ARTICLE pubs.acs.org/Langmuir
Regulating the Size and Stabilization of Lipid Raft-like Domains and Using Calcium Ions as Their Probe Or Szekely, Yaelle Schilt, Ariel Steiner, and Uri Raviv* The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, 91904 Jerusalem, Israel
bS Supporting Information ABSTRACT: We apply a means to probe, stabilize, and control the size of lipid raft-like domains in vitro. In biomembranes the size of lipid rafts is ca. 1030 nm. In vitro, mixing saturated and unsaturated lipids results in microdomains, which are unstable and coalesce. This inconsistency is puzzling. It has been hypothesized that biological line-active surfactants reduce the line tension between saturated and unsaturated lipids and stabilize small domains in vivo. Using solution X-ray scattering, we studied the structure of binary and ternary lipid mixtures in the presence of calcium ions. Three lipids were used: saturated, unsaturated, and a hybrid (1-saturated2-unsaturated) lipid that is predominant in the phospholipids of cellular membranes. Only membranes composed of the saturated lipid can adsorb calcium ions, become charged, and therefore considerably swell. The selective calcium affinity was used to show that binary mixtures, containing the saturated lipid, phase separated into large-scale domains. Our data suggests that by introducing the hybrid lipid to a mixture of the saturated and unsaturated lipids, the size of the domains decreased with the concentration of the hybrid lipid, until the three lipids could completely mix. We attribute this behavior to the tendency of the hybrid lipid to act as a line-active cosurfactant that can easily reside at the interface between the saturated and the unsaturated lipids and reduce the line tension between them. These findings are consistent with a recent theory and provide insight into the self-organization of lipid rafts, their stabilization, and size regulation in biomembranes.
’ INTRODUCTION Lipid domains in biological membranes, often called rafts, have a typical diameter of 1030 nm.1,2 Unless stabilized by proteins, the rafts are transient3 and form due to the lateral segregation of different lipids, driven by their intermolecular interactions. The composition4 and thickness58 of the domains differ from that of the membrane matrix. In vivo, rafts are rich in sphingolipids, saturated phospholipids, and cholesterol. The lateral order in the rafts is higher than in the surrounding bilayer, typically composed of unsaturated lipids with phosphatidylcholine (PC) headgroups.5,914 Sphingolipids and cholesterol, often found in rafts, are essential for the biological functions of raft-embedded proteins.9 The rafts serve as relay stations in intracellular signaling3,9 and play a role in apical protein transport. They also ensure specificity and fidelity during signal transduction9 and are conceived as part of a mechanism for the intracellular trafficking of lipids and lipidanchored proteins.12,14 Other cellular processes involving lipid rafts include bacterial and viral cell targeting,15 insulin-stimulated glucose transport,16 stabilization of microtubules,17 axon growth and guidance,18 cell apoptosis,19 and amyloid-β oligomerization.20 Model membrane studies provide insight into the possible behavior of lipid rafts in cells.21 Binary mixtures containing an unsaturated lipid with a low melting temperature, Tm, and a saturated lipid with a high Tm usually do not form nanoscale liquid domains.22 They may form, however, domains of a solidlike gel phase (i.e., solid-ordered or Lβ phase), which coexist with domains of a fluid phase23 (i.e., liquid-disordered or Lα phase). r 2011 American Chemical Society
The line tension at the interface between the lipids results from membrane curvature and chain packing mismatch,24,25 which separates these domains. To form nanoscale rafts, the interfacial line tension should be close to zero.7 Without stabilizing agents, nanoscopic and microscopic domains are unstable and eventually coalesce into phase-separated macroscopic domains to reduce the interfacial line tension.6,7,2426 The saturated lipid 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) and the unsaturated lipid 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC) phase separate.22 Examples of other phase-separating lipid mixtures, their domain size, and probing methods are given in section 1 of the Supporting Information. Theory predicts27 that a line-active component in cell membranes can stabilize domains of a particular size over biologically useful time scales. A hybrid lipid, containing a saturated and an unsaturated tails, may act as a line-active component at saturated/unsaturated interfaces. The hybrid lipid 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) is therefore likely to preferentially go to the interface between the two coexisting bulk phases in a DOPC/DPPC mixture and lower the interfacial line tension, without affecting the properties of the lipid molecules within the domains. This line tension reduction is expected because the saturatedunsaturated tail orientation of Received: August 8, 2011 Revised: October 26, 2011 Published: November 08, 2011 14767
dx.doi.org/10.1021/la203074q | Langmuir 2011, 27, 14767–14775
Langmuir
ARTICLE
Figure 1. An illustration of the arrangement of the lipids in the binary and ternary mixtures. The unsaturated lipid, DOPC, is blue, the saturated lipid, DPPC, is red, and the hybrid lipid, POPC, is half red and half blue. In the binary mixtures the lipids phase separate. POPC can sit at the interface between the DPPC-rich domain and the DOPC-rich domain. When POPC is added, the domain size decreases (middle cartoon). When the molar ratio between the saturated, unsaturated, and the hybrid lipid crosses 1:1:1 (left cartoon), the three lipids can completely mix. The relative size of the different lipids and the way they are incorporated in the membrane are based on our interpretation of the findings presented in this paper.
POPC at the interface is energetically favorable, and hence sufficient to overcome the entropy loss in bringing the hybrid lipid molecules to the interface, from a state of mixing.27 The predicted domain sizes under those conditions range from tens to hundreds of nanometers.28 In our earlier study,29 we demonstrated, in agreement with previous studies,3032 that Ca2+ ions preferentially adsorb onto dipolar membranes composed of saturated zwitterionic (PC) lipids such as DPPC. The adsorbed ions charge the initially neutral membranes, and their lamellar repeat distance, D, increases from ca. 6 nm to a significantly larger distance, determined by the concentration of the added salt. The Ca2+ affinity of the unsaturated lipid DOPC or the hybrid lipid POPC is low, and their D barely changes when CaCl2 is added. The PC headgroup has a dipole moment of ca. 619 D (depending on its orientation3335). Saturated lipids adsorb the ions readily as they have a smaller area per headgroup, and by adsorbing the Ca2+ ions there is a net gain of ion-dipole interactions, with little entropic cost.29 The unsaturated and hybrid lipids have larger areas per headgroup, and their headgroups can freely rotate about their own location. The adsorption of Ca2+ ions limits this free rotation, costs entropy, and is therefore less favorable than the adsorption onto saturated lipids. In the present study, we use the selectivity of Ca2+ binding and solution small-angle X-ray scattering (SAXS) to investigate how the concentration of the hybrid lipid POPC controls the size of domains in binary mixtures of the segregating lipids DOPC and DPPC. We used the different binding affinities29 of Ca2+ ions to the three lipids to probe the structure of the different lipidic populations in the ternary mixtures as a function of their composition. Using this unique and noninvasive approach, our findings suggest that POPC stabilizes and regulates the size of lipid raft-like domains in vitro and can even dissolve the domains
and ideally mix the three lipids when its concentration is sufficiently high (Figure 1). This study is the experimental support for the hypothesis that line-active cosurfactants reduce the interfacial line tension between lipid domains in vitro. It is possible that a similar mechanism operates in vivo. In the future, we hope to improve our model by studying the effect of cholesterol that is an important component of cellular membranes. DPPC has its biological correspondence in sphingolipids, which are the high melting temperature lipids that predominate membrane ordered domains in cells. Our method should be easily used to study domain formation in mixtures containing, for example, sphingomyelin instead of DPPC.
’ MATERIALS AND METHODS Highly purified water (Barnstead Nanopure Diamond) with resistivity of 18.1 MΩ 3 cm and total organic compounds (TOC) of 1 ppb or less was used for preparing solutions. Chloroform (assay 99.8%) was purchased from J.T. Baker (Holland). Salt solutions were prepared by diluting a 1 M standard CaCl2 salt solution, purchased from SigmaAldrich Co. (St. Louis, MO). Lyophilized DOPC, DPPC, and POPC (>99% pure, according to the manufacture data) were used without further purification (Avanti Polar Lipids, Inc., Alabaster, AL). Phospholipid mixture solutions at total lipid concentration of 15 mg/(mL of salt solution), with 5 or 10 mM CaCl2, were prepared29 and measured using solution SAXS setups36 and then analyzed using the software X+ developed in our laboratory,37,38 as described in our earlier studies29,3638 and in sections 2 and 3 of the Supporting Information. The lipids were first mixed in chloroform in glass vials, transferred into the quartz capillary used for the SAXS measurements, or remained in the glass vials. The samples were then dried (in the capillaries or in the glass vials), and CaCl2 solutions were added to the dried lipid. Samples in glass vials were vortex mixed and then transferred into the quartz capillaries. The capillaries were flamed-sealed and centrifuged at 6000g at 25 °C, 14768
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir
ARTICLE
for 90 min, using a SIGMA 1-15PK centrifuge with a capillary rotor. No matter how the samples were prepared, and lipid pellets were obtained, toward which the X-ray beam was aligned (see section 2 in the Supporting Information). The 2D scattering patterns were radially integrated.39 The scattering intensity, I, as a function of the magnitude of the momentum transfer vector, q, was then fit to a form-factor model of a stack of infinite flat uniform slabs with varying electron density to describe the electron density distribution of the lamellar stack, where τhead is the headgroup layer thickness and τtail is the carbon chain region thickness (Figure S1). Details about this model were given elsewhere.29,38 The structure factor peaks revealed the repeat distance, D, of the lamellar phases (see section 3 of the Supporting Information).
’ RESULTS Mixing the unsaturated DOPC and the saturated DPPC lipids, at ambient room temperature, results in their phase separation.7,24,25 As a consequence, there is an interface between the two phases and that interface carries an energy cost, which is the line tension that develops at the interface. The hybrid lipid, POPC, has a saturated tail that is identical to those of DPPC and an unsaturated tail that is identical to those of DOPC. POPC could therefore act as a line-active surfactant and reduce the line tension between the immiscible lipids, thereby considerably shrinking the domain size of the two phases. Theory27,28 suggests that under certain conditions the hybrid lipid may facilitate complete mixing of the three lipids. To experimentally test this hypothesis, we took advantage of the different adsorption affinities of DOPC, POPC, and DPPC to Ca2+ ions. In our earlier study,29 we demonstrated that in the presence of CaCl2 (at concentration of 1 mM or more) DPPC membranes adsorb Ca2+ ions readily and the membrane behaves as a charged membrane. The adsorption of Ca2+ ions results in a multilamellar phase that swells to a large repeat distance, D. For example, for DPPC in pure water, D = 6.3 ( 0.1 nm, but in the presence of 5 mM CaCl2, D swells to 26 ( 2 nm. DOPC and POPC membranes do not adsorb these ions (at the salt concentrations used here); thus, their D values, determined by the balance between van der Waals attraction, hydration, and undulation repulsions, remain at 6.2 ( 0.2 nm for DOPC and 6.3 ( 0.2 nm for POPC, under those salt concentrations. Phase Separation in Binary Lipid Mixtures. Taking advantage of the differences in the D values of the different lipids when CaCl2 is added, we started by preparing binary mixtures of DOPC and DPPC at different DOPC molar fractions, χDOPC. We then added 5 or 10 mM CaCl2 solutions to the mixtures and measured D, using SAXS. Figure 2 shows the resulting D values as a function of χDOPC. Each data set corresponds to the average of several repeated measurements of the same sample at different times and of similar samples prepared and measured at different times. The error bars are thus statistical errors. The data clearly show the coexistence of two phases, which are most likely composed of a DOPC-rich phase and a DPPCcalcium-complexrich phase, indicated as P1 and P2 in Figure 2, respectively. Charged membranes repel each other, and their lamellar phase swells in pure water or in salt solutions with Debye screening length, λD, larger than ca. 1 nm. D of charged membranes increases monotonically when the lipid volume fraction decreases.40,41 At low χDOPC, the D of the P2 phase (dominated by the DPPC calcium complex that forms a charged membrane) increases when mixed with DOPC. When χDOPC is greater than ca. 0.2, D does not change, within the scatter. We attribute the initial increase in D to
Figure 2. Repeat lamellar spacing, D, as a function of DOPC molar fraction, χDOPC, of binary DOPCDPPC mixtures, in 5 mM (solid symbols) and 10 mM (open symbols) CaCl2 solutions. In all the mixtures the final total lipid concentration was 15 mg/(mL of salt solution) that is ca. 1.5 wt % lipid, and the samples were prepared in a glass vial. The lipid mixture microphase separates into two coexisting lamellar phases. P1 is the lamellar phase dominated by DOPC, and P2 is the lamellar phase dominated by the DPPCcalcium complex, as appears from the corresponding single component phases. Error bars are statistical errors.
the decrease in the DPPC volume fraction, ϕDPPC, as χDOPC increases (note that the total lipid volume fraction, ϕ, remains constant). On the other hand, when ϕDPPC decreases, the amount of free Ca2+ ions increases; hence, λD shortens and acts to lower D. When χDOPC is greater than ca. 0.2, the two opposing effects nearly balance each other; hence, D weakly depends on the binary mixture composition. If P2 were to contain some DOPC, the membrane charge density would have decreased and this could lower D. The observed increase in D suggests that already from low χDOPC DOPC and DPPC barely mix, in agreement with earlier reports, claiming that the two lipids do not even occupy the same vesicle.24,25,4244 At very low or very high χDOPC the two components are expected to be miscible due to the entropy of a small amount of one phase in the other. Phase separation is therefore observed at a finite range of concentration and not at the lowest (0.9) χDOPC. We then mixed DPPC with the hybrid lipid, POPC. POPC does not adsorb Ca2+ ions in the conditions used in this study.29 Thus, the lamellar repeat distance, D, of POPC should remain small at 6.3 ( 0.2 nm. Figure 3 shows the D values of the two coexisting phases as a function of the POPC molar fraction, χPOPC. There is a slight decrease in the D of the P1 phase, which is shown (Figure S2) and discussed on an expanded scale in section 4 of the Supporting Information. The data suggest that there is some mixing of POPC and DPPC in the P1 phase. We attribute the rise of the mean bilayer thickness, when the molar fraction of DPPC increases, to an incomplete insertion of DPPC into the POPC bilayer, due to their size mismatch. There is a slight increase in the lamellar repeat distance of the P2 phase that is most likely dominated by DPPC, which adsorbs the Ca2+ ions. Above χPOPC = 0.2, within the scatter, D does not vary with χPOPC. This observation indicates, as explained above, that the two lipids hardly mix at 0.2 e χPOPC e 0.9. 14769
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir
Figure 3. D as a function of POPC molar fraction, χPOPC, of binary POPCDPPC mixtures, in 5 mM (solid symbols) and 10 mM (open symbols) CaCl2 solutions. The final total lipid concentration in all the mixtures was 15 mg/(mL of salt solution), and the samples were prepared in a glass vial. Two distinct lamellar phases coexist. Based on the single-component phases, P1 is the lamellar phase dominated by POPC and P2 is the lamellar phase dominated by the DPPCcalcium complex.
Figure 4. D as a function of χPOPC, of ternary mixtures, prepared in capillaries, with 5 mM (solid symbols) or 10 mM (open symbols) CaCl2 solutions. The DOPC:DPPC molar ratio was fixed at 1:2 in (a) and at 1:4 at (b), and the amount POPC was adjusted as needed. The final total lipid concentration in all the mixtures was 15 mg/(mL of salt solution).
Ternary Mixtures of POPC, DPPC, and DOPC. DPPC and DOPC phase separate (Figure 2) due to the difference in the structure and packing of their tails, which leads to line tension at the interface between domains. Theroy27,28 suggests that the hybrid lipid, POPC, however, can sit between the domains and act as a line tension reducing agent because of its hybrid character. The following experiments examined this concept. We mixed DPPC and DOPC, at a fixed molar ratio, with different amounts of the hybrid lipid POPC and added 5 or 10 mM CaCl2 solutions to the lipid mixture. Figure 4 shows the measured D values, as a function of the POPC molar fraction, χPOPC, of the two phases at constant DOPC:DPPC molar ratio of
ARTICLE
1:2 and 1:4. Figure S4 in section 5 of the Supporting Information presents the D values of the two phases at a constant DOPC: DPPC molar ratio of 1:1, in the two preparation methods (vials and capillaries, see Materials and Methods), as a function of χPOPC. The ternary mixtures separated into two lamellar phases: membranes that did not adsorb Ca2+, hence their D values were small (the P1 phase), and membranes that adsorbed Ca2+ ions, hence their D values were significantly larger (the P2 phase). Figure S3 in section 4 of the Supporting Information demonstrates that the ternary P1 phases have different lipid compositions than the pure lipid phases or the binary P1 phases. The samples that were prepared in glass vials were vortex mixed and are expected to be closer to equilibrium. Yet, the samples prepared directly in quartz capillaries (Figure S4a) exhibited the same behavior as the samples in vials (Figure S4b), suggesting that flow of ions and water was free and the system was most likely at or near equilibrium. No matter how the ternary mixtures were prepared, D of the P2 phase was similar and decreased monotonically with χPOPC, at a given DOPC:DPPC molar ratio. The molar fractions of DPPC and DOPC in the binary mixtures, for which χPOPC = 0, in Figure S4 and Figure 4, correspond to the plateau region of the P2 phases in Figure 2. Under those conditions, D (of the P2 phases) did not change (at a given salt concentration) and was in agreement, within the scatter, with the binary mixture set of measurements. Nearly all the DPPC molecules that adsorbed calcium probably went to the ternary P2 phase as there was no evidence for any additional charged, DPPC containing, phase. The results of the ternary mixtures (Figure 4) are dramatically different than those of the binary mixtures (Figures 2 and 3), suggesting miscibility of the ternary mixture over a wide range of compositions due to the effect of the hybrid lipid. When the DPPC-rich P2 phase of the binary mixtures was diluted (by increasing χPOPC or χDOPC and keeping the total lipid concentration constant), its D increased and leveled off (Figures 2 and 3). In contrast, when we diluted the ternary P2 phase (containing most of the added DPPC), by adding the hybrid lipid, POPC, fixing the DOPC:DPPC molar ratio, and keeping the total lipid concentration constant (Figure 4), its D monotonically decreased with χPOPC. Even at low χPOPC the effect occurs. In section 7 of the Supporting Information we show that lowering the membrane charge density weakens the electrostatic repulsion between bilayers and decreases D. Figure 4 shows that D decreases with χPOPC. This observation suggests that the membrane charge density decreased with χPOPC. To monotonically reduce the membrane charge density of the ternary P2 phase, all the three lipids composing it and particularly a significant amount of the added POPC should have gone into the P2 phase. Because D increased (or leveled off) with increasing χPOPC in the POPCDPPC binary mixture (Figure 3), the ternary P2 phase most likely contains a significant amount of DOPC. As both DOPC and POPC do not adsorb Ca2+ ions (under the conditions of our experiments), the membrane charge density decreased when the molar fraction of POPC, in the ternary P2 phase, increased.
’ DISCUSSION Phospholipids with different saturation levels of the hydrocarbon chains phase separate (Figures 2 and 3) due to their different packing properties and line tension forms at the interface between 14770
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir
Figure 5. Fitted and estimated size parameters of the binary mixtures as a function of lipid compostion. The headgroup and tail thicknesses were fitted (symbols) using a form-factor model of a stack of infinite flat uniform slabs with varying electron density. The radially integrated solution X-ray scattering curves were fitted to the parameters of the form factor, using our analysis software X+.37 We present DOPCDPPC mixtures (a) and POPCDPPC mixtures (b), in 5 mM (solid symbols) or 10 mM CaCl2 (open symbols) solutions. The fitted solid data points are an average of samples prepared in capillaries and samples prepared in vials. The estimated structural parameters were calculated based on the pure-component parameters, assuming ideal lipid mixing (solid curves) or complete phase separation (broken curves).
the domains. The tails of DPPC are fully saturated and adopt an all-trans conformation, while each unsaturated tail of DOPC has a double bond in a cis conformation, leading to a geometrical kink, enforcing a loose tail packing. Although the hybrid lipid, POPC, has only one kinked tail, under our conditions (Figure 3) POPC phase separates from DPPC, in agreement with a grazing incidence X-ray diffraction study,45 a wide-angle X-ray scattering study,46 and differential calorimetry scanning measurements combined with 2H nuclear magnetic resonance experiments.47 In marked contrast to the binary P2 phases (the DPPC-rich phases) that their lamellar repeat distance, D, increases with decreasing χDPPC (Figures 2 and 3), D of the ternary P2 phase (Figure 4 and Figure S4) decreases with decreasing χDPPC. This observation can only be explained by a ternary interaction between the three lipids, which cannot occur in any of the binary mixtures. In the ternary mixtures the three lipids mix over a wide range of concentrations due to the effect of the hybrid lipid that reduces the line tension between the saturated (DPPC) and unsaturated (DOPC) lipids. As the hybrid and unsaturated lipids went into the ternary P2 phase and did not adsorb Ca2+ ions, the membrane charge density decreased, hence D decreased with increasing χPOPC (decreasing χDPPC) (Figure 4). Phase Separation or Mixing? Determining the membranes’ structural parameters is essential for investigating the states of the lipid mixtures. We were able to analyze both form and structure factors when Ca2+ adsorbed onto the membranes in the P2 phases, using a model of a stack of infinite flat uniform slabs with varying electron density, Iuniform bilayer (q,τhead,τtail,ΔFhead,ΔFtail), as described.29,37,38 The model parameters are the lipid headgroup thickness, τhead, the lipid tail thickness, τtail, and the corresponding electron density contrasts with respect to the solvent, ΔFhead and ΔFtail. Figure 5 presents the mean form-factor size parameters of the binary DOPCDPPC and POPCDPPC mixtures, obtained from the fit. All the points correspond to the average structural
ARTICLE
size parameters, τhead and τtail, of the two preparation methods (samples prepared in glass vials and samples prepared directly in the quartz capillary). The structural size parameters do not differ between the preparation methods (Figure S5 in section 6 of the Supporting Information). Figure S6 in section 6 of the Supporting Information presents the corresponding mean electron density of the tails and the heads in the binary mixtures. The electron densities decrease with the molar fraction of DOPC or POPC and reach a constant level above a molar fraction of ca. 0.5. The reduction in the electron density is consistent with the increase in the size of the headgroups observed in Figure 5 that we attribute to an incomplete insertion of the DOPC or POPC lipid domains into the DPPC-rich bilayers, in the P2 phase. To check if the lipid mixtures ideally mix or phase separate, we estimated the expected membrane structural parameters as follows. If the lipids uniformly mix in the bilayers, the bilayer structural parameters (both size and electron density) should be a linear combination of the pure components. For example, if we have a binary lipid mixture of DOPC and DPPC, assuming they mix well, the ideal-mixing model structural size parameters will be DOPC þ χDPPC τDPPC τmix tail ¼ χDOPC τtail tail
¼ χDOPC τDOPC þ ð1 χDOPC ÞτDPPC tail tail
ð1Þ
and DOPC τmix þ χDPPC τDPPC head ¼ χDOPC τ head head
¼ χDOPC τDOPC þ ð1 χDOPC ÞτDPPC head head
ð2Þ
mix τmix tail and τhead are the effective tail and head layer thicknesses of the mixture, in the slab model (i.e., the hydrocarbon chain lipid and the headgroup regions). τlipid tail and τhead are the corresponding pure lipid thicknesses, extracted from fitting the scattering curves of the respective pure lipids. A similar linear combination was applied for the electron density contrasts of the ideally mix mixed lipids, ΔFmix head and ΔFtail . The expected scattering intensity in this case is
uniform mix mix mix IðqÞideal_mixture ¼ Ibilayer ðq, τmix head , τ tail , ΔFhead , ΔFtail Þ
ð3Þ
The solid lines in Figure 5 show the estimated structural size parameters of each mixture composition, assuming the lipids ideally mixed. On the other hand, if the lipids phase separate, the scattering intensity (i.e., the square of scattering amplitude) is a linear combination of the scattering intensities of each phase. Although phase separation at finite temperature results in impure phases, here we use the expected scattering intensities of the pure lipid phases as bounds in our models. In the binary DOPC DPPC mixture, for example, we get uniform ðqÞ Iphase_separated_mixture uniform DOPC DOPC ðq, τDOPC , ΔFDOPC Þ ¼ χDOPC Ibilayer head , τ tail head , ΔFtail uniform DPPC DPPC DPPC þ χDPPC Ibilayer ðq, τDPPC head , τ tail , ΔFhead , ΔFtail Þ
ð4Þ
We used X+37 to calculate the form-factor model for each pure lipid separately, based on its structural parameters, obtained from the analysis of the corresponding separate measurements. We then calculated Iuniform phase_separated_mixture(q) as a linear combination of the molar fractions, for each mixture composition (eq 4). The 14771
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir
ARTICLE
calculated form factors were then fitted, using X+, to models of Iuniform bilayer (q,τhead,τtail,ΔFhead,ΔFtail) to extract the predicted structural parameters, τhead and τtail, of mixtures that completely phase separated to their pure components (Figure 5, broken curves). We realize that this assumption (eq 4) is only theoretical as at finite temperatures phase separation results in impure phases and there could be other possible phases. This situation will result in a deviation of the scattering intensity from the linear combination in eq 4. Nevertheless, our estimate captures the extreme expected limits of the system: uniform mixing versus total phase separation. The headgroup thicknesses obtained by fitting the measured scattering curves of the binary mixtures to Iuniform bilayer (q,τhead, τtail,ΔFhead,ΔFtail) (Figure 5, solid circles) deviated quite largely from both estimates. The large deviation from the solid line (ideal mixing) suggests that the binary lipid mixtures do not uniformly mix at any of the composition used in this study. For the DOPCDPPC binary mixtures (Figure 5a), there is a reasonable fit for the phase separation case (broken curve) above a DOPC molar fraction of 0.5. This suggests that in those compositions the mixture separate into phases that are nearly pure, in agreement with earlier studies.22 As noted above, at ambient room temperature the phases could not be pure for entropic reasons. At lower DOPC molar fractions, the phase separation is partial, as the data deviate from the broken curve. As suggested above, an incomplete insertion of the DOPC into the DPPC bilayer, in the P2 phase, is consistent with the observed thicknesses and electron densities. For the POPCDPPC binary mixtures (Figure 5b and Figure S5), the data fit the phase separation broken curve only at a very high POPC content (χPOPC > 0.8), and the majority of the samples behaved as the DOPCDPPC binary mixtures at χDOPC < 0.5. Both binary mixtures may well mix at very high χPOPC or χDOPC; however, the contrast between the results of Figures 2 and 3 and Figure 4, together with the results of Figure 5, suggests that this may be the case only when χPOPC or χDOPC exceeds 0.95. For comparison, we included in Figure 5 and Figure S6 the data points of the binary mixtures in 10 mM CaCl2 solutions. The structural parameters of these membranes are in agreement, within the scatter, with the parameters of the membranes that were dissolved in 5 mM CaCl2 solutions. These results show that the calcium ions weakly contribute to the electron density profiles obtained from the fit to the measured scattering curves and have immeasurable effect on the behavior of the lipids. The Ca2+ ions are therefore a noninvasive and unique probe for investigating the phase behavior of zwitterionic lipid mixtures. Our study is at ambient room temperature and in the absence of cholesterol. Nevertheless, it is of interest to note that in the presence of cholesterol, above a critical temperature, mixtures of the hybrid and saturated lipids might have finite domains because the hybrid can also act as a line-active component between itself and the saturated lipid.48 The effect of cholesterol is biologically relevant and interesting, however, beyond the scope of the current study. In Figure 6, we analyzed the ternary mixture form factors, in a similar way, to examine their mixing behavior. In the ternary mixtures, the structural parameters of the uniform mixing states are linear combinations of the pure lipids’ parameters:
Figure 6. Fitted (symbols) and calculated (curves) headgroup and tail thicknesses (obtained as in Figure 5) as a function of χPOPC of the ternary lipid mixutres in 5 (solid symbols) or in 10 mM CaCl2 (open symbols) solutions. The DOPC:DPPC molar ratios were 1:1 (a), 1:2 (b), and 1:4 (c). As in Figure 5, all the fitted data points correspond to the average of the two preparation methods (capillaries and vials). The estimated parameters were calculated based on the pure component parameters, assuming ideal lipid mixing (solid curves) or complete phase separation (broken curves).
DOPC POPC þ χDPPC τDPPC τmix head ¼ χDOPC τ head head þ χPOPC τhead
¼ χDOPC τDOPC þ χDPPC τDPPC head head þ ð1 χDOPC χDPPC ÞτPOPC head
The expected scattering intensity of the ideally mixed ternary mixtures is given by eq 3, using the size parameters of eqs 5 and 6 and the analogous electron density expressions. The scattering intensity of the mixtures that demixed was assumed to be a linear combination of the pure lipids’ scattering intensities: uniform ðqÞ Iphase_separated_mixture uniform DOPC DOPC ¼ χDOPC Ibilayer ðq, τDOPC , ΔFDOPC Þ head , τ tail head , ΔFtail uniform DPPC DPPC DPPC ðq, τDPPC þ χDPPC Ibilayer head , τ tail , ΔFhead , ΔFtail Þ
DOPC þ χDPPC τDPPC þ χPOPC τPOPC τmix tail ¼ χDOPC τ tail tail tail
uniform POPC POPC POPC þ χPOPC Ibilayer ðq, τPOPC head , τtail , ΔFhead , ΔFtail Þ
ð7Þ
¼ χDOPC τDOPC þ χDPPC τDPPC tail tail þ ð1 χDOPC χDPPC ÞτPOPC tail
ð6Þ
ð5Þ
We then analyzed the ternary mixtures data as we did for the binary mixtures. As noted above, at finite temperatures used in 14772
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir our experiments, impure phases are expected and therefore many more options could be considered. The assumption that we have a linear combination of pure phases, however, serves only to obtain an extreme theoretical limit. Note that at χPOPC = 0 both calculations (broken and solid curves in Figure 6) do not coincide with the structural parameters fitted from the scattering data. Those data points are taken from Figure 5a (DOPC DPPC binary mixtures with molar ratios of 1:1, 1:2, and 1:4), where these deviations were already observed and discussed. As the POPC molar fraction in the ternary mixture increases, the data better fit the uniform mixing state (using eq 3 with the parameters of eqs 5 and 6). The headgroup thicknesses extracted from the measurements of the ternary mixtures with constant DOPC:DPPC molar ratio of 1:1 (Figure 6a) follow the solid line (representing the uniform mixture) already when χPOPC is above ca. 0.3. For the other two series, uniform mixing takes place only when χPOPC exceeds ca. 0.5 (Figure 6b,c). If the P2 phases have a composition that is similar to the total lipid molar ratios, then ideal mixing is obtained when the molar ratios exceed 1:1:1 (Figure 6a), 1:2:3 (Figure 6b), and 1:4:5 (Figure 6c) DOPC: DPPC:POPC. The 1:1:1 molar ratio is likely to generate ideal mixing as the membrane can be composed of alternating saturated, hybrid, and unsaturated lipids (Figure 1). Under those conditions the membrane is expected to be in the liquid-disordered Lα phase. Wide-angle X-ray scattering measurements on ternary mixtures with constant DOPC:DPPC molar ratio of 1:1 (Supporting Information section 8, Figure S9b) suggest that the lateral correlation length, L, in which the lipids are in lateral positional correlation, decreased from ca. 14 ( 3 nm down to ca. 2 nm (the typical size of the Lα phase), when crossing the expected idealmixing molar ratio of 1:1:1 (or χPOPC = 1/3). At χPOPC e 1/3 the addition of POPC decreased the D spacing between membranes (Figure 4), without affecting L as, within the scatter of Figure S9b, L of the Lβ (DPPC-rich) gel phase did not alter. These results, however, are somewhat inconclusive and should be treated with care, as DOPCDPPC binary mixtures also showed a similar decrease in L when crossing χDOPC = 1/2 (Figure S9a), in contrast to the analysis in Figure 5a (see Supporting Information section 8). Complementary, high-resolution methods should be developed to directly measure the domain size of those mixtures and clarify this issue. The marked contrast between Figure 4 and Figures 2 and 3 and the analysis presented in Figures 5 and 6 suggest that the hybrid lipid, POPC, changes D in a manner that is consistent with decreasing the domain size in the ternary mixture, with increasing χPOPC. To mix well, higher χDPPC requires higher χPOPC (Figure 6). At lower χPOPC, the bilayers are likely to contain larger domains that are not well incorporated into the P2 membrane and results in bilayers with larger mean thickness and lower electron density (see Figure S7 in section 6 of the Supporting Information). The data are consistent with domain sizes that monotonically decrease with increasing χPOPC (Figure 1). Those domains, however, should be sufficiently small and well distributed to form a single charged membrane phase (P2) with a decreasing mean membrane charge density, as D monotonically decreases with χPOPC (Figure 4 and Figure S4). In contrast, D of the binary mixtures increases or levels off with decreasing χDPPC, showing that entropy alone is insufficient, under the conditions of our experiments, to mix well the DPPC lipid with neither DOPC nor POPC, in most of the range of intermediate molar ratios. In general, the expected tail thicknesses are less sensitive to the bilayer structure of the binary and ternary mixtures. Nevertheless,
ARTICLE
before the ternary mixtures ideally mix and the headgroup thicknesses are higher than the expected values, the data show that the tails are below their expected values and their electron density is somewhat higher. This result suggests that under those conditions the lipid tails do not uniformly overlap; hence, the lipids partly extend from the bilayer and appear as membranes with larger headgroups (Figure 1). Figure 6 shows that the calcium concentration weakly affects the size parameters at a given membrane composition. Yet, at higher DPPC molar fractions, the bilayers contain more adsorbed calcium ions, and the headgroups appear thicker and their electron density is higher (Figure S7). At lower DPPC molar fractions the bilayers contain less calcium ions and the headgroups go back to their original size. When the molar fraction of DPPC decreases in the membrane, the domains and hence the calcium ions are farther apart, their mean size decreases, and their mean contribution to the electron density decreases. Under those conditions, the effective membrane charge density decreases. Our data and analysis are consistent with the hypothesis that the hybrid lipid (POPC) helps to mix the otherwise phaseseparated saturated (DOPC) and unsaturated (DPPC) lipids, stabilizes the domains, and controls their size. As the amount of POPC in the system increases, it lowers the line tension in the system, and the lipids can mix well. Because POPC and DOPC weakly bind calcium ions, fewer ions adsorbed onto the membrane surfaces that contained larger amounts of POPC and DOPC, and the mean membrane surface charge density decreased. The lower membrane charge density attenuated the electrostatic repulsion and led to the decrease in the measured lamellar repeat distance, D, with increasing χPOPC (Figure 4 and Figure S4). This result is consistent with stabilization of smaller domains in the presence of the hybrid lipid POPC, which most likely sits at the interface between the saturated and unsaturated lipids (i.e., at the boundary of the raft-like domain) and lowers the line tension between them (Figure 1). To account for the measured reduction of D on the P2 phases in the ternary mixtures, we calculated the expected mean membrane charge density and free Ca2+ ion concentrations (and hence λD), based on the literature49 binding constant of calcium to DPPC, Ka = 37 M1, and assuming that the composition of the P2 membrane is similar to the total mixture composition (a rather crude assumption). Under those assumptions, we estimated the interaction curves at different membrane charge densities, as we did in our earlier study29 and as explained in section 7 of the Supporting Information. In Figure S8, we compare the minima of the interaction curves with the measured dW values, where dW = D δ, and δ is the head-to-head distance (membrane thickness) obtained from the data analysis of each mixture. The dW that corresponds to the minima of the free energy curves decreased with χPOPC. The absolute values of the estimated dw, however, were higher than the measured ones and the reduction of the estimated dW was not as steep as that of the measured values. This result indicates that the reduction of the mean surface charge density decreases the lamellar repeat distance, but the composition of the P2 phase differs from the total lipid molar composition. For instance, the DPPC molecules may exhibit different partitioning between P1 and P2, and their concentration in P2 will be lower. The lower DPPC membrane concentration results in weaker cation adsorption and a steeper decrease in the repeat distances. There could also be a disordered phase, composed of lipid vesicles and tubes, in coexistence with P1 and P2, which can apply osmotic pressure on the lamellar 14773
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir phases, causing their interlamellar spacing to decrease more than the theory predicts.29 Since we have a five-component system (salt, three types of lipids and water), the maximum number of phases, according to Gibbs’ phase rule, is 3. As we saw no evidence of a third ordered (lamellar) phase and neither of the lamellar phases ideally swelled (i.e., they did not fill out the entire space), a third phase is expected. The two lamellar phases should, therefore, coexist with a disordered lipid phase. Moreover, the charges in our membranes are nonuniformly distributed and concentrated in the DPPC-rich domains. The interaction between patchy charged surfaces is likely to be less repulsive compared with the uniformly charged surfaces in our estimates due to correlation effects.50 Finally, we should recall that all lipid mixtures contained water and CaCl2; hence, the total number of components in the mixtures was either 4 or 5. Under those conditions, both types of mixtures could assume nonhorizontal tie lines in the phase diagram. As the mixtures microphase separated we had no good way to separate the phases and measure their exact compositions. This information could provide further insight into the observed behavior of our lipid mixtures.
’ CONCLUSIONS Lipid rafts in model and biological membranes have been heavily studied. Yet, regulating the size of rafts, in vitro, remained a challenge. A unique approach for studying the phase behavior of lipid mixtures has been developed and applied to suggest how to stabilize and regulate the size of lipid raft-like domains in vitro. Using the higher affinity of Ca2+ ions to saturated (DPPC) compared with unsaturated (DOPC or POPC) dipolar membranes and solution X-ray scattering, we showed that Ca2+ ions can noninvasively probe the phase behavior and structure of saturated and unsaturated zwitterionic lipid mixtures. Our data suggest that by adding the hybrid lipid POPC to the binary mixture of DPPC and DOPC, stable small domains form. Moreover, our data analysis is consistent with the decrease of the DPPC domain size with increasing the molar fraction of POPC in dipolar ternary lipid mixtures. We attribute this behavior to the reduction in the line tension that the hybrid lipid can induce by adsorbing to the phase boundaries between the saturated and the unsaturated lipid domains, thus inhibiting large-scale phase separation in the ternary mixtures. ’ ASSOCIATED CONTENT
bS
Supporting Information. Section 1: examples of phaseseparating lipids, their domain size, and probing methods; section 2: detailed sample preparation procedures; section 3: details about the small- and wide-angle X-ray scattering measurements; section 4: analysis of the P1 phase that did not adsorb Ca2+; section 5: comparing the different preparation methods of POPC, DPPC, and DOPC ternary mixtures; section 6: the electron density profiles of the lipid mixtures; section 7: the interaction between bilayers in the ternary P2 phases; section 8: wide-angle X-ray scattering measurements. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail
[email protected], tel 972-2-6586030, fax 972-25618033.
ARTICLE
’ ACKNOWLEDGMENT We thank the SOLEIL synchrotron, SWING beamline, Elettra, 5.2 L SAXS beamline, ESRF, beamlines BM26B, and ID02 as some of the data presented in the paper were acquired there. We thank S. A. Safran for introducing us to the subject and for critical reading of this manuscript. We thank S. A. Safran, D. Harries, R. Brewster, L. Addadi, and P. A. Pincus for helpful discussions. This project was supported by the Israel Science Foundation, The Human Frontiers Science Program Organization (Career Development Award, CDA 0059/2006), the USIsrael Binational Science Foundation, the Farkash center, and the James Frank program. O.S. acknowledges support from the Samuel and Lottie Rudin Foundation fellowships. U.R. acknowledges support from Alon Fellowship for young investigators. We also thank the Safra, Wolfson, and Rudin foundations for supporting our laboratory. ’ REFERENCES (1) Pralle, A.; Keller, P.; Florin, E. L.; Simons, K.; H€orber, J. K. H. Sphingolipid-Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells. J. Cell Biol. 2000, 148 (5), 997–1007. (2) de Almeida, R. F. M.; Loura, L. M. S.; Fedorov, A.; Prieto, M. Lipid Rafts have Different Sizes Depending on Membrane Composition: A Time-resolved Fluorescence Resonance Energy Transfer Study. J. Mol. Biol. 2005, 346 (4), 1109–1120. (3) Edidin, M. Membrane cholesterol, protein phosphorylation, and lipid rafts. Sci. STKE 2001, 2001 (67), PE1. (4) Lipowsky, R. Domains and Rafts in Membranes Hidden Dimensions of Selforganization. J. Biol. Phys. 2002, 28 (2), 195–210. (5) Binder, W. H.; Barragan, V.; Menger, F. M. Domains and Rafts in Lipid Membranes. Angew. Chem., Int. Ed. 2003, 42 (47), 5802–5827. (6) Tokumasu, F.; Jin, A. J.; Feigenson, G. W.; Dvorak, J. A. Nanoscopic Lipid Domain Dynamics Revealed by Atomic Force Microscopy. Biophys. J. 2003, 84 (4), 2609–2618. (7) Kuzmin, P. I.; Akimov, S. A.; Chizmadzhev, Y. A.; Zimmerberg, J.; Cohen, F. S. Line Tension and Interaction Energies of Membrane Rafts Calculated from Lipid Splay and Tilt. Biophys. J. 2005, 88 (2), 1120–1133. (8) Akimov, S. A.; Kuzmin, P. I.; Zimmerberg, J.; Cohen, F. S. Lateral tension increases the line tension between two domains in a lipid bilayer membrane. Phys. Rev. E 2007, 75 (1), 011919. (9) Simons, K.; Ikonen, E. Functional rafts in cell membranes. Nature 1997, 387 (6633), 569–572. (10) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Lipid Rafts Reconstituted in Model Membranes. Biophys. J. 2001, 80 (3), 1417–1428. (11) Veatch, S. L.; Keller, S. L. Organization in Lipid Membranes Containing Cholesterol. Phys. Rev. Lett. 2002, 89 (26), 268101. (12) Edidin, M. The State of Lipid Rafts: From Model Membranes to Cells. Annu. Rev. Biophys. Biomol. Struct. 2003, 32 (1), 257–283. (13) Lipowsky, R.; Dimova, R. Domains in membranes and vesicles. J. Phys.: Condens. Matter 2003, 15 (1), S31–S45. (14) Lingwood, D.; Simons, K. Lipid Rafts As a Membrane-Organizing Principle. Science 2010, 327 (5961), 46–50. (15) Carrasco, M.; Amorim, M. J.; Digard, P. Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane. Traffic 2004, 5 (12), 979–992. (16) Chiang, S. H.; Baumann, C. A.; Kanzaki, M.; Thurmond, D. C.; Watson, R. T.; Neudauer, C. L.; Macara, I. G.; Pessin, J. E.; Saltiel, A. R. Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 2001, 410 (6831), 944–948. (17) Palazzo, A. F.; Eng, C. H.; Schlaepfer, D. D.; Marcantonio, E. E.; Gundersen, G. G. Localized stabilization of microtubules by integrinand FAK-facilitated Rho signaling. Science 2004, 303 (5659), 836–839. (18) Kamiguchi, H. The region-specific activities of lipid rafts during axon growth and guidance. J. Neurochem. 2006, 98 (2), 330–335. 14774
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
Langmuir (19) Takahashi, E.; Inanami, O.; Ohta, T.; Matsuda, A.; Kuwabara, M. Lipid raft disruption prevents apoptosis induced by 2-chloro-20 -deoxyadenosine (Cladribine) in leukemia cell lines. Leuk. Res. 2006, 30 (12), 1555–1561. (20) Hattori, C.; Asai, M.; Onishi, H.; Sasagawa, N.; Hashimoto, Y.; Saido, T. C.; Maruyama, K.; Mizutani, S.; Ishiura, S. BACE1 interacts with lipid raft proteins. J. Neurosci. Res. 2006, 84 (4), 912–917. (21) London, E. How principles of domain formation in model membranes may explain ambiguities concerning lipid raft formation in cells. Biochim. Biophys. Acta, Mol. Cell Res. 2005, 1746 (3), 203–220. (22) Veatch, S. L.; Keller, S. L. Separation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol. Biophys. J. 2003, 85 (5), 3074–3083. (23) Lee, A. G. Lipid phase transitions and phase diagrams II. Mixtures involving lipids. Biochim. Biophys. Acta, Rev. Biomembr. 1977, 472 (34), 285–344. (24) Lentz, B. R.; Barenholz, Y.; Thompson, T. E. Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 2. Two-component phosphatidylcholine liposomes. Biochemistry 1976, 15 (20), 4529–4537. (25) Elliott, R.; Katsov, K.; Schick, M.; Szleifer, I. Phase separation of saturated and mono-unsaturated lipids as determined from a microscopic model. J. Chem. Phys. 2005, 122 (4), 044904. (26) Yethiraj, A.; Weisshaar, J. C. Why Are Lipid Rafts Not Observed In Vivo? Biophys. J. 2007, 93 (9), 3113–3119. (27) Brewster, R.; Pincus, P. A.; Safran, S. A. Hybrid Lipids as a Biological Surface-Active Component. Biophys. J. 2009, 97 (4), 1087–1094. (28) Brewster, R.; Safran, S. A. Line Active Hybrid Lipids Determine Domain Size in Phase Separation of Saturated and Unsaturated Lipids. Biophys. J. 2010, 98 (6), L21–L23. (29) Szekely, O.; Steiner, A.; Szekely, P.; Amit, E.; Asor, R.; Tamburu, C.; Raviv, U. The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. Langmuir 2011, 27, 7419–7438. (30) Lau, A. L. Y.; McLaughlin, A. C.; MacDonald, R. C.; McLaughlin, S. G. A. The Adsorption of Alkaline Earth Cations to Phosphatidyl Choline Bilayer Membranes: A Unique Effect of Calcium. In Bioelectrochemistry: Ions, Surfaces, Membranes; American Chemical Society: Washington, DC, 1980; pp 4956. (31) Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P. Adsorption of Divalent Cations to a Variety of Phosphatidylcholine Bilayers. Biochemistry 1981, 20 (7), 1771–1777. (32) Huster, D.; Arnold, K.; Gawrisch, K. Strength of Ca2+ Binding to Retinal Lipid Membranes: Consequences for Lipid Organization. Biophys. J. 2000, 78 (6), 3011–3018. (33) Seelig, J.; Gally, H.-U.; Wohlgemuth, R. Orientation and flexibility of the choline head group in phosphatidylcholine bilayers. Biochim. Biophys. Acta, Biomembr. 1977, 467 (2), 109–119. (34) Shepherd, J. C. W.; B€uldt, G. Zwitterionic dipoles as a dielectric probe for investigating head group mobility in phospholipid membranes. Biochim. Biophys. Acta, Biomembr. 1978, 514 (1), 83–94. (35) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Preferred conformation and molecular packing of phosphatidylethanolamine and phosphatidylcholine. Biochim. Biophys. Acta, Rev. Biomembr. 1981, 650 (1), 21–51. (36) Nadler, M.; Steiner, A.; Dvir, T.; Szekely, O.; Szekely, P.; Ginsburg, A.; Asor, R.; Resh, R.; Tamburu, C.; Peres, M.; Raviv, U. Following the structural changes during zinc-induced crystallization of charged membranes using time-resolved solution X-ray scattering. Soft Matter 2011, 7 (4), 1512–1523. (37) Ben-Nun, T.; Ginsburg, A.; Szekely, P.; Raviv, U. X+: a comprehensive computationally accelerated structure analysis tool for solution X-ray scattering from supramolecular self-assemblies. J. Appl. Crystallogr. 2010, 43 (6), 1522–1531. (38) Szekely, P.; Ginsburg, A.; Ben-Nun, T.; Raviv, U. Solution X-ray Scattering Form Factors of Supramolecular Self-Assembled Structures. Langmuir 2010, 26 (16), 13110–13129. (39) Hammersley, A. P.; Svensson, S. O.; Hanfland, M.; Fitch, A. N.; Hausermann, D. Two-dimensional detector software: From real detector to idealised image or two-theta scan. High Pressure Res. 1996, 14 (46), 235–248.
ARTICLE
(40) Deme, B.; Dubois, M.; Gulik-Krzywicki, T.; Zemb, T. Giant collective fluctuations of charged membranes at the lamellar-to-vesicle unbinding transition. 1. Characterization of a new lipid morphology by SANS, SAXS, and electron microscopy. Langmuir 2002, 18 (4), 997–1004. (41) Deme, B.; Dubois, M.; Zemb, T. Giant collective fluctuations of charged membranes at the lamellar-to-vesicle unbinding transition. 2. Equation of state in the absence of salt. Langmuir 2002, 18 (4), 1005–1013. (42) Ladbrooke, B. D.; Chapman, D. Thermal analysis of lipids, proteins and biological membranes a review and summary of some recent studies. Chem. Phys. Lipids 1969, 3 (4), 304–356. (43) Curatolo, W.; Radhakrishnan, R.; Gupta, C. M.; Khorana, H. G. Photoactivatable carbene-generating phospholipids: Physical properties and use in detection of phase separations in lipid mixtures. Biochemistry 1981, 20 (5), 1374–1378. (44) de Almeida, R. F. M.; Borst, J.; Fedorov, A.; Prieto, M.; Visser, A. J. W. G. Complexity of Lipid Domains and Rafts in Giant Unilamellar Vesicles Revealed by Combining Imaging and Microscopic and Macroscopic Time-Resolved Fluorescence. Biophys. J. 2007, 93 (2), 539–553. (45) Ziblat, R.; Leiserowitz, L.; Addadi, L. Crystalline Domain Structure and Cholesterol Crystal Nucleation in Single Hydrated DPPC: Cholesterol:POPC Bilayers. J. Am. Chem. Soc. 2010, 132 (28), 9920–9927. (46) Caffrey, M.; Feigenson, G. W. Influence of metal ions on the phase properties of phosphatidic acid in combination with natural and synthetic phosphatidylcholines: an x-ray diffraction study using synchrotron radiation. Biochemistry 1984, 23 (2), 323–331. (47) Curatolo, W.; Sears, B.; Neuringer, L. J. A calorimetry and deuterium NMR study of mixed model membranes of 1-palmitoyl-2oleylphosphatidylcholine and saturated phosphatidylcholines. Biochimica et Biophysica Acta (BBA) - Biomembranes 1985, 817 (2), 261–270. (48) Yamamoto, T.; Brewster, R.; Pincus, P. A.; Safran, S. A. In Line Activity of Hybrid Lipids: Stabilization of Rafts?, Biological Membranes Are We All on The Same Raft? International Student Workshop on Lipid Domains, Weizmann Institute of Science, Rehovot, Israel, 2010; Weizmann Institute of Science: Rehovot, Israel, 2010; p 11. (49) Satoh, K. Determination of Binding Constants of Ca2+, Na+, and Cl Ions to Liposomal Membranes of Dipalmitoylphosphatidylcholine at Gel Phase by Particle Electrophoresis. Biochim. Biophys. Acta, Biomembr. 1995, 1239 (2), 239–248. (50) Lukatsky, D. B.; Safran, S. A. Universal reduction of pressure between charged surfaces by long-wavelength surface charge modulation. Europhys. Lett. 2002, 60 (4), 629–635.
14775
dx.doi.org/10.1021/la203074q |Langmuir 2011, 27, 14767–14775
