Letter pubs.acs.org/JPCL
Hydrostatic Pressure Promotes Domain Formation in Model Lipid Raft Membranes David L. Worcester†,§ and Michael Weinrich*,†,‡ †
NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg 20899, Maryland, United States Eunice Kennedy Shriver National Center of Child Health and Human Development, National Institutes of Health, 31 Center Drive, Bethesda 20892, Maryland, United States § Department of Physiology and Biophysics, University of California, Irvine 92697, California, United States ‡
S Supporting Information *
ABSTRACT: Neutron diffraction measurements demonstrate that hydrostatic pressure promotes liquid-ordered (Lo) domain formation in lipid membranes prepared as both oriented multilayers and unilamellar vesicles made of a canonical ternary lipid mixture for which demixing transitions have been extensively studied. The results demonstrate an unusually large dependence of the mixing transition on hydrostatic pressure. Additionally, data at 28 °C show that the magnitude of increase in Lo caused by 10 MPa pressure is much the same as the decrease in Lo produced by twice minimum alveolar concentrations (MAC) of general anesthetics such as halothane, nitrous oxide, and xenon. Therefore, the results may provide a plausible explanation for the reversal of general anesthesia by hydrostatic pressure.
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anesthesia, we investigated the effects of hydrostatic pressure on a canonical Lo/Ld domain forming lipid mixture. The results show that hydrostatic pressure is very effective in producing Lo domain formation and reversing lipid mixing. The Clapeyron equation dP/dT = ΔH/TΔV (where P is pressure, T is temperature, ΔH is the change in enthalpy, and ΔV is the change in volume) can be used to describe pressure changes of the mixing transition. The results demonstrate surprisingly large values of dT/dP over the broad temperature range of the transition. Neutron diffraction was used to study oriented multilayers of dipalmitoylphosphatidylcholine (DPPC)/dioleoylphosphatidylcholine (DOPC)/cholesterol (2/2/1 molar ratio) in D2O at pressures up to 50 MPa. At atmospheric pressure, two diffraction peaks, corresponding to Lo and Ld domains, were easily measured for both first and second orders of diffraction at temperatures at or below 31 °C, as in previous work.3,4 Samples were fully hydrated in D2O, which provided high contrast with the undeuterated lipids and large structure factors for the first and second orders. Higher diffraction orders are reduced by D2O and intensity was insufficient to readily observe these. After allowing the sample to anneal overnight, the temperature was gradually raised until the two first order peaks became one peak at 34 °C. Hydrostatic pressure of 10.3 MPa resulted in broadening of this first-order diffraction peak, with a small
nhomogeneity in biological membranes has gained considerable interest with the observations that cholesterol-rich regions of plasma membranes selectively incorporate certain membrane proteins and thereby regulate membrane processes such as transport and signaling.1 Model ternary lipid membranes of unsaturated and saturated phospholipids mixed with cholesterol exhibit coexisting liquid ordered (Lo) and disordered (Ld) domains and are thought to resemble properties of mammalian plasma membranes. The formation and nature of the domains have been extensively studied to establish domain sizes and phase diagrams based on composition and temperature.2 The demixing transitions occur over an unusually broad range of temperatures, spanning 10 °C or more. This is in distinct contrast with the sharp main transition (gel to Ld) of single phospholipids. We have recently shown that hydrophobic compounds, including halothane, nitrous oxide, and xenon, which are used for inhalation anesthesia, promote mixing and shift the demixing transition by several degrees to lower temperatures.3,4 Thus, inhalation anesthetics increase lipid mixing and decrease the amount of Lo (raft) phase. While much work has focused on inhalation anesthetics as ligands which bind directly to protein ion channels,5 there is still no satisfactory explanation or consensus for the mechanisms of general anesthesia and the remarkable effect of hydrostatic pressure in reversing general anesthesia. Pressures above 10 MPa (∼100 atm) reverse general anesthesia in tadpoles6 and mice;7 however, changes of enzyme activities or protein structural changes require pressures at least an order of magnitude greater.8,9 Because hydrostatic pressure reverses © XXXX American Chemical Society
Received: September 25, 2015 Accepted: October 22, 2015
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DOI: 10.1021/acs.jpclett.5b02134 J. Phys. Chem. Lett. 2015, 6, 4417−4421
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The Journal of Physical Chemistry Letters shoulder, but gave clear separation of second-order diffraction peaks. With increasing pressure, both the first- and secondorder diffraction peaks became increasingly separated (Figures 1a,b). This was immediately reversible upon lowering the pressure. Effects of pressure were measured at 23, 28, 31, and 34 °C to span the temperature range of the mixing transition. For small changes in the unit cell dimension, the change in ratio of first-order peak areas (Lo/Ld) approximates the change in the ratio of the mass in the two domains and is decreased by both increasing temperature and application of anesthetics.3,4 As illustrated in Figure 1c,d, ratios for both first- and secondorder peaks decrease with temperature but increase monotonically with increasing pressure at all temperatures. For both firstand second-order ratios, comparing equal ratios at different temperatures gives (dT/dP) ≈ − (0.2 °C/MPa). Another way to evaluate the effects of pressure on raft forming lipids is by comparison with the effects of inhalation anesthetics. The linear fits to the first order ratios at 28 and 31 °C in Figure 1c give an increase of 17.6 and 25% in Lo/Ld for a 10 MPa change in pressure, respectively. This is much the same (in magnitude) as the decrease in Lo/Ld given by our previous data 3,4 for the effects of twice a minimum alveolar concentration of the general anesthetics halothane, nitrous oxide, and xenon. For example, xenon and nitrous oxide produced 15 and 17% reductions in Lo/Ld for 2 MAC, respectively, at 28 °C (from figure 3a,b in ref 4). At each temperature and pressure, d spacings were determined by linear fits using first- and second-order peak positions and the Bragg equation for at least two separate measurements. To place the d spacings in context, they are plotted in Figure 2a together with data redrawn from earlier neutron diffraction pressure studies on DPPC-cholesterol and egg phosphatidylcholine (EPC) multilamellar vesicles.10 In the previous work, the d spacings for DPPC-cholesterol and EPC increase by ∼0.1 Å per 10 MPa. The pressure-induced changes in d spacing for the DPPC/DOPC/cholesterol system are larger (0.2 to 0.4 Å per 10 MPa), which reflects changes in the composition of the domains (demixing) that was not present in the one- and two-component samples of the previous work. The d spacing for the Ld domain in the present work actually decreases (−0.04 to −0.2 Å per 10 MPa) with pressure. Because pure Ld bilayers increase in thickness with pressure10 it is concluded that the observed decrease in d spacing for Ld domains in ternary mixtures reflects the transfer of certain lipids, DPPC, and cholesterol, out of the Ld domains. Because d spacings as well as peak intensities were affected by pressure, membrane density profiles were calculated by Fourier synthesis for each temperature and pressure to determine the bilayer thicknesses of the domains.11 Figure 2b displays these bilayer thicknesses as a function of pressure. The slopes are very similar to the slopes for d spacings. Increase in bilayer thickness with increasing pressure is a distinctive feature of many lipid bilayer membranes. In these cases, bilayer compressibility normal to the membrane is negative and results from large, positive in-plane compressibility. Lipid molecular areas decrease with pressure, and closer molecular packing therefore results in increased bilayer thickness. In ternary lipid mixtures such as studied here, demixing and domain formation or growth also occurs, resulting in large thickness increase for liquid-ordered domains and a distinctive thickness decrease (rather than just smaller increase) for liquid-disordered domains. Demixing effects are therefore dominant and the symmetry between the Lo and Ld data in Figure 2a,b largely reflects the reciprocal
Figure 1. (a) First-order Bragg diffraction peaks from DOPC/DPPC/ cholesterol (2/2/1 molar ratio) multilayers on silicon substrate in D2O at 34 °C. Neutron wavelength is 5 Å. The momentum transfer (q) is normal to the plane of the multilayers. Intensity is total neutron counts. Pressures are relative to atmospheric pressure (1 MPa ≈ 10× atmospheric pressure). There is only one peak at atmospheric pressure and 34 °C. As pressure increases, the peak broadens until clear separation occurs at 20 MPa. The first peak corresponds to the liquid ordered phase and the second peak corresponds to the liquid disordered phase. (b) Second-order Bragg diffraction peaks for the same system. Increasing separation between the peaks with increasing 4418
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normal to the bilayer planes.12 This is a remarkable feature at high hydration and must be due to interbilayer interactions that depend on area per lipid so that 2D domains of like composition (e.g., Lo and Lo) will stack and form homogeneous 3D domains.13 Diffraction techniques will not readily detect different domains without such stacking, and this could be the case when domains become small. To investigate whether the data from multilayers reflect a physiologically relevant effect that can occur in single membranes, we used small-angle neutron scattering (SANS) to measure the effect of hydrostatic pressure on mixing/ demixing transitions in small unilamellar vesicles (SUVs). To detect domains in the SUVs we employed the contrast matching technique first developed and employed by Pencer14 for SANS studies of lipid domains. The DPPC in the lipid mixture had both fatty acid chains deuterated, and the D2O/ H2O mixture was selected to have the same neutron scattering length density as the lipid mixture when fully mixed at 33 °C so that the scattering intensity at this temperature is nearly minimum; however, domains smaller than ∼10 nm are hard to detect by SANS,15 and physiological domains may be smaller than this. As demixing occurs at lower temperatures, contrast with both types of domains increases, giving increased scattering. Figure 3a displays the raw intensity data obtained from vesicles at 25 °C. As pressure is increased from atmospheric pressure to 31 MPa, there is a corresponding increase in scattering, indicating the growth of two distinct domains with different scattering length densities. Quantification of the domain separation is best accomplished using the scattering invariant Q,14,16,17 Q = ∫ Iq2 dq, and under match conditions is a function of the relative proportions of the two domains.17 The relationship between temperature, pressure, and the scattering invariant Q is illustrated in Figure 3b. At atmospheric pressure and 31 °C, detection of separate domains is barely visible and the signal grows progressively larger as temperature is decreased, corresponding well to previous SANS measurements on this mixture.16 In fact, our measurements reproduced the data of ref 16 almost exactly. At 25 °C measurements at different pressures demonstrate the same antagonism between temperature and pressure, as seen in the multilayer experiment. There is a nonlinear increase in the scattering invariant Q up to 10 MPa. But at 21 and 31 MPa we see a fairly constant relationship between temperature and pressure: − 1 °C ≈ 4 MPa, according to which, increasing the pressure by 4 MPa produces the same change in the invariant scattering function as a 1 °C reduction in temperature. Thus, measurements on the mixing transition of both the multilayer and unilamellar vesicles give values of dT/dP of −0.2 to −0.3 °C/MPa. These values are in the same range as those observed for the gel to liquid crystal transition for DPPC18 and other saturated phospholipids.19 We chose the lipid mixture for the current study because it is widely used as a model lipid raft mixture and because it has a broad mixing transition with coexisting Lo and Ld phases through a wide temperature range.2 Values of dT/dP for the gel to liquid crystal transition in saturated phospholipids change very little with the acyl chain length,19 and the mixing transitions of different ternary raft forming mixtures are qualitatively similar between the different mixtures, albeit with different transition midpoints.20 By the Clapeyron equation, the similar values of dT/dP mean that the ΔV/ΔS values are similar, as expected if there are similar
Figure 1. continued pressure is more evident than in the first order peaks. (c) Ratios of the areas of first-order Bragg peaks as a function of pressure at four temperatures. Dotted lines are drawn to guide the eye. Solid lines are linear fits (Supporting Information). At 34 °C and atmospheric pressure, the ratio of Lo to Ld is zero. (d) Ratios of the areas of the second-order Bragg peaks as a function of pressure at four temperatures.
Figure 2. (a) d spacing for the Lo and Ld phases as a function of pressure for different temperatures. Error bars are smaller than the heights of the symbols. Solid lines are linear fits. Dotted lines are drawn to guide the eye. Dashed lines are redrawn from ref 10 for a DPPC/cholesterol mixture and egg phosphatidylcholine (EPC), as indicated on the graph. Note that the d spacing for the Lo phase increases with increasing pressure more steeply than that of the DPPC/cholesterol binary mixture, while the spacing for the Ld phase actually decreases with increasing pressure. The nonlinear portion of the trace at 34 °C makes clear that this unusual decrease in d spacing is matched by a corresponding increase in the Lo phase, reflecting a transfer of lipid between these two phases. (b) Widths of the Lo and Ld bilayer lipid phases from Fourier reconstructions as a function of pressure and temperature.
compositional changes for the two domains, which result from transfer of certain lipids from one domain to the other. This conclusion is confirmed by the SANS experiment described below. Measurements of Bragg peaks corresponding to different compositional domains in multilayers require that 2D domains in individual bilayers stack in a domain-homogeneous way 4419
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domains3,4 have physiological significance. There is strong evidence of the association of signaling proteins, including some ion channels, with lipid rafts,1,29 and lipid rafts have been shown to be involved in the organization of neurotransmitter receptor complexes on postsynaptic membranes,30−33 including those involved in nociception.33 Rapid trafficking of glutamatergic receptors in and out of the region of post synaptic density is well established and appears crucial for the regulation of excitatory synapses.34−36 One implication of the pressure results reported here, combined with our3,4 and other’s37 demonstrations of the effects of anesthetics on liquid-ordered (raft) domains, is that inhalation anesthetics could disperse postsynaptic receptors, which are normally gathered at synapses where they are available to released neurotransmitters. By dispersal, receptors are effectively inhibited because less neurotransmitter is available to them outside the synaptic region. Receptor dispersal would act in addition to receptor inhibition by direct binding of anesthetic to receptors. Thus, these two mechanisms may work together to diminish synaptic transmission. The former is expected to be responsible for pressure reversal of anesthesia because pressure reversal of the latter has been difficult to demonstrate38 (see also Note in Supporting Information). It is concluded that a plausible explanation of the phenomenon of pressure reversal of general anesthesia is provided by lipid Lo domain formation and that Lo domain dissolution is a mechanism of anesthetic action that works in addition to receptor inhibition by direct anesthetic binding. This is based on the physical properties of complex lipid mixtures that are significant for the lipid/protein mixtures of biological membranes, especially at synapses.
Figure 3. (a) Small-angle neutron scattering intensity versus q for D62-DPPC/DOPC/cholesterol (2/2/1 molar ratio) unilamellar vesicles in D2O/H2O (45%) at 25 °C matched to the scattering length for vesicles at atmospheric pressure at 33 °C: ▼, 0 MPa; ▲, 10 MPa; ●, 21 MPa; ■, 31 MPa. Detector at 5 m, neutron wavelength 6 Å. (b) Scattering invariant Q (∫ Iq2 dq) for the SANS data as a function of pressure and temperature. Dotted line is drawn to guide the eye through the points at 0 MPa. The arrow indicates ΔT, while ΔP is the MPa value at 25 °C and the ratio of these values gives dT/ dP, assuming linearity. The dT/dP values thus obtained are 0.26, 0.24, and 0.20 °C/MPa, from top to bottom.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.5b02134. Detailed materials and methods, coefficients to the linear fits to Figures 1 and 2, phase diagram, and notes. (PDF)
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AUTHOR INFORMATION
Corresponding Author
volume/disorder changes in the hydrocarbon regions in both cases. Previous investigators used differential scanning calorimetry and electron spin resonance to examine the main transitions of pure, saturated phospholipids18 and binary mixtures of saturated phospholipids with cholesterol21 as well as mixing transitions in binary mixtures of saturated phospholipids which differ in fatty acid chain lengths by at least four carbons.22 Studies also examined the antagonistic effects of temperature and pressure on membrane fluidity, for which dT/dP is the change in temperature required to offset the effects of a change in pressure. Such studies of synaptic and myelin fractions of goldfish brain23 and liquid crystalline lipid bilayers24 gave dT/ dP values in the range of 0.13 to 0.21 °C/MPa. Several groups25−28 previously observed antagonistic effects of pressure and anesthetics on transitions between gel and liquid crystal states in these model systems. Extending these studies to more physiological systems requires unsaturated as well as saturated phospholipids and in place of gel states, Lo domains. Our demonstrations that hydrostatic pressure of physiologically relevant magnitude reverses domain mixing in both multilayers and unilamellar vesicles of a ternary lipid raft mixture, suggest that anesthetic effects found for lipid
*Phone 301-402-4201. E-mail
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Science Foundation and the NIST Center for Neutron Research for access to neutron scattering facilities and Juscellino Leao for providing and helping with pressure equipment. We thank Boualem Hammouda and Jun Liu for help with SANS. Support to D.L.W. was provided by U.S. National Institutes of Health Grant GM 86685 (to Stephen H. White). We thank Dr. Sergey Bezrukov for useful discussions. The identification of any commercial product or trade name does not imply any endorsement or recommendation by the National Institute of Standards and Technology.
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REFERENCES
(1) Lingwood, D.; Simons, K. Lipid Rafts as a Membrane-Organizing Principle. Science 2010, 327, 46−50. (2) Feigenson, G. W. Phase Diagrams and Lipid Domains in Multicomponent Lipid Bilayer Mixtures. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 47−52. 4420
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(24) Chong, P. L.; Weber, G. Pressure Dependence of 1,6-Diphenyl1,3,5-Hexiatriane Flourescence in Single-Component Liposomes. Biochemistry 1983, 22, 5544−5550. (25) Kamaya, H.; Ueda, I.; Moore, P. S.; Eyring, H. Antagonism Between High Pressure and Anesthetics In The Thermal PhaseTransition of Dipalmitoyl Phosphatidylcholine Bilayer. Biochim. Biophys. Acta, Biomembr. 1979, 550, 131−7. (26) Mountcastle, D. B.; Biltonen, R. L.; Halsey, M. J. Effect of Anesthetics and Pressure on the Thermotropic Behavior of Multilamellar Dipalmitoylphosphatidylcholine Liposomes. Proc. Natl. Acad. Sci. U. S. A. 1978, 75, 4906−10. (27) Trudell, J. R.; Hubbell, W. L.; Cohen, E. N.; Kendig, J. J. Pressure Reversal of Anesthesia: The Extent of Small-Molecule Exclusion From Spin-Labeled Phospholipid Model Membranes. Anesthesiology 1973, 38, 207−11. (28) Trudell, J. R.; Payan, D. G.; Chin, J. H.; Cohen, E. N. The Antagonistic Effect of an Inhalation Anesthetic and High Pressure on the Phase Diagram of Mixed Dipalmitoyl-Dimyristoylphosphatidylcholine Bilayers. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 210−3. (29) Head, B. P.; Patel, H. H.; Insel, P. A. Interaction of Membrane/ Lipid Rafts with the Cytoskeleton: Impact on Signaling and Function: Membrane/Lipid Rafts, Mediators of Cytoskeletal Arrangement and Cell Signaling. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 532−45. (30) Delint-Ramirez, I.; Fernandez, E.; Bayes, A.; Kicsi, E.; Komiyama, N. H.; Grant, S. G. In Vivo Composition of NMDA Receptor Signaling Complexes Differs Between Membrane Subdomains and is Modulated By PSD-95 and PSD-93. J. Neurosci. 2010, 30, 8162−70. (31) Delint-Ramirez, I.; Salcedo-Tello, P.; Bermudez-Rattoni, F. Spatial Memory Formation Induces Recruitment of NMDA Receptor and PSD-95 to Synaptic Lipid Rafts. J. Neurochem. 2008, 106, 1658− 68. (32) Swulius, M. T.; Farley, M. M.; Bryant, M. A.; Waxham, M. N. Electron Cryotomography of Postsynaptic Densities During Development Reveals a Mechanism of Assembly. Neuroscience 2012, 212, 19− 29. (33) Pristera, A.; Okuse, K. Building Excitable Membranes: Lipid Rafts and Multiple Controls on Trafficking of Electrogenic Molecules. Neuroscientist 2012, 18, 70−81. (34) Choquet, D.; Triller, A. The Dynamic Synapse. Neuron 2013, 80, 691−703. (35) Huganir, R. L.; Nicoll, R. A. AMPARs and Synaptic Plasticity: The Last 25 Years. Neuron 2013, 80, 704−17. (36) Renner, M.; Choquet, D.; Triller, A. Control of the Postsynaptic Membrane Viscosity. J. Neurosci. 2009, 29, 2926−37. (37) Gray, E.; Karslake, J.; Machta, B. B.; Veatch, S. L. Liquid General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles. Biophys. J. 2013, 105, 2751−9. (38) Franks, N. P. Molecular Targets Underlying General Anaesthesia. Br. J. Pharmacol. 2006, 147 (Suppl 1), S72−81.
(3) Weinrich, M.; Nanda, H.; Worcester, D. L.; Majkrzak, C. F.; Maranville, B. B.; Bezrukov, S. M. Halothane Changes the Domain Structure of a Binary Lipid Membrane. Langmuir 2012, 28, 4723−8. (4) Weinrich, M.; Worcester, D. L. Xenon and Other Volatile Anesthetics Change Domain Structure in Model Lipid Raft Membranes. J. Phys. Chem. B 2013, 117, 16141−7. (5) Franks, N. P.; Lieb, W. R. Molecular Mechanisms of General Anaesthesia. Nature 1982, 300, 487−93. (6) Johnson, F. H.; Flagler, E. A. Hydrostatic Pressure Reversal of Narcosis in Tadpoles. Science 1950, 112, 91−2. (7) Lever, M. J.; Miller, K. W.; Paton, W. D.; Smith, E. B. Pressure Reversal of Anaesthesia. Nature 1971, 231, 368−71. (8) Boonyaratanakornkit, B. B.; Park, C. B.; Clark, D. S. Pressure Effects on Intra- and Intermolecular Interactions Within Proteins. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 2002, 1595, 235− 49. (9) Girard, E.; Marchal, S.; Perez, J.; Finet, S.; Kahn, R.; Fourme, R.; Marassio, G.; Dhaussy, A. C.; Prange, T.; Giffard, M.; Dulin, F.; Bonnete, F.; Lange, R.; Abraini, J. H.; Mezouar, M.; Colloc’h, N. Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure. Biophys. J. 2010, 98, 2365−73. (10) Braganza, L. F.; Worcester, D. L. Structural Changes in Lipid Bilayers and Biological Membranes Caused Hydrostatic Pressure. Biochemistry 1986, 25, 7484−8. (11) Worcester, D. L.; Franks, N. P. Structural Analysis of Hydrated Egg Lecithin and Cholesterol Bilayers. II. Neutron Diffraction. J. Mol. Biol. 1976, 100, 359−78. (12) Mills, T. T.; Tristram-Nagle, S.; Heberle, F. A.; Morales, N. F.; Zhao, J.; Wu, J.; Toombes, G. E.; Nagle, J. F.; Feigenson, G. W. Liquid-Liquid Domains in Bilayers Detected by Wide Angle X-Ray Scattering. Biophys. J. 2008, 95, 682−90. (13) Tayebi, L.; Ma, Y.; Vashaee, D.; Chen, G.; Sinha, S. K.; Parikh, A. N. Long-Range Interlayer Alignment Of Intralayer Domains In Stacked Lipid Bilayers. Nat. Mater. 2012, 11, 1074−80. (14) Pencer, J.; Mills, T.; Anghel, V.; Krueger, S.; Epand, R. M.; Katsaras, J. Detection of Submicron-Sized Raft-Like Domains in Membranes by Small-Angle Neutron Scattering. Eur. Phys. J. E: Soft Matter Biol. Phys. 2005, 18, 447−58. (15) Petruzielo, R. S.; Heberle, F. A.; Drazba, P.; Katsaras, J.; Feigenson, G. W. Phase Behavior and Domain Size in SphingomyelinContaining Lipid Bilayers. Biochim. Biophys. Acta, Biomembr. 2013, 1828, 1302−13. (16) Masui, T.; Urakami, N.; Imai, M. Nano-Meter-Sized Domain Formation in Lipid Membranes Observed by Small Angle Neutron Scattering. Eur. Phys. J. E: Soft Matter Biol. Phys. 2008, 27, 379−89. (17) Pencer, J.; Mills, T. T.; Kucerka, N.; Nieh, M. P.; Katsaras, J. Small-Angle Neutron Scattering to Detect Rafts and Lipid Domains. Methods Mol. Biol. 2007, 398, 231−44. (18) Albon, N.; Sturtevant, J. M. Nature of the Gel to Liquid Crystal Transition of Synthetic Phosphatidylcholines. Proc. Natl. Acad. Sci. U. S. A. 1978, 75, 2258−60. (19) Ichimori, H.; Hata, T.; Matsuki, H.; Kaneshina, S. Barotropic Phase Transitions and Pressure-Induced Interdigitation on Bilayer Membranes of Phospholipids with Varying Acyl Chain Lengths. Biochim. Biophys. Acta, Biomembr. 1998, 1414, 165−74. (20) Veatch, S. L.; Keller, S. L. Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol. Biophys. J. 2003, 85, 3074−3083. (21) Shimshick, E. J.; McConnell, H. M. Lateral Phase Separations in Binary Mixtures of Cholesterol and Phospholipids. Biochem. Biophys. Res. Commun. 1973, 53, 446−51. (22) Mabrey, S.; Sturtevant, J. M. Investigation of Phase Transitions of Lipids and Lipid Mixtures by Sensitivity Differential Scanning Calorimetry. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 3862−6. (23) Chong, P. L.; Cossins, A. R.; Weber, G. A Differential Polarized Phase Fluorometric Study of the Effects of High Hydrostatic Pressure Upon the Fluidity of Cellular Membranes. Biochemistry 1983, 22, 409−15. 4421
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