fatty acid linked tothe sn-2 position of 14:0/14:0-PC, or the N-linked positionof 14:0-SM. This is a more direct determination of acyl-chain order compared to the DPH anisotropy measurements.The other benefit of deuterium measurements is that infor-mation is obtained for selected segments of the acyl-chain,whereas DPH anisotropy only yields average informationfrom the hydrophobic part of the bilayer membrane. Fig. 2A shows representative 2H spectra of multilamellar lipo-somes of either 14:0/14:0(d27)-PC or 14:0(d27)-SM.

To be able to determine order parameter profiles for theSM and PC bilayers the 2H spectra were deconvolutedinto q ¼ 0� spectra through dePaking (35,38,39). The deute-rium order profile for the perdeuterated acyl-chain wascalculated from the dePaked spectra at each measuredtemperature, and data for 14:0/14:0(d27)-PC is shown inFig. 3 A. It can be seen that the order parameter of carbons2–8 are undistinguishable from the spectra, only more distalcarbons experienced increased disorder. This is consistentwith previous studies on 14:0/14:0(d27)-PC (40,41).

The order parameter profiles of 14:0(d27)-SM at differenttemperatures are shown in Fig. 3 B. The assignment of thepeaks in the 14:0(d27)-SM spectra was based on publishedresults with 16:0(d31)-SM (27), assuming that shorteningthe acyl-chain by two carbons does not affect the interfacialorientation of the acyl-chains. Except at 34�C, no clearplateau region was obtained in the order parameter profilesof 14:0(d27)-SM. Instead, the acyl-chain order appeared todecrease with carbon distance from the interface, as previ-ously observed for 16:0(d31)-SM (27). The chain order at

FIGURE 2 2H-NMR spectrum of unaligned 14:0/

14:0(d27)-PCor14:0(d27)-SM liposomes. (A)Original

measurements for PC and SM. (B) dePaked spectra

for the same samples.

Biophysical Journal 100(11) 2633–2641

FIGURE 4 Average deuterium order parameter (SCD) versus temperature

for 14:0/14:0(d27)-PC and 14:0(d27)-SM bilayers. The average order param-

eter was calculated for carbons 2–8 in PC and 3–8 in SM. The lines shown

are polynomial fits to the data, and are given to guide the eye.

FIGURE 3 Deuterium order parameter (SCD) in 14:0/14:0(d27)-PC or

14:0(d27)-SM. The calculated SCD values are given for PC (upper panel)

and SM (lower panel) as a function of carbon number.

2636 Lonnfors et al.

the interface was higher in 14:0-SM bilayers than in 14:0/14:0-PC bilayers at selected temperatures. This is quantifiedin Fig. 4. For both lipids the chain order was lowered withincreasing temperature. We can conclude that lipid orderdetermined by either DPH anisotropy or deuterium solid-state NMR yield the same results in our system.

A

B

FIGURE 5 CTL partitioning into phospholipid bilayers as a function of

DPH (A) or CTL anisotropy (B). The relative and average acyl-chain order

was determined from DPH anisotropy at different temperatures (see Fig. 1),

or from CTL anisotropy (see Materials andMethods). CTL partitioning into

phospholipid bilayers was determined at the indicated temperatures. The Kx

of CTL is plotted against DPH or CTL anisotropy. Each value is the average

from two-to-three determinations5 SE. The lines shown are linear regres-

sion fits to the data, and are given to guide the eye.

CTL equilibrium partitioning

To determine how CTL equilibrium partitioning wasaffected by bilayer acyl-chain order and phospholipidtype, we measured CTL partitioning between cyclodextrinand different unilamellar bilayers at different temperatures,and correlated the Kx with bilayer acyl-chain order.

Based on the relative bilayer acyl-chain order as reportedby DPH anisotropy, it can be seen in Fig. 5 A that the affinityof CTL to bilayers was lower for PC-containing membranesas compared with bilayers prepared from 14:0-SM atcomparable bilayer acyl-chain order. This difference in Kx

was clearly seen at different degrees of bilayer acyl-chainorder, and appeared to become more accentuated as thebilayer acyl-chain order increased (lower temperature).Changing the length of the sn-1 or the sn-2 acyl-chain inthe PC did not alleviate the difference in affinity seenbetween SM and PC, suggesting that acyl-chain match (ormismatch) was not significantly responsible for the differ-ence in bilayer affinity of CTL.

Biophysical Journal 100(11) 2633–2641

CTL orientation in bilayers is much more restricted thanDPH orientation, because the sterol is adjacent to phospho-lipid acyl-chains, and because the b-hydroxyl will be posi-tioned close to the membrane/water interface. DPH isdominantly hydrophobic and is not expected to have the

FIGURE 7 van’ t Hoff plot for CTL association with 14:0-SM and 14:0/

14:0-PC bilayers. Values shown are the mean 5 SE from two average

values determined at each indicated temperature (1/T). The lines are linear

regression fits to each set of data.

Sterol/Phospholipid Interaction 2637

same orientation in the bilayer as CTL (42). We thereforealso determined CTL anisotropy at different temperaturesand for different bilayer systems, and correlated this withCTL partitioning. When we plotted the CTL anisotropyvalues against the corresponding CTL partitioning coeffi-cient, we could clearly see that even at identical CTL anisot-ropy values, the CTL Kx was higher in 14:0-SM-containingbilayers as compared to 14:0/14:0-PC-containing bilayers(Fig. 5 B).

Finally, when CTL Kx was plotted against the deuteriumorder parameter obtained from 2H-NMR measurements, itagain became clear that at comparable acyl-chain order,the CTL Kx was markedly higher with 14:0-SM bilayersas compared to 14:0/14:0-PC bilayers (Fig. 6). CTL Kx

was plotted against the average deuterium order parameterobtained from carbon/deuterium pairs 2–8 for the PC and3–8 for the SM, because this segment is most likely involvedin interactions with adjacent sterol. Also, the difference inCTL Kx for 14:0/14:0-PC and 14:0-SM bilayers becomemarkedly more prominent as the deuterium order parameterincreased.

To further analyze the binding of CTL to 14:0-SM and14:0/14:0-PC bilayers, van’ t Hoff plots were made fromthe Kx determined at different temperatures. From the plotsshown in Fig. 7 one can see that the binding of CTL to bothphospholipid bilayers was an exothermic process (as ln K isrising with 1/T). This was expected as binding of cholesterolto both POPC and PSM bilayers has been shown to beexothermic (43). From the slope of the plots one can obtainDHo(SM) ¼ �43.5 kJ/mol and DHo(PC) ¼ �35.2 kJ/mol.From the intercepts, one obtains DSo(SM) ¼ �110.4 J/moland DSo(PC) ¼ �93.5 J/mol. This indicates that the bindingof CTL to the SM bilayers is enthalpically more favorableand entropically less favorable than CTL binding to the PC

FIGURE 6 CTL partitioning into phospholipid bilayers as a function of

bilayer acyl-chain order parameter (SCD). CTL partitioning into phospho-

lipid bilayers was determined at the indicated temperatures. The Kx of

CTL is plotted against the average SCD order parameter for carbons 2–8

(PC) or 3–8 (SM). Each value is the average from two-to-three determina-

tions 5 SE. The lines shown are linear regression fits to the data, and are

given to guide the eye.

bilayers in the examined temperature range. Hence, theobserved higher affinity of the sterol for the SM bilayersmust be due to enthalpic contributions.

DISCUSSION

The affinity of cholesterol or other sterols for phospholipidbilayers is the net result of many competing influences.Cholesterol may prefer to interact with a specific phospho-lipid (e.g., a saturated one with a large headgroup) and thesefavorable interactions would be expected to result in anincreased sterol affinity for bilayers made of such phospho-lipids (8,44,45). Cholesterol may also have an aversion forinteracting with certain phospholipids (e.g., polyunsaturatedphospholipids (46,47)), and would thus be forced to interactwith phospholipids it might not otherwise prefer. We have inthis study focused on the partitioning of CTL betweencyclodextrins and vesicles prepared from one-componentphospholipid bilayers. We wanted to determine whetherbilayer acyl-chain order was the sole determinant of sterolaffinity when sterol partitioning into PC or SM bilayerswas compared. Before discussing the results obtained forPC and SM bilayers, let us first critically discuss the exper-imental conditions used in our assays.

CTL was used as a cholesterol mimic in our study,because its fluorescence properties are useful and allowfor experimental setups not possible with, e.g., radiolabeledcholesterol (19,34,48,49). CTL is not chemically identicalto cholesterol, because its B- and C-rings contain additionaldouble bonds, which render the sterol slightly more polar(30,31). The conjugated double bonds are also likely toaffect the planarity of the sterol as compared to that ofcholesterol (50). A change in the ring-system planarityand/or polarity is likely to affect sterol/acyl-chain interac-tion in bilayers (51). However, CTL is known to partitioninto liquid-order domains, to condense phospholipidpacking, and increase acyl-chain order (19,21,34,53), thus

Biophysical Journal 100(11) 2633–2641

2638 Lonnfors et al.

showing cholesterol-like membrane effects. AlthoughCTL’s affinity for cyclodextrins and bilayer membranes ismost likely slightly different from cholesterol’s (thusaffecting absolute partitioning coefficients), it has beenshown that CTL’s relative partitioning between cyclodex-trins and bilayer membranes is comparable to that seen forradiolabeled cholesterol (19,34).We therefore concludethat results obtained for CTL are representative of thebehavior of cholesterol.

The types of phospholipids we chose for the study arecommon in cell membranes of most cells; however, themolecular species selected are only minor species(13,54,55). We chose molecular species with tetradecanoylor pentadecanoyl acyl-chains in the sn-1or sn-2 positions(PCs), or with tetradecanoyl in the N-linked position ofSM. These fairly short acyl-chains render the respectivephospholipids fluid in a temperature window (25–45�C)which is suitable with regard to the fluorescence propertiesof CTL. Water and oxygen-induced quenching of CTL fluo-rescence emission becomes excessive at elevated tempera-tures. Furthermore, 14:0/14:0-PC and 14:0-SM are alsofairly comparable molecules with regard to shape andhydrophobic length, because the 14:0 chain in sn-1 of PCis of comparable length with the long-chain base of SM,considering that the glycerol backbone in PC adds threecarbons to its effective length. The molecules used are nothighly asymmetric and are not expected to give rise to inter-digitation (56).

We can now consider how acyl-chain order in the bilayermembranes has been determined. The simple way was tomeasure DPH anisotropy in bilayer membranes as a temper-ature-function. The order of the acyl chains will affectthe dynamic behavior of DPH and this is reported in theanisotropy function. DPH anisotropy measurements havesuccessfully been used to demonstrate temperature andcholesterol-induced changes in bilayer acyl-chain order(57–61). A more direct way to determine acyl-chain orderis to measure the deuterium order parameter (SCD) of thelipids under study (62). We did this for 14:0/14:0(d27)-PCand 14:0(d27)-SM at different temperatures. Looking atFig. 4, we can, for instance, see that the 14:0(d27)-SM bilayerhad to be at a 10–12�C higher temperature than the corre-sponding 14:0/14:0(d27)-PC bilayer, to yield equal SCD(comparisons made at SCD of 0.20 or 0.19). With DPHanisotropy, equal hri was obtained in 14:0/14:0-PC and14:0-SM bilayers when the SM bilayer was at a 11–13�Chigher temperature (comparisons made at hri of 0.12 and0.10). These results suggest that both methods give qualita-tively comparable acyl-chain order results. Thus, our resultsare consistent with the conclusion by Heyn (63) that theorder parameters obtained from deuterium NMR and fluo-rescence depolarization (of DPH) show surprisingly goodcorrelation.

When the CTL partitioning was determined at differenttemperatures for bilayers containing either a PC with tetra-

Biophysical Journal 100(11) 2633–2641

decanoyl or pentadecanoyl acyl-chains in the sn-1or sn-2positions (PC), or an SM with tetradecanoyl in the N-linked,and the Kx was plotted against the DPH anisotropy function(Fig. 3), we clearly show that at equal DPH anisotropy theKx of CTL was highest for SM bilayers and lowest for14:0/14:0-PC bilayers. If a 15:0 fatty acid was linked toeither sn-1 or sn-2, the measured acyl-chain order parameterwas slightly higher (Fig. 1), and the Kx versus DPH anisot-ropy was intermediate between 14:0/14:0-PC and 14:0-SM.These results infer that:

1. CTL (and therefore cholesterol) interacts more favorablywith SM than PC at equal acyl-chain order parameter.

2. Chain length (14:0 or 15:0 in PC) only had a moderateeffect on CTL (and cholesterol) interaction and bilayersterol affinity.

Similar results were largely obtained when CTL Kx wasplotted against the CTL anisotropy (Fig. 4) or the deuteriumorder parameter (Fig. 5 A).The SCD average values used inFig. 5 A were taken from the order parameter of the acyl-chain segment most likely to interact with the sterol(carbons 2–8 in PC and 3–8 in SM). However, even ifthe order parameter of all carbons had been included inthe average, a plot very similar to Fig. 5 A would still beobtained (data not shown). It can also be noted that thedifference in Kx between PC and SM appeared to increasemarkedly when the bilayer acyl-chain order parameterincreased (temperature decreased). This difference mayresult from temperature-related effects (stabilization atdecreased temperatures) on the hydrogen-bonding networkwhich is different in SM and PC bilayers and which is likelyto affect the measured CTL partitioning coefficient.

It is clear that the higher affinity that CTL/cholesterolshows for SM bilayers as compared to PC bilayers withsimilar degree of ordering must derive from the structuraldifferences between the two phospholipids. Based on theirfunctional groups, SM will have more possibilities to formhydrogen bonds than PC. Whereas PCs can only be accep-tors for hydrogen bonding, SM can form both inter- andintramolecular hydrogen bonds with other phospholipidsand with cholesterol, a fact that also shows up in MD simu-lations (26). Intermolecular SM-SM hydrogen bonds areexpected to affect the dynamics in SM bilayers, decreasingboth lateral diffusion rates and rotational motions, which inturn should indirectly also strengthen interactions betweenSM and cholesterol. Such differences in dynamics areprobably also contributing to the observed difference inacyl-chain order between 14:0/14:0-PC and 14:0-SM.

The importance of hydrogen bonding between cholesteroland SM for their interaction has been debated (15).However, several MD simulations suggest that hydrogenbonds form between cholesterol and SM, and they wouldtherefore be expected to result in some stabilization of inter-action not seen in a comparable cholesterol/PC system. Ithas also been observed that methylation of the OH-group

Sterol/Phospholipid Interaction 2639

or the NH-function in SM almost completely abolishesfavorable interaction between sterol and SM (55). Hence,we find it likely that hydrogen bonding and interfacialinteractions contribute to the stabilization of sterol/SMassociation. In recent studies it has been observed that theaffinity of cholesterol/CTL for SM-containing bilayerswould largely be determined by the higher chain order inthese samples (8,19). In these studies the bilayers werecomposed of POPC and SM in addition to the sterol. It ispossible that the hydrogen bonding between SM and choles-terol was less important in these samples because hydrogenbonding between PC and SM may have dominated, in linewith what has been observed in MD simulations ofPOPC:PSM:cholesterol bilayers (16). In this study, wherepure SM bilayers were compared to pure PC bilayers, thehydrogen bonds between SM and cholesterol can be ex-pected to be more frequent.

Although SM and PC share a common headgroup (phos-phocholine), its dynamic and orientational properties maybe different for SM and PC. Headgroup properties areknown to markedly affect cholesterol/phospholipid interac-tion (20,44,64). In MD simulations of PSM and DPPC bila-yers, small differences in headgroup orientation have beenobserved (26). It was observed that the SM headgroup hadtwo orientational distributions of which the most commonone is tilted 15� toward the interior of the bilayer. NMRstudies of SM headgroups have also indicated that theconformation of the SM phosphocholine headgroup differsfrom that in PC (65–67). It has been suggested that thiscould lead to better shielding of sterols from water, therebymaking sterol/SM interaction more favorable (16). Thesame MD simulation study also indicated that cholesterolhave a higher tendency to form charge pairs with the phos-phocholine headgroup of SM than with PC (16).

Another possible contributing factor to the observeddifference in partitioning at equal (bulk) acyl-chain order isthe possibly different local ordering of acyl-chains by CTL.Suppose CTL orders its neighboring SM acyl-chains morethan it orders the neighboring PC acyl-chains, then this localorder would influence the partitioning process. Because it isimpossible to measure the local order, we measured insteadhow the inclusion of 33 mol % cholesterol affected DPHanisotropy in 14:0/14:0-PC and 14:0-SMbilayers at differenttemperatures but at equal average order (see Fig. S1 in theSupporting Material). At temperatures where the acyl-chainorder was equal in the absence of sterol, the ordering effect ofcholesterol was slightly higher on SM bilayers than on PCbilayers. Hence, the higher affinity of CTL for SM bilayerscould have been partially due to larger ordering effects ofthe sterol on the neighboring SM acyl-chains. However, ascholesterol displayed only a slightly larger ordering effecton SM acyl-chains, it is clear that other factors also musthave contributed to the higher affinity.

The thermodynamic parameters for CTL binding to 14:0/14:0-PC and 14:0-SM bilayers obtained from the van’ t Hoff

plots show that insertion of CTL in the bilayers is entropi-cally unfavorable. This can be explained by the increasedorder in the bilayers as a result of interactions with thesterol. The finding that CTL association with SM bilayerswas more unfavorable than with PC bilayers can be rational-ized by the observation that SM bilayers are slightly moreordered by sterol inclusion. The exothermic enthalpiesobtained from the van’ t Hoff plots show that binding ofCTL to phospholipid bilayers was enthalpically driven, asexpected based on previously reported data (43). Isothermaltitration calorimetry and molecular dynamics simulationshave previously shown that the transfer of cholesterolfrom fluid POPC to fluid SM bilayers is enthalpically driven(43,68). The transfer of CTL from 14:0/14:0-PC to 14:0-SMbilayers would also be enthalpically driven, as the bindingof CTL to 14:0-SM bilayers was more exothermic thanbinding to 14:/14:0-PC bilayers. The enthalpic gain of trans-ferring of CTL from 14:0/14:0-PC to 14:0-SM bilayerscould be due to a conformational enthalpy change but alsoto increased formation of hydrogen bonds when the sterolis moved to the SM bilayer.

We conclude that sterol/phospholipid interaction is mark-edly influenced by bilayer acyl-chain order, and that CTL orcholesterol prefers to interact with ordered phospholipids.However, when the bilayer acyl-chain order is equal, CTLand cholesterol appears to prefer interacting with SM overPC. This seems to be in part due to a larger ordering effectof sterols on SM acyl-chains, an effect that may be broughtabout by stronger interactions between SM and sterols.Stronger interactions between SM and CTL could be dueto better shielding from water by the SM headgroup andcharge pairing between this and the sterol. Hydrogenbonding between the sterol and SM is also most likely tobe an important stabilizing factor that is supported byenthalpically driven transfer of CTL from 14:0/14:0-PC to14:0-SM bilayers and the fact that, in PC:SM mixtures,the affinity of sterols was influenced by the acyl-chain order.

SUPPORTING MATERIAL

One figure is available at http://www.biophysj.org/biophysj/supplemental/

S0006-3495(11)00518-2.

This study was supported by generous grants from the Sigrid Juselius foun-

dation (to J.P.S.), Academy of Finland (to T.K.M.N.), the Foundation of

Abo Akademi University (to J.P.S.), and the Netherlands Organization for

Scientific Research (NWO-TOP grant No. 700-54-303 to J.P.F.D.).

REFERENCES

1. Yeagle, P. L. 1985. Cholesterol and the cell membrane. Biochim. Bio-phys. Acta. 822:267–287.

2. Yeagle, P. L., A. D. Albert, ., J. Frye. 1990. Cholesterol dynamics inmembranes. Biophys. J. 57:413–424.

3. Ohvo-Rekila, H., B. Ramstedt,., J. P. Slotte. 2002. Cholesterol inter-actions with phospholipids in membranes. Prog. Lipid Res. 41:66–97.

Biophysical Journal 100(11) 2633–2641

2640 Lonnfors et al.

4. Haines, T. H. 2001. Do sterols reduce proton and sodium leaks throughlipid bilayers? Prog. Lipid Res. 40:299–324.

5. Cooper, R. A. 1978. Influence of increased membrane cholesterol onmembrane fluidity and cell function in human red blood cells.J. Supramol. Struct. 8:413–430.

6. Wolf, C., K. Koumanov,., P. J. Quinn. 2001. Cholesterol favors phaseseparation of sphingomyelin. Biophys. Chem. 89:163–172.

7. Simons, K., and W. L. Vaz. 2004. Model systems, lipid rafts, and cellmembranes. Annu. Rev. Biophys. Biomol. Struct. 33:269–295.

8. Halling, K. K., B. Ramstedt,., T. K. Nyholm. 2008. Cholesterol inter-actions with fluid-phase phospholipids: effect on the lateral organiza-tion of the bilayer. Biophys. J. 95:3861–3871.

9. Bunge, A., P. Muller, ., D. Huster. 2008. Characterization of theternary mixture of sphingomyelin, POPC, and cholesterol: supportfor an inhomogeneous lipid distribution at high temperatures.Biophys. J. 94:2680–2690.

10. Barenholz, Y. 1984. Sphingomyelin-lecithin balance in membranes:composition, structure, and function relationships. In Physiology ofMembrane Fluidity. M. Shinitzky, editor. CRC Press, Boca Raton,FL. 131–174.

11. Ramstedt, B., and J. P. Slotte. 1999. Interaction of cholesterol withsphingomyelins and acyl-chain-matched phosphatidylcholines: a com-parative study of the effect of the chain length. Biophys. J. 76:908–915.

12. van Duyl, B. Y., D. Ganchev, ., J. A. Killian. 2003. Sphingomyelinis much more effective than saturated phosphatidylcholine in excludingunsaturated phosphatidylcholine from domains formed with choles-terol. FEBS Lett. 547:101–106.

13. Barenholz, Y., and T. E. Thompson. 1980. Sphingomyelins in bilayersand biological membranes. Biochim. Biophys. Acta. 604:129–158.

14. Boggs, J. M. 1987. Lipid intermolecular hydrogen bonding: influenceon structural organization and membrane function. Biochim. Biophys.Acta. 906:353–404.

15. Guo, W., V. Kurze,., J. A. Hamilton. 2002. A solid-state NMR studyof phospholipid-cholesterol interactions: sphingomyelin-cholesterolbinary systems. Biophys. J. 83:1465–1478.

16. Aittoniemi, J., P. S. Niemela, ., I. Vattulainen. 2007. Insight into theputative specific interactions between cholesterol, sphingomyelin, andpalmitoyl-oleoyl phosphatidylcholine. Biophys. J. 92:1125–1137.

17. Kucerka, N., D. Marquardt, ., J. Katsaras. 2010. Cholesterol in bila-yers with PUFA chains: doping with DMPC or POPC results in sterolreorientation and membrane-domain formation. Biochemistry.49:7485–7493.

18. Bennett, W. F., J. L. MacCallum, ., D. P. Tieleman. 2009. Molecularview of cholesterol flip-flop and chemical potential in differentmembrane environments. J. Am. Chem. Soc. 131:12714–12720.

19. Nystrom, J. H., M. Lonnfors, and T. K. Nyholm. 2010. Transmembranepeptides influence the affinity of sterols for phospholipid bilayers.Biophys. J. 99:526–533.

20. Bjorkbom, A., T. Rog, ., J. P. Slotte. 2010. Effect of sphingomyelinheadgroup size on molecular properties and interactions with choles-terol. Biophys. J. 99:3300–3308.

21. Bjorkqvist, Y. J. E., T. K. M. Nyholm,., B. Ramstedt. 2005. Domainformation and stability in complex lipid bilayers as reported by choles-tatrienol. Biophys. J. 88:4054–4063.

22. Mattjus, P., and J. P. Slotte. 1996. Does cholesterol discriminatebetween sphingomyelin and phosphatidylcholine in mixed monolayerscontaining both phospholipids? Chem. Phys. Lipids. 81:69–80.

23. Maulik, P. R., and G. G. Shipley. 1996. N-palmitoyl sphingomyelinbilayers: structure and interactions with cholesterol and dipalmitoyl-phosphatidylcholine. Biochemistry. 35:8025–8034.

24. Ramstedt, B., and J. P. Slotte. 1999. Comparison of the biophysicalproperties of racemic and d-erythro-N-acyl sphingomyelins.Biophys. J. 77:1498–1506.

25. McMullen, T. P., R. N. Lewis, and R. N. McElhaney. 1993. Differentialscanning calorimetric study of the effect of cholesterol on the thermo-

Biophysical Journal 100(11) 2633–2641

tropic phase behavior of a homologous series of linear saturatedphosphatidylcholines. Biochemistry. 32:516–522.

26. Niemela, P., M. T. Hyvonen, and I. Vattulainen. 2004. Structure anddynamics of sphingomyelin bilayer: insight gained through systematiccomparison to phosphatidylcholine. Biophys. J. 87:2976–2989.

27. Mehnert, T., K. Jacob, ., K. Beyer. 2006. Structure and lipid interac-tion of N-palmitoylsphingomyelin in bilayer membranes as revealed by2H-NMR spectroscopy. Biophys. J. 90:939–946.

28. Cohen, R., Y. Barenholz, ., A. Dagan. 1984. Preparation and charac-terization of well defined d-erythro sphingomyelins. Chem. Phys.Lipids. 35:371–384.

29. Rouser, G., S. Fleischer, and A. Yamamoto. 1970. Two dimensionalthen layer chromatographic separation of polar lipids and determina-tion of phospholipids by phosphorus analysis of spots. Lipids.5:494–496.

30. Fischer, R. T., F. A. Stephenson, ., F. Schroeder. 1984. d5,7,9(11)-Cholestatrien-3 b-ol: a fluorescent cholesterol analogue. Chem. Phys.Lipids. 36:1–14.

31. Ohvo-Rekila, H., B. Akerlund, and J. P. Slotte. 2000. Cyclodextrin-catalyzed extraction of fluorescent sterols from monolayer membranesand small unilamellar vesicles. Chem. Phys. Lipids. 105:167–178.

32. Jaikishan, S., A. Bjorkbom, and J. P. Slotte. 2010. Sphingomyelinanalogs with branched N-acyl-chains: the position of branchingdramatically affects acyl-chain order and sterol interactions in bilayermembranes. Biochim. Biophys. Acta. 1798:1987–1994.

33. Lakowicz, J. R. 1999. Principles of Fluorescence Spectroscopy. KluwerAcademic/Plenum Publishers, New York.

34. Nyholm, T. K., P. M. Grandell, ., J. P. Slotte. 2010. Sterol affinityfor bilayer membranes is affected by their ceramide content and theceramide chain length. Biochim. Biophys. Acta. 1798:1008–1013.

35. Davis, J. H. 1983. The description of membrane lipid conformation,order and dynamics by 2H-NMR. Biochim. Biophys. Acta. 737:117–171.

36. Huang, C., and S. Li. 1999. Calorimetric and molecular mechanicsstudies of the thermotropic phase behavior of membrane phospho-lipids. Biochim. Biophys. Acta. 1422:273–307.

37. Koynova, R., and M. Caffrey. 1998. Phases and phase transitions of thephosphatidylcholines. Biochim. Biophys. Acta. 1376:91–145.

38. Sternin, E., M. Bloom, and A. L. Mackay. 1983. De-Pake-ing of NMRspectra. J. Magn. Reson. 55:274–282.

39. McCabe, M. A., and S. R. Wassall. 1997. Rapid deconvolution of NMRpowder spectra by weighted fast Fourier transformation. Solid StateNucl. Magn. Reson. 10:53–61.

40. Krajewski-Bertrand, M. A., A. Milon,., G. Ourisson. 1992. The inter-action of various cholesterol ‘ancestors’ with lipid membranes:a 2H-NMR study on oriented bilayers. Biochim. Biophys. Acta.1105:213–220.

41. Petrache, H. I., S. W. Dodd, and M. F. Brown. 2000. Area per lipid andacyl length distributions in fluid phosphatidylcholines determined by2H NMR spectroscopy. Biophys. J. 79:3172–3192.

42. Pebay-Peyroula, E., E. J. Dufourc, and A. G. Szabo. 1994. Locationof diphenyl-hexatriene and trimethylammonium-diphenyl-hexatrienein dipalmitoylphosphatidylcholine bilayers by neutron diffraction.Biophys. Chem. 53:45–56.

43. Tsamaloukas, A., H. Szadkowska, and H. Heerklotz. 2006. Thermody-namic comparison of the interactions of cholesterol with unsaturatedphospholipid and sphingomyelins. Biophys. J. 90:4479–4487.

44. Huang, J., J. T. Buboltz, and G. W. Feigenson. 1999. Maximum solu-bility of cholesterol in phosphatidylcholine and phosphatidylethanol-amine bilayers. Biochim. Biophys. Acta. 1417:89–100.

45. Niu, S. L., and B. J. Litman. 2002. Determination of membrane choles-terol partition coefficient using a lipid vesicle-cyclodextrin binarysystem: effect of phospholipid acyl-chain unsaturation and headgroupcomposition. Biophys. J. 83:3408–3415.

Sterol/Phospholipid Interaction 2641

46. Brzustowicz, M. R., V. Cherezov, ., S. R. Wassall. 2002. Molecularorganization of cholesterol in polyunsaturated membranes: microdo-main formation. Biophys. J. 82:285–298.

47. Wassall, S. R., M. R. Brzustowicz, ., W. Stillwell. 2004. Order fromdisorder, corralling cholesterol with chaotic lipids. The role of polyun-saturated lipids in membrane raft formation. Chem. Phys. Lipids.132:79–88.

48. Hyslop, P. A., B. Morel, and R. D. Sauerheber. 1990. Organization andinteraction of cholesterol and phosphatidylcholine in model bilayermembranes. Biochemistry. 29:1025–1038.

49. Schroeder, F., G. Nemecz, ., T. E. Thompson. 1988. Fluorescenceproperties of cholestatrienol in phosphatidylcholine bilayer vesicles.Biophys. Chem. 32:57–72.

50. Slotte, J. P. 1992. Substrate specificity of cholesterol oxidase fromStreptomyces cinnamomeus—a monolayer study. J. Steroid Biochem.Mol. Biol. 42:521–526.

51. Rog, T., I. Vattulainen,., M. Karttunen. 2008. Comparison of choles-terol and its direct precursors along the biosynthetic pathway: effects ofcholesterol, desmosterol and 7-dehydrocholesterol on saturated andunsaturated lipid bilayers. J. Chem. Phys. 129:154508.

52. Reference deleted in proof.

53. Scheidt, H. A., P. Muller,., D. Huster. 2003. The potential of fluores-cent and spin-labeled steroid analogs to mimic natural cholesterol.J. Biol. Chem. 278:45563–45569.

54. Valsecchi, M., L. Mauri,., S. Sonnino. 2007. Ceramide and sphingo-myelin species of fibroblasts and neurons in culture. J. Lipid Res.48:417–424.

55. Blom, T. S., M. Koivusalo, ., E. Ikonen. 2001. Mass spectrometricanalysis reveals an increase in plasma membrane polyunsaturatedphospholipid species upon cellular cholesterol loading. Biochemistry.40:14635–14644.

56. McIntosh, T. J., S. A. Simon, ., C. H. Huang. 1992. Structure andcohesive properties of sphingomyelin/cholesterol bilayers. Biochem-istry. 31:2012–2020.

57. Bjorkbom, A., H. Ohvo-Rekila, ., J. P. Slotte. 2010. Characterizationof membrane properties of inositol phosphorylceramide. Biochim.Biophys. Acta. 1798:453–460.

58. Hunter, G. W., and T. C. Squier. 1998. Phospholipid acyl-chain rota-

tional dynamics are independent of headgroup structure in unilamellarvesicles containing binary mixtures of dioleoyl-phosphatidylcholine

and dioleoyl-phosphatidylethanolamine. Biochim. Biophys. Acta.1415:63–76.

59. Jaikishan, S., A. Bjorkbom, and J. P. Slotte. 2010. Phosphatidyl alco-

hols: effect of head group size on domain forming properties and inter-actions with sterols. Biochim. Biophys. Acta. 1798:1615–1622.

60. Lentz, B. R. 1993. Use of fluorescent probes to monitor molecularorder and motions within liposome bilayers. Chem. Phys. Lipids.

64:99–116.

61. Mitchell, D. C., and B. J. Litman. 1998. Molecular order and dynamicsin bilayers consisting of highly polyunsaturated phospholipids.

Biophys. J. 74:879–891.

62. Stockton, G. W., and I. C. Smith. 1976. A deuterium nuclear magneticresonance study of the condensing effect of cholesterol on egg phos-

phatidylcholine bilayer membranes. I. Perdeuterated fatty acid probes.Chem. Phys. Lipids. 17:251–263.

63. Heyn, M. P. 1979. Determination of lipid order parameters and rota-tional correlation times from fluorescence depolarization experiments.FEBS Lett. 108:359–364.

64. Huang, J., and G. W. Feigenson. 1999. A microscopic interaction

model of maximum solubility of cholesterol in lipid bilayers.Biophys. J. 76:2142–2157.

65. Malcolm, I. C., J. C. Ross, and J. Higinbotham. 2005. A study of the

headgroup motion of sphingomyelin using 31P NMR and an analyti-cally soluble model. Solid State Nucl. Magn. Reson. 27:247–256.

66. Schmidt, C. F., Y. Barenholz, and T. E. Thompson. 1977. A nuclear

magnetic resonance study of sphingomyelin in bilayer systems.Biochemistry. 16:2649–2656.

67. Siminovitch, D. J., and K. R. Jeffrey. 1981. Orientational order in

the choline headgroup of sphingomyelin: a 14N-NMR study. Biochim.Biophys. Acta. 645:270–278.

68. Zhang, Z., L. Lu, and M. L. Berkowitz. 2008. Energetics of cholesteroltransfer between lipid bilayers. J. Phys. Chem. B. 112:3807–3811.

Biophysical Journal 100(11) 2633–2641