Letter pubs.acs.org/Langmuir
Push−Pull Mechanism for Lipid Raft Formation Martin R. Krause,† Trevor A. Daly,† Paulo F. Almeida,‡ and Steven L. Regen*,† †
Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, United States Department of Chemistry and Biochemistry, University of North Carolina Wilmington, North Carolina 28403, United States
‡
S Supporting Information *
ABSTRACT: A quantitative assessment has been made of the interaction between exchangeable mimics of 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol in the liquid-ordered (l0) and the liquid-disordered (ld) states using the nearest-neighbor recognition (NNR) method. This assessment has established that these lipids mix ideally in the l0 phase (i.e., they show no net attraction or repulsion toward each other) but exhibit repulsive interactions in the ld phase. The implications of these findings for the interactions between unsaturated phospholipids and cholesterol in eukaryotic cell membranes are briefly discussed.
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INTRODUCTION The lipid raft hypothesis has emerged in recent years as a general organizing principle for the structure of eukaryotic cell membranes.1−3 Since it was first proposed, interest in developing a fundamental understanding of lipid−lipid interactions in model membranes has intensified.4 In particular, the notion that sphingolipids associate with cholesterol to form transient domains in eukaryotic cell membranes and that these domains are involved in signal transduction is gaining acceptance.3 Additionally, the belief that the liquid-ordered (l0) and liquid-disordered (ld) states are good working models of lipids rafts and the surrounding “sea” of fluid lipids, respectively, has also become popular.5 Although it is clear that “high-melting” lipids (having gel to liquid-crystalline phase -transition temperatures of Tm ≥ 37 °C) have an affinity for cholesterol in the l0 phase, whether unsaturated “low-melting” phospholipids are also capable of associating with cholesterol in the l0 or ld phases has not been firmly established. In lipid bilayers made from polyunsaturated phospholipids such as 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), neutron diffraction experiments have shown that cholesterol lies horizontally between the two monolayer leaflets, reflecting its “aversion” for a side-by-side interaction with the highly disordered, kinked acyl chains.6 However, the introduction of 50 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC), which is the major unsaturated lipid found in eukaryotic membranes, results in cholesterol having an “upright” orientation, lying parallel to the phospholipids with its hydroxyl group located at the membrane/water interface.6 Taken together, these findings indicate that cholesterol has a greater preference for becoming a nearest-neighbor of POPC than DAPC. Further analyses, based on fluorescence resonance energy transfer experiments in combination with Monte Carlo simulations as well as isothermal titration calorimetry measurements, indicate that the interaction between POPC and cholesterol is unfavorable in the ld phase.4,7 The affinity that © 2014 American Chemical Society
POPC may have for cholesterol in the l0 phase is unknown. In bilayers of POPC containing 30 mol % cholesterol, which is thought to lie in the l0 phase, the free energy of interaction was estimated to be close to zero, indicating no net attraction or respulsion.4,8−10 In contrast, a recent simulation of the l0−ld phase boundary in ternary mixtures of 1,2-distearoyl-sn-glycero3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol using pairwise interactions suggests that DOPC has an affinity for cholesterol in the l0 phase.11 Here, we provide a quantitative assessment of the interaction between exchangeable mimics of POPC and cholesterol in the l0 and ld phases using the nearest-neighbor recognition (NNR) method. To our knowledge, these are the first direct experimental measurements of the interaction between any low-melting phospholipid and sterol in the l0 and ld states. The NNR technique is a chemical method that probes lipid mixing on the molecular level.12,13 In brief, NNR measurements quantify the thermodynamic tendency of exchangeable monomers to become nearest neighbors of one another. Typically, two lipids of interest (A and B) are transformed into exchangeable dimers (homodimers AA and BB and heterodimer AB) through the introduction of a disulfide bond. The lipids are then allowed to undergo monomer interchange via thiolate−disulfide exchange (Figure 1).14 Here, the resulting equilibrium is governed by a constant, K, where K = [AB]2/ ([AA][BB]). When lipid monomers A and B mix ideally, this is revealed by an equilibrium constant that equals 4.0. Preferred homoassociations are reflected by values of K that are less than 4.0; favored heteroassociations are indicated for K > 4.0. If one takes statistical considerations into account, the nearestReceived: February 7, 2014 Revised: March 9, 2014 Published: March 12, 2014 3285
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cholesterol.13 Third, the nearest-neighbor interaction free energy between trace amounts of 1 and 2 in host membranes made from DPPC and cholesterol is indistinguishable from those found in membranes made exclusively from 1 and 2.19 Also, the temperature dependence of this free energy in both membranes is indistinguishable.19 These results indicate that the two membranes provide the same average microenvironment around 1 and 2 and that they contain similar proportions of ld and l0 phases at a given temperature. Fourth, the conversion from the ld to the l0 phase observed by NNR in membranes made exclusively from 1 and 2 occurs exactly over the same range of sterol concentrations as that found for DPPC/cholesterol, as determined by a variety of methods.13,20 This provides further evidence that the interactions between 1 and 2 are very similar to those between DPPC and cholesterol.
Figure 1. Stylized illustration showing exchangeable homodimers, AA and BB, the corresponding heterodimer, AB, and the equation that describes the dimer equilibrium.
neighbor interaction free energy between A and B is then given by ωAB = −1/2 RT ln(K/4).4 Many methods have been used to investigate cholesterol− phospholipid interactions such as differential scanning calorimetry, fluorescence resonance energy transfer, isothermal titration calorimetry, analyses of phase diagrams, and cyclodextrin-mediated partitioning. Compared to all of these methods, the NNR method has the distinct advantage of measuring lipid−lipid interactions in a direct manner that is independent of other experiments or calculations.4,15 Additionally, the NNR method is extremely sensitive and can detect interaction free energies down to tens of calories per mole. Despite the introduction of disulfide linkages and differences in headgroup composition, these exchangeable lipids have proven to be excellent mimics of their natural counterparts. For example, that fact that 1 and 2 are excellent mimics of 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol, respectively, is strongly supported by several lines of evidence (Chart 1). First, DPPC and 1, having identical acyl
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METHODS AND MATERIALS
Detailed procedures for the synthesis of 2-3 and 3-3 are described in the Supporting Information section; 2-2 was synthesized using methods similar to those previously reported.13 Thin films of lipid were prepared by evaporating a chloroform solution containing 0.30 μmol of 2-3 and varying amounts of DPPC, 4, and cholesterol under a stream of argon. (See the Supporting Information for exact compositions.) After the thin film was dried overnight under reduced pressure (0.4 mmHg), 2.0 mL of a 10 mM Tris-HCl buffer (10 mM Tris, 150 mM NaCl, 2 mM NaN3, 1 mM EDTA, pH 7.4) was added to each of the dried films. The mixtures were then vortex mixed every 5 min for 30 s over a time span of 30 min with intermittent incubation at 60 °C. The dispersions were then subjected to six freeze/thaw cycles (liquid nitrogen/60 °C water bath) and extruded 20 times through a 200-nm-pore-diameter polycarbonate filter (Nuclepore, Whatman Inc.) using argon at a pressure of ca. 100 psi. Subsequently, a 60 μL aliquot of 1.68 μM monensin in Tris-HCl buffer was added to aid in the pH equilibration across the membrane. After the liposomal dispersions (1600 μL) were heated to 45 °C and the oxygen was removed by purging with argon, thiolate−disulfide interchange reactions were initiated by adding threo-dithiothreitol (15 μL of a 19.8 mM solution in pH 7.4 Tris buffer, 1 equiv with respect to the disulfide content) and sufficient amounts of 0.1 M NaOH (50 μL) to bring the pH to 7.4 at 45 °C. Aliquots (250 μL) were withdrawn as a function of time, and the exchange reactions were quenched by adding 25 μL of 8.3 M acetic acid with vortex mixing of the test tubes containing these aliquots. The quenched aliquots were then quickly frozen using liquid nitrogen and stored at −20 °C until HPLC analysis was carried out. To each thawed aliquot was added 1000 μL of CHCl3/CH3OH (2/ 1 v/v) and aldrithiol-2 (i.e., 2,2′-dipyridyldisulfide, 37 μL of a 10 mM solution in CHCl3). The mixtures were then vortex mixed and centrifuged, and the aqueous phase was removed using a Pasteur pipet. The organic phases were concentrated under reduced pressure using a Savant SVC-100 SpeedVac concentrator equipped with a cold trap and a vacuum pump (∼1 h at 0.4 Torr). The residual lipids were dissolved in 20 μL of CHCl3 and 80 μL of the HPLC mobile phase. The samples were subsequently analyzed by HPLC using a C18 reverse-phase column. This analysis was done in an isocratic mode using a mobile phase consisting of 760 mL of ethanol, 120 mL of deionized water, 100 mL of hexane, and 10 mL of 1 M aqueous N(n-Bu)4OAc. The flow rate was 0.9 mL/min, and the column temperature was 31 °C. Detection was made at 203 nm. Values of K, where K = [2-3]/[2-2][33], were calculated from peak areas obtained from HPLC chromatograms using appropriate calibration curves.
Chart 1
chains, exhibit nearly identical gel to liquid-crystalline phasetransition temperatures (Tm), which are 41.5 and 41.9 °C, respectively, and enthalpies (ΔH), which are 8.7 and 9.3 kcal/ mol phospholipid.12 This is exactly analogous to the situation for phosphatidylcholines and phosphatidylglycerols where nearly identical melting behavior and ideal mixing is observed despite a difference in their headgroup structure and charge.16 Conversely, lipids with the same acyl chains but different headgroups, resulting in different Tm and ΔH values, do not mix ideally (e.g., phosphatidylcholines and phosphatidylethanol amines17,18). Second, sterol mimic 2 shows nearly identical monolayer behavior and condensing power to that found with
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RESULTS AND DISCUSSION To gain insight into the interaction between cholesterol and POPC, we have carried out NNR measurements using exchangeable mimics 2 and 3 in 200 nm unilamellar liposomes. As discussed previously, the cis-cyclopropyl moiety locks in a 3286
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“kink” in the acyl chain and circumvents the configurational instability associated with double bonds under NNR conditions.21 For the same reason, lipid 4 was chosen as a nonexchangeable mimic of POPC. It should be noted that the replacement of a cis double bond with a cis-cylopropyl moiety does not greatly affect the physical properties of the phospholipid. Thus, 4 has a Tm of −10 °C, which is very similar to the Tm value of −3 °C for POPC.17,22,23 Also, as demonstrated previously, 3 has monolayer properties that are nearly identical to those of POPC; both have limiting areas of ∼80 Å2/phospholipid.21 Phospholipid 4 was obtained by direct acylation of 1palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine with dihydrosterculic acid (not shown). Homodimer 3-3 was then synthesized using an improved procedure as outlined in Scheme 1. In brief, 4 was converted, enzymatically, to the
Figure 2. Plot of generalized polarization vs temperature for liposomes made from the following molar percentages of lipids: (□) DPPC/ DPPG/cholesterol, 57.5/2.5/40; (○) DPPC/DPPG/cholesterol, 95/ 2.5/2.5; (▲) 4/DPPG/cholesterol, 95/2.5/2.5; (Δ) POPC/DPPG/ cholesterol, 95/2.5/2.5. (Inset:) GP for membranes containing (▲) 4 and (Δ) POPC as a function of the mole fraction of cholesterol at ca. 40 °C.
Scheme 1
except that all of the GP values are substantially reduced. The replacement of POPC with 4 affords a very similar profile, except that the GP values are slightly higher, implying that the bilayer is slightly more compact. With increasing concentrations of cholesterol, the GP values for the POPC- and 4-based membranes increase continuously, which is expected on the basis of cholesterol’s condensing effect (Figure 2 and Supporting Information).26 Also, both membranes show a similar dependency of their GP values on the mole fraction of cholesterol present. Although a detailed phase diagram for POPC/cholesterol has not yet been firmly established, bilayers containing increasing amounts of cholesterol experience a continuous transition from the ld to the l0 phase.8−10 Using established NNR procedures, we then investigated the mixing of 2 with 3 in the ld phase with bilayers of 4 and their mixing in the l0 phase in bilayers of cholesterol-rich DPPC at 45 °C.27 In brief, we found that the mixing of 2 with 3 was virtually ideal in the l0 phase but that these same two lipids repel each other (ωAB = +160 ± 30 cal/mol) in the ld phase (Table 1). In
corresponding ethanolamine, 5.24 Subsequent condensation with di-3-thiobis[succinimidyl propionate] (DSP) afforded 3-3. The requisite heterodimer, 2-3, was prepared by condensing 5 with N-[1-(carboxyethyldithio)-2-ethyl]cholesteryl carbamate (CEDCC, not shown). Finally, homodimer, 2-2 (Tm ≈ −10 °C) was obtained using methods similar to those previously described.13 Before carrying out NNR measurements, we compared the phase behavior of bilayers made from POPC, 4, and DPPC as a function of temperature and cholesterol content. To do this, we incorporated the phase-sensitive probe, Laurdan, and determined generalized polarization (GP) values.25 Here, GP = (I440 − I490)/(I440 + I490), where I440 and I490 represent the fluorescence emission intensities at these wavelengths (λex = 350 nm). The values of GP reflect the polarity surrounding the Laurdan moiety and are sensitive to changes in phase. For each type of membrane, a small quantity (2.5 mol %) of 1,2dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) has been added to match the net negative charge that is introduced by the exchangeable phospholipid, 3, for related membranes used in NNR experiments. At low cholesterol concentration (2.5 mol %), DPPC shows the expected gel-to-fluid phase transition with a Tm of ca. 41 °C (Figure 2). When 40 mol % cholesterol is included in the bilayer, which maintains the l0 phase from 30 to 55 °C, the GP values decrease, modestly, with increasing temperature.20 Bilayers of POPC that contained 2.5 mol % cholesterol show a similar temperature profile to that of cholesterol-rich DPPC
Table 1. Nearest-Neighbor Interactions of 2 with 3a phase
cholesterol (mol %)
ldb (ld/l0)c,d (ld/l0)d,e l0f
2.5 20.0 40.0 40.0
ωAB (cal/mol)
K 2.4 2.7 3.4 4.0
± ± ± ±
0.25 0.27 0.39 0.10
160 120 52 0.0
± ± ± ±
30 33 38 7.9
a Measurements were made at 45 °C. b4/2/3 (95/2.5/2.5 mol/mol/ mol). c4/cholesterol/2/3 (77.5/17.5/2.5/2.5 mol/mol/mol/mol). d Assigned to the ld/l0 coexistence region. e4/cholesterol/2/3 (57.5/ 37.5/2.5/2.5 mol/mol/mol/mol). fDPPC/cholesterol/2/3 (57.5/ 37.5/2.5/2.5 mol/mol/mol/mol).
addition, increasing amounts of cholesterol in bilayers of 4 (i.e., 20 and 40 mol %) resulted in a steady shift toward the ideal mixing of 2 with 3, but these interactions were still unfavorable. This is in sharp contrast to the interactions between 1 and 2, which are characterized by ωAB ≈ 0 cal/mol in the ld phase and ωAB = −260 ± 6.3 cal/mol (strong association) in the l0 phase at 45 °C.27 The ideal mixing that we have found for 2 and 3 in the l0 phase and the repulsive interactions between these same two 3287
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lipids in the ld phase represents the first direct insight into the mixing of a low-melting phospholipid with a sterol in fluid bilayers. Moreover, the values of these nearest-neighbor interaction free energies are in excellent agreement with those previously determined from indirect analysis or are estimated from differences in interactions between lipid pairs. Thus, in the ld phase, where the POPC−cholesterol interaction free energy was estimated to be ωAB = +200 cal/mol (between ca. 25−37 °C), we have now found it to be +160 cal/mol at 45 °C.7 In the l0 phase, the estimate was ωAB ≈ 0, which is exactly what we have now determined.4 This agreement adds further confidence to the relevance of these NNR results because they are fully consistent with lipid−lipid interactions determined by a variety of other methods using natural phospholipids and cholesterol. Because polyunsaturated analogues have more kinks than 3, the present findings lead us to conclude that unsaturated phospholipids in general are unlikely to exhibit any net attraction for cholesterol in fluid bilayers. The repulsive interactions that we have measured between 2 and 3 in the ld phase, together with our previous finding of associative interactions between 1 and 2 in the l0 phase, provide a simple explanation of the known ability of low-melting, unsaturated phospholipids to stabilize ordered domains formed from high-melting, saturated phospholipids and cholesterol.28−30 Specifically, one now expects that low-melting phospholipids will “push” cholesterol out of disordered regions as high-melting phospholipids “pull” it into ordered domains. The larger the number of kinks that a low-melting phospholipid has, the stronger its push and the greater its effectiveness in stabilizing ordered domains.30
(2) Ahmed, S. N.; Brown, D. A.; London, E. On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 1997, 36, 10944−10953. (3) Lingwood, D.; Simons, K. Lipid rafts as a membrane-organizing principle. Science 2010, 327, 46−50. (4) Almeida, P. F. F. Thermodynamics of lipid interactions in complex bilayers. Biochim. Biophys. Acta 2009, 1788, 72−85. (5) Kaiser, H.-J.; Lingwood, D.; Levental, I.; Sampaio, J. L.; Kalvodova, L.; Rajendran, L.; Simons, K. Order of lipid phases in model and plasma membranes. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16645−16650. (6) Kucerka, N.; Marquardt, D.; Harroun, T. A.; Nieh, M.-P.; Wassall, S. R.; deJong, D. H.; Schafer, L. V.; Marrink, S. J.; Katsaras, J. Cholesterol in bilayers with PUFA chains: doping with DMPC or POPC results in sterol reorientation and membrane-domain formation. Biochemistry 2010, 49, 7485−7493. (7) Tsamaloukas, A.; Szadkowska, H.; Heerklotz, H. Nonideal mixing in multicomponent lipid/detergent systems. J. Phys.: Condens. Matter 2006, 18, S1125−S1138. (8) Veatch, S. L.; Keller, S. L. Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys. Rev. Lett. 2005, 94, 148101. (9) Zhao, J.; Wu, J.; Shao, H.; Kong, F.; Jain, N.; Hunt, G.; Feigenson, G. W. Phase studies of model biomembranes: macroscopic coexistence of L(alpha) + L(beta) with light-induced coexistence of L(alpha) + L(ordered) phases. Biochim. Biophys. Acta 2007, 1768, 2777−2786. (10) Marsh, D. Cholesterol-induced fluid membrane domains: a compendium of lipid-raft ternary phase diagrams. Biochim. Biophys. Acta 2009, 1788, 2114−2123. (11) Dai, J.; Alwarawrah, M.; Ali, M. R.; Feigenson, G. W.; Huang, J. A molecular view of the cholesterol condensing effect in DOPC lipid bilayers. J. Phys. Chem. B 2011, 115, 1662−1671. (12) Kristovitch, S. M.; Regen, S. L. Nearest-neighbor recognition in phospholipid membranes: a molecular-level approach to the study of membrane suprastructure. J. Am. Chem. Soc. 1992, 114, 9828−9835. (13) Sugahara, M.; Uragami, M.; Yan, X.; Regen, S. L. The structural role of cholesterol in biological membranes. J. Am. Chem. Soc. 2001, 123, 7939−7940. (14) Bang, E.-K.; Lista, M.; Sforazzini, G.; Sakai, N.; Matile, S. Poly(disulfide)s. Chem. Sci. 2012, 3, 1752−1763. (15) Niu, S.-L.; Litman, B. J. Determination of membrane cholesterol partition coefficient using lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition. Biophys. J. 2002, 83, 3408−3415. (16) Findlay, E. J.; Barton, P. G. Phase behavior of synthetic phosphatidylglycerols and binary mixtures with phosphatidylcholines in the presence and absence of calcium ions. Biochemistry 1978, 17, 2400−2405. (17) Marsh, D. Handbook of Lipid Bilayers, 2nd ed.; CRC Press: Boca Raton, FL, 2013. (18) Huang, C.; Wang, Z. Q.; Lin, H. N.; Brumbaugh, E. E.; Li, S. Interconversion of bilayer phase transition temperatures between phosphatidylethanolamines and phosphatidylcholines. Biochim. Biophys. Acta 1994, 1189, 7−12. (19) Zhang, J.; Cao, H.; Jing, B.; Almeida, P. F.; Regen, S. L. Cholesterol-phospholipid association in fluid bilayers: a thermodynamic analysis from nearest-neighbor recognition measurements. Biophys. J. 2006, 91, 1402−1406. (20) Sankaram, M. B.; Thompson, T. E. Cholesterol-induced fluidphase immiscibility in membranes. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8686−8690. (21) Jing, B.; Tokutake, N.; McCullough, D. H., III; Regen, S. L. A quantitative assessment of the influence of permanent kinks on the mixing behavior of phospholipids in cholesterol-rich bilayers. J. Am. Chem. Soc. 2004, 126, 15344−15345. (22) Cevc, G., Ed. Phospholipids Handbook; Marcel Dekker: New York, 1993.
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CONCLUSIONS This new physical principle, the push−pull mechanism, whereby cholesterol is pushed away from low-melting phospholipids and pulled toward high-melting lipids, must also be operating in biological membranes. Thus, the present findings, together with previous results from indirect analyses, establish a fundamentally new role that unsaturated phospholipids must play in eukaryotic cell membranes: to support the formation of lipid rafts through repulsive interactions with cholesterol.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures and characterization data. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the National Science Foundation (CHE-1145500).
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REFERENCES
(1) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature 1997, 387, 569−572. 3288
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(23) Koynova, R.; Caffrey, M. Phases and phase transitions of the phosphatidylcholines. Biochim. Biophys. Acta 1998, 1376, 91−145. (24) Akoka, S.; Meir, C.; Tellier, C.; Belaud, C.; Poignant, S. Synthesis of N15 enriched phospholipids by transphosphatidylation. Synth. Commun. 1985, 15, 101−107. (25) Parassi, T.; DiStefano, M.; Loiero, M.; Ravagnan, G.; Gratton, E. Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe. Biophys. J. 1994, 66, 120−132. (26) Hung, W. C.; Lee, M. T.; Chen, F. Y.; Huang, H. W. The condensing effect of cholesterol in lipid bilayers. Biophys. J. 2007, 92, 3960−3967. (27) Turkyilmaz, S.; Almeida, P. F.; Regen, S. L. Effects of Isoflurane, halothane and chloroform on the interactions and lateral organization of lipids in the liquid-ordered phase. Langmuir 2011, 27, 14380− 14385. (28) Bakht, O.; Pathak, P.; London, E. Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterolcontaining ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms. Biophys. J. 2007, 93, 4307−4318. (29) Wassall, S. R.; Brzustowicz, M. R.; Shaikh, S. R.; Cherezov, V.; Caffrey, M.; Stillwell, W. Order from disorder, corralling cholesterol with chaotic lipids: the role of polyunsaturated lipids in membrane raft formation. Chem. Phys. Lipids 2004, 132, 79−88. (30) Shaikh, S. R.; Cherezov, V.; Caffrey, M.; Stillwell, W.; Wassall, S. R. Interaction of cholesterol with a docosahexaenoic acid-containing phosphatidylethanolamine: trigger for microdomain/raft formation? Biochemistry 2003, 42, 12028−12037.
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