Article pubs.acs.org/Langmuir
Exchangeable Mimics of DPPC and DPPG Exhibiting Similar NearestNeighbor Interactions in Fluid Bilayers Masaru Mukai and Steven L. Regen* Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, United States S Supporting Information *
ABSTRACT: The interactions between an exchangeable mimic of 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), Phos(±), with an exchangeable mimic of cholesterol, Chol, have been analyzed in fluid bilayers by means of nearest-neighbor recognition measurements. These interactions have been found to be very similar to those of an exchangeable mimic of 1,2dipalmitoyl-sn-glycerol-3-phospho-(1′rac-glycerol) (DPPG), Phos(−), interacting with Chol. Thus, both phospholipids have a similar preference for becoming nearest-neighbors of Chol in the liquidordered (l0) phase, and both mix, ideally, with Chol in the liquid-disordered (ld) phase. These findings, together with the almost negligible screening effects found for the latter, provide strong evidence that electrostatic forces play a minor role in the preference that both phospholipids have in becoming a favored nearest-neighbor of Chol. They also imply that the main driving force for forming the liquid-ordered phase, and for defining the lateral organization of this phase, is an intrinsic affinity that high-melting lipids and cholesterol have for each other.
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INTRODUCTION
The two-dimensional organization of lipids in biological membranes is believed to play a central role in a variety of fundamental processes such as signal transduction and trafficking.1−3 This hypothesis has led to a large number of investigations that have been aimed at gaining insight into the mixing behavior of lipids, especially in well-defined liquidordered (l0) and liquid-disordered (ld) bilayers.4−9 The former is thought to mimic putative “lipid rafts” (i.e., domains in cell membranes that are rich in cholesterol and high-melting sphingolipids), while the latter is thought to mimic the surrounding “sea” of low-melting phospholipids. Recently, evidence has begun to emerge indicating that unfavorable interactions between cholesterol and kinked phospholipids in the ld phase can contribute, significantly, to the formation of the l0 phase.10,11 However, the underlying forces that are involved in creating the l0 phase have remained unclear. Despite a considerable number of experimental and theoretical studies of the liquid-ordered phase, its precise two-dimensional organization is unknown.12 In this regard, the role that electrostatic forces play in defining the time-averaged lateral organization of cholesterol and high-melting lipids in such assemblies has yet to be established. At the molecular level, one can imagine that two zwitterionic phospholipids that are separated by an uncharged cholesterol molecule in a tightly packed liquid-ordered state could be more stable than an analogous assembly in which the two zwitterionic lipids are nearest-neighbors (Figure 1A). Specifically, such a separation could minimize headgroup repulsion by reducing unfavorable dipole−dipole (electrostatic) and steric interactions.13 Although headgroup repulsion between zwitterionic lipids lies intermediate between that of ionic and nonionic lipids, it is conceivable that such interactions could be strong © 2015 American Chemical Society
Figure 1. Stylized illustration showing (A) two zwitterionic lipids that are separated by a cholesterol molecule in equilibrium with an arrangement having the two lipids as nearest-neighbors, and (B) similar assemblies involving anionic lipids.
enough to play a major role in defining the two-dimensional structure of the l0 phase.13 If electrostatic forces were, in fact, important, then substitution of a zwitterionic lipid with a negatively charged analog should lead to even stronger headgroup repulsions and a greater prefererence for cholesterol becoming a nearestneighbor of this analog; i.e., the equilibrium shown in Figure 1B should be shfted further to the “left”. However, if screening effects under physiologically relevant conditions were sufficient to eliminate electrostatic repulsion between these negatively charged lipids, then they must also be sufficient to eliminate any electrostatic repulsion between zwitterionic analogs.14 Thus, if K1 = K2, then the lateral organization of the l0 phase (and possibly lipid rafts) should be dominated by forces other than Received: June 22, 2015 Revised: October 23, 2015 Published: November 4, 2015 12674
DOI: 10.1021/acs.langmuir.5b03174 Langmuir 2015, 31, 12674−12678
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electrostatic forces; e.g., hydrophobic and steric interactions (Figure 1). We have become keenly interested in this question of electrostatic forces in the l0 phase as the result of several nearest-neighbor recognition (NNR) studies that we have reported in which a negatively charged, exchangeable phospholipid, Phos(−) was used to mimic DPPC as well a DPPG. In particular, we felt the need to firmly establish that the strong preference for Phos(−) becoming a nearest-neighbor of Chol in the l0 phase was not due electrostatic forces. More specifically, we sought to rule out the possibility that unfavorable electrostatic forces between neighboring Phos(−) molecules were the main driving force for Phos(−) becoming a favored nearest-neighbor of Chol.
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Article
RESULTS AND DISCUSSION
Nearest-Neighbor Interactions. In this investigation, we compared the nearest-neighbor interactions between Phos(±) with Chol with those previously recorded for Phos(−) and Chol in l0 and ld bilayers by means of the nearest-neighbor recognition (NNR) measurements (Chart 1).15 Chart 1
EXPERIMENTAL SECTION
Nearest-Neighbor Recognition Measurements. Thin films of lipid were prepared by evaporating chloroform solutions containing varying amounts of DPPC, cholesterol, Chol-Chol, Phos(±)-Phos(±), Phos(±)-Chol, and 2 under a stream of argon. After drying a given thin film 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. The mixtures were then vortexmixed 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). Subsequently, a 60 μL aliquot of 1.68 μM monensin in TRIS-HCl buffer was added to aid the pH equilibration across the membrane. After the liposomal dispersions (1600 μL) were heated to 45 °C and the oxygen removed by purging with argon, sufficient amounts of 0.1 M NaOH were added (typically using 50 μL) to return the pH to 7.4; i.e., a slight lowering of the pH was caused by heating. Aliquots (400 μL) were withdrawn as a function of time and the exchange reactions quenched by adding a small volume of 0.5 M acetic acid (50 and 100 μL, homodimer and heterodimer experiment, respectively) 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 1500 μL of CHCl3/ CH3OH (2/1, v/v) and Aldrithiol-2 (i.e., 2,2′-dipyridyldisulfide, 60 μL of a 10 mM solution in CHCl3). The mixtures were then vortex-mixed, centrifuged, and the aqueous phase 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 vacuum pump (∼1 h at 0.4 Torr). The residual lipids were dissolved in 20 μL of CHCl3/CH3OH (2/1, v/v) 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, 100 mL of deionized water, 100 mL of hexane, and 12 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 260 nm. Values of K, where K = [Phos(±)-Chol]2/ [Chol-Chol][Phos(±)-Phos(±)] were calculated from peak areas obtained from HPLC chromatograms using appropriate calibration curves.
As discussed previously, NNR experiments take “snapshots” of nearest-neighbor preferences by measuring the thermodynamic tendency of lipids to become nearest-neighbors of one another.15 Experimentally, two exchangeable lipids of interest (e.g., Phos(−) and Chol) are converted into homodimers Phos(−)−Phos(−) and Chol−Chol, and the corresponding heterodimer, Phos(−)−Chol, via the introduction of a disulfide bond (Chart 1). By allowing the monomers to undergo exchange via thiolate−disulfide interchange, an equilibrium mixture is established that defines an equilibrium constant, K, and their nearest-neighbor interactions (Figure 2).16 When the
Figure 2. Homodimers Phos(−)−Phos(−) and Chol−Chol in equilibrium with the heterodimer, Phos(−)−Chol.
lipids mix, ideally, this is indicated by an equilibrium constant equaling 4.0 (eq 1). Favorable homoassociations are indicated by values of K that are less than 4.0, and favorable heteroassociations are indicated by values of K that are greater than 4.0. A nearest-neighbor interaction free energy, ωPhos(−)‑Chol, can be defined as the net interaction between Phos(−) and Chol), which is equal to −1/2 RT ln (K/4). Here, since the heterodimer is statistically favored over each homodimer by a factor of 2, the value of K is divided by 4. Also, a factor of 1/2 is included to take into account the fact that two dimers are involved in the equilibrium. What distinguishes the NNR method from all other experimental approaches (e.g., differential scanning calorimetry, fluorescence resonance energy transfer, isothermal titration 12675
DOI: 10.1021/acs.langmuir.5b03174 Langmuir 2015, 31, 12674−12678
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liquid-crystalline phase transition temperature that are very similar to those of DPPC.19 Thiolate−disulfide interchange reactions were carried out at 45 °C in multilamellar vesicles made from DPPC that were either rich in cholesterol (l0 phase) or devoid of this sterol (ld phase).18,20 Because dimers of Phos(±) were found to be unstable to reducing conditions (i.e., dithiothreitol) and to extended reaction times (i.e., >100 h), all exhange reactions were were initiated by the use of 2 and were carried out for a maximum of 48 h. To maintain an equimolar quantity of exchangeable phospholipid and sterol, the inclusion of an appropriate amount of Phos(±)-Phos(±) was included in all liposomes. It should be noted that these conditions are in contrast to our normal NNR experiments, which start with pure heterodimer, which ensures that all monolayer leaflets have an equivalent amount of exchangeable phospholipid and exchangeable sterol. When a nonuniform distribution of homodimers exists among the monolayer leaflets, the values of K tend to be low, artificially. In Table 1 are shown the values of K that were calculated as a function of time for liposomes in the l0 phase that were,
calorimetry, analyses of phase diagrams, and cyclodextrinmediated partitioning) is that it measures lipid−lipid interactions in a direct manner and is independent of other experiments or calculations.15 One caveat with the NNR method, however, is that the lipids that are used are unnatural. Because of its relative ease of synthesis, we have made extensive use of Phos(−) as a mimic of 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) as well as 1,2-dipalmitoylsn-glycero-3-phosphoglycerol (DPPG).15 As we have argued, previously, although PCs and PGs differ in their headgroup charge, several lines of evidence have supported the use of Phos(−) as a mimic of DPPC and DPPG. Specifically, all three of these lipids have identical acyl chains and very similar gel to liquid-crystalline phase transition temperatures (41−42 °C), enthalpies, and monolayer behavior at the air/water interface. This is exactly analogous to the situation found for nonexchangeable PCs and PGs, where identical acyl chains lead to nearly identical melting behavior and ideal mixing.17 Since Chol has nearly identical monolayer behavior and condensing properties as cholesterol, we have made extensive use of it as a mimic of this natural sterol.15 Finally, the fact that the nearest-neighbor free energies that characterize Phos(−) interacting with Chol in host membranes made from DPPC and cholesterol are indistinguishable from those measured in membranes made exclusively from Phos(−) and Chol at various temperatures implies that the two membranes provide the same average microenvironment around these exchangeable lipids.15 It also implies that these membranes contain similar proportions of the ld and l0 phase at a given temperature. However, direct experimental proof that the interaction between Phos(−) and Chol is similar to the interaction between a zwitterionic analog and Chol has been sorely missing. In Scheme 1 the method of synthesis that was used to prepare the required heterodimer, Phos(±)-Chol, is shown.
Table 1. Calculated Values of K for [Phos(±)/Chol] at Different Times in the lo Phase Starting from HeterodimerRich Liposomesa time (h)
trial 1
trial 2
trial 3
0 6 24 48
10.5 9.6 8.9 8.8
11.2 10.1 10.0 9.5
11.6 9.6 8.9 8.4
average 10.8 9.8 9.3 8.9
± ± ± ±
0.7 0.3 0.6 0.6
a NNR reactions carried out in the liquid-ordered state at 45 °C using liposomes that were made from [lipid, μmol]: [DPPC, 6.9], [cholesterol, 4.5], [Phos(±)-Phos(±), 0.08], [Phos(±)-Chol, 0.15], [2, 0.15].
Scheme 1 Table 2. Calculated Values of K for [Phos(±)/Chol] at Different Times in the lo Phase Starting from HomodimerRich Liposomesa time (h)
trial 1
trial 2
trial 3
0 6 24 48
4.5 5.5 5.6 6.5
5.8 7.6 6.3 7.2
6.8 6.4 6.7 7.1
average 5.7 6.5 6.2 7.0
± ± ± ±
1.2 1.1 0.6 0.4
a NNR reactions carried out in the liquid-ordered state at 45 °C using liposomes that were made from [lipid, μmol]: [DPPC, 6.9], [cholesterol, 4.5], [Phos(±)-Phos(±), 0.15], [2, 0.30].
initially, richer in heterodimer; Table 2 shows corresponding values in the l0 phase in liposomes that were richer, initially, in homodimer. As expected, increased reaction times lead to a decrease in K for the former and an increase for the latter. Because the exchangeable monomers are more uniformly distributed in heterodimer-rich experiments, we regard the value of K equaling 8.9 ± 0.6 to be close to the true equilibrium value. This, in fact, compares favorably with K = 9.2 ± 0.2, which has been found for the mixing of Phos(−) with Chol.21 Most importantly, both sets of data in Tables 1 and 2 clearly show that there is a strong preference for Phos(±) becoming a favored nearest-neighbor of Chol.
Thus, a 2-pyridyl-2-dithioethyl analog of DPPC (i.e., 1) was reacted with an exchangeable form of cholesterol (2) in chloroform to give the requisite heterodimer. Synthetic methods that were used for preparing Chol-Chol, Phos(−)Phos(−), and Phos(±)-Phos(±) were similar to those described elsewhere. 15,18 As previously demonstrated, Phos(±)-Phos(±) has monolayer behavior and a gel to 12676
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Table 6. Calculated Values of K for [Phos(−)/Chol] at Different Times in the lo Phase Starting from HomodimerRich Liposomesa
In Tables 3 and 4 are listed the values of K for the mixing of Phos(±) with Chol in the ld phase starting from heterodimerTable 3. Calculated Values of K for [Phos(±)/Chol] at Different Times in the ld Phase Starting from HeterodimerRicha time (h)
trial 1
trial 2
trial 3
0 6 24 48
6.9 6.5 4.4 4.3
4.5 3.8 3.8 3.4
7.2 6.8 5.9 3.5
average 6.2 5.7 4.7 3.7
± ± ± ±
time (h)
trial 1
trial 2
trial 3
average
6 24 48
7.1 --8.3
6.9 9.2 9.7
8.2 8.5 9.4
7.4 ± 0.7 8.8 ± 0.5 9.0 ± 0.5
NNR reactions carried out in the liquid-ordered state at 45 °C using liposomes that were made from [lipid, μmol]: [DPPC, 6.9], [cholesterol, 4.5], [Phos(−)-Phos(−), 0.15], [Chol−Chol, 0.15]. Exchange reactions were initiated by the addition of DTT.21 a
1.5 1.7 1.1 0.5
NNR reactions carried out in the liquid-disordered state at 45 °C using liposomes that were made from [lipid, μmol]: [DPPC, 11.4], [Phos(±)-Phos(±), 0.08], [Phos(±)-Chol, 0.15], [2, 0.15].
a
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CONCLUSIONS The fact that the interactions between Phos(±) and Chol are very similar to those of Phos(−) with Chol, together with the almost negligible screening effects found for the latter, provides strong evidence that electrostatic forces play a minor role in the preference that both phospholipids have in becoming a favored nearest-neighbor of Chol. At the same time, these findings imply that the main driving force for forming the liquid-ordered phase, and for defining its lateral organization, is an intrinsic affinity that high-melting lipids and cholesterol have for each other. Two caveats should be noted when extrapolating these findings to lipid rafts. First, this study has been limited to single lo and ld phases because they are well-defined and because, even in these simplest of model systems, the contributions made from electrostatic forces have been uncertain. How electrostatic forces may influence nearest-neighbor interactions in more complex systems that more closely mimic putative lipid rafts (e.g., membranes made from a low melting phospholipid, a high-melting phospholipid, and cholesterol) where there is microscopic l0−ld phase separation remains to be established.22,23 Second, although we and others have made extensive use of saturated and unsaturated phospholipids in model studies, it is conceivable that the presence of amide moiety in sphingolipids could play a major role in the organization of lipid rafts.24,25 Further NNR studies in more complex systems are expected to help unravel the mystery surrounding the formation and two-dimensional structure of lipid rafts.
Table 4. Calculated Values of K for [Phos(±)/Chol] at Different Times in the ld Phase Starting from HomodimerRicha time (h)
trial 1
trial 2
trial 3
0 6 24 48
1.9 2.5 4.8 5.2
1.7 3.0 5.3 4.9
1.6 2.3 5.2 4.8
average 1.8 2.6 5.1 5.0
± ± ± ±
0.2 0.4 0.3 0.2
NNR reactions carried out in the liquid-ordered state at 45 °C using liposomes that were made from [lipid, μmol]: [DPPC, 11.4], [Phos(±)-Phos(±), 0.15], [2, 0.30]. a
rich and homodimer-rich exchangeable lipids, respectively. As expected, the mixing between these two exchangeable lipids in this phase is close to random. The reason for the slightly elevated value of K (5.0 ± 0.2) coming from the liposomes that were richer in homodimer is not presently clear. However, the fact that K reaches a value of 3.7 ± 0.5 from the “heterodimerrich side” clearly indicates that the mixing of these two lipids is close to random, which is what was found for Phos(−) mixing with Chol, where K was 3.9 ± 0.3.21 Finally, to gain insight into the possible contributions made by screening effects in the l0 phase, we have carried out NNR measurements for Phos(−)/Chol using 10 mM instead of 150 mM NaCl (Tables 5 and 6). The closeness of the 48 h values Table 5. Calculated Values of K for [Phos(−)/Chol] at Different Times in the lo Phase Starting from HeterodimerRich Liposomes Using 10 mM NaCla time (h)
trial 1
trial 2
trial 3
average
6 24 48
13.1 11.0 10.3
15.2 10.7 10.5
12.1 11.5 10.8
13.5 ± 1.6 11.1 ± 0.4 10.6 ± 0.2
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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.langmuir.5b03174. Methods used for the synthesis of Phos(±)-Chol and NNR data (PDF)
NNR reactions carried out in the liquid-ordered state at 45 °C using liposomes that were made from [lipid, μmol]: [DPPC, 6.9], [cholesterol, 4.5], [Phos(−)-Chol, 0.30]. Exchange reactions were initiated by the addition of DTT.21 a
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. coming from the heterodimer-rich membranes (10.6 ± 0.6) and homodimer-rich membranes (9.0 ± 0.5) relative to the recorded value of 9.2 ± 0.2 found using 150 mM NaCl, clearly indicates that contributions made by screening effects to the mixing of Phos(−) with Chol are close to negligible.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the National Science Foundation (CHE-1145500). 12677
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