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Net Interactions That Push Cholesterol Away From Unsaturated Phospholipids Are Driven By Enthalpy Chang Wang, Paulo F. Almeida, and Steven L. Regen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00983 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Biochemistry

Net Interactions That Push Cholesterol Away From Unsaturated Phospholipids Are Driven By Enthalpy Chang Wang,† Paulo F. Almeida*‡ and Steven L. Regen*† †Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, United States and ‡Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, North Carolina 28403, United States

ABSTRACT:

The exchangeable unsaturated phospholipids c1-Phos and c3-Phos, which bear one

and three permanent kinks in their acyl chains, are mimics of the biologically important, lowmelting phosphatidylcholines (PC) having one and three cis double bonds in their sn-2 chains (i.e., 16:0,18:1 PC and 16:0,18:3 PC, respectively. The net interaction between an exchangeable form of cholesterol (Chol) with c1-Phos and with c3-Phos has been investigated using the nearest-neighbor recognition method. These interactions were found to be unfavorable in both cases having a positive free energy, ω, for replacing like by unlike nearest-neighbor contacts. The values for this free energy (or interaction parameter) were ω = +165 cal/mol between Chol and c1-Phos, and ω = +395 cal/mol between Chol and c3-Phos. We now report the temperature dependence of these interactions in liquid-disordered bilayers. Their experimentally determined temperature dependencies, in combination with Monte Carlo simulations, revealed that the interaction parameter ω is dominated in both cases by enthalpy. These findings have important implications for the distribution of lipids in natural membranes and for the formation of lipid rafts.

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INTRODUCTION A complete understanding of how mammalian cell membranes function at the molecular level requires detailed insight into their two-dimensional organization. At present, such knowledge may best be described as primitive.1,2 Even for the most popular of models—the lipid raft hypothesis—its key features have yet to be established. Thus, although it is widely believed that cholesterol combines with sphingolipids to form transient domains, the actual sizes, lifetimes and functioning of these domains have not been defined.3-8 Moreover, the thermodynamic forces that drive lipid raft formation, are poorly understood. To date, virtually all of the attention has focused on attractive interactions between cholesterol and sphingolipids (or high-melting phospholipid surrogates).9-12 However, several reports have now appeared that have drawn attention to the importance of low-melting, polyunsaturated phospholipids influencing the lateral organization of cholesterol-rich membranes.13-19 Ten years ago we showed that the interactions between a mimic of cholesterol (i.e., Chol) and a mimic of the high-melting phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (i.e., Phos) are strongly favorable (“attractive”) in liquid-ordered (Lo) bilayers and are dominated by enthalpy (Chart 1).20 Recently, we discovered that unfavorable ("repulsive") interactions between Chol and a mimic of a polyunsaturated, low-melting phospholipid (c 3Phos) in liquid-disordered (Ld) bilayers are even stronger than the attractive interactions between Chol and Phos.21 This discovery came from a series of nearest-neighbor recognition (NNR) measurements in model membranes. Currently, Lo and Ld bilayers are considered to be the best working models of lipid rafts and the fluid sea of phospholipid that surrounds them, respectively.


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Chart 1 Na O P O

O O

O

N H

O

P

S O

O O O

O

HN O

S

O Na O P O O

N H O

O

O

O

S

O

O

O

O

c3-Phos

Chol

Phos

N H

O

S

O

O

O

Na O

c1 -Phos

Nearest-neighbor recognition measurements are unique in that they directly quantify nearestneighbor interactions in lipid bilayers.22 In a typical NNR experiment, thiol-based lipid monomers A and B are generated via partial reduction of disulfide-bridged heterodimers (AB) and are allowed to undergo monomer-dimer interchange with the remaining dimers (Figure 1).23

S S S

S S

SH DTT

S S

S S

S

SH

AA S S

+ S

AB S S

S S

AB

S S S

BB

Equilibrium Product Mixture Figure 1. Stylized illustration of dimer equilibration via thiolate-disulfide interchange.


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These exchange reactions leads to an equilibrium mixture of dimers, AA + BB ⇌ 2 AB, which is governed by the equilibrium constant K = [AB]2/([AA][BB]). When equilibrium is reached, the nearest-neighbor interaction free energy, ω, between A and B is then defined by ω = εAB − ½ (εAA − εBB), where ε, in general, are free energies.24 An interaction is considered to be

repulsive if ω > 0, which means that unlike lipids tend to separate from each other. The free energy that characterizes this repulsive interaction is then be related to the equilibrium constant by ω = −½ RT ln(K/4) through the quasi-chemical (QC) approximation.24-26 Thus, by use of the NNR method we found that Chol was attracted to the DPPC mimic (Phos) in the Lo phase with a free energy corresponding to ω≈ −250 cal/mol of phospholipid at 45°C.20,27 In contrast, Chol was repelled by c3-Phos in the Ld phase to a degree in which ω≈ +400 cal/mol of phospholipid.21 Reducing the number of permanent kinks in the exchangeable phospholipid to one (c1-Phos), mimicking 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), led to a weaker repulsive interaction with Chol corresponding to ω≈ +160 cal/mol of phospholipid.28 We have termed this combination of repulsive and attractive interactions as a "push/pull mechanism" that drives the formation of lipid rafts. Thus, cholesterol is pushed out of the fluid

Ld phase by the repulsive interactions with disordered polyunsaturated phospholipids and pulled into the Lo phase by the attractive interactions with ordered, saturated phospholipids.28-30 We also posited that polyunsaturated phospholipids are likely to be major contributors to the formation of lipid rafts.21 Attractive interactions between cholesterol and high-melting lipids are a likely consequence of van der Waals forces that take advantage of the rigidity and the complementary shape of such lipids. Not surprisingly, therefore, the attraction between Chol and Phos (DPPC) in the lo phase was found to be driven by enthalpy, with ΔhAB=−2000 cal/mol.20 However, the thermodynamic origin of the stronger repulsive interaction between Chol and c3-Phos in the Ld phase is


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Biochemistry

unknown. Intuitively, one might expect that the repulsive interactions between polyunsaturated lipids (which are mimicked by c3-Phos) and cholesterol would be driven by entropy since the disordered chains of the phospholipid would lose conformational freedom when placed next to a rigid molecule such as cholesterol. By pushing away a rigid sterol molecule as a nearestneighbor and replacing it with a flexible phospholipid, a similarly flexible phospholipid is now free to adopt a larger number of conformations (Figure 2).

Figure 2. Stylized illustration showing unfavorable sterol-phospholipid interactions driven by conformational entropy and/or enthalpy.

Alternatively, sterol—phospholipid repulsion could arise because the enthalpy of interaction between two phospholipids is lower than between a phospholipid and cholesterol. It is also unclear whether the very small value of ω, which characterizes the slight repulsion between Chol and c1-Phos in the Ld phase is due to a low enthalpy and low entropy of interaction or due to large enthalpy-entropy compensations, as is often the case in biochemical molecules having many degrees of freedom; hence the need for the present investigation. MATERIALS AND METHODS Nearest-Neighbor Recognition Experiments. To prepare LUVs, thin films of lipid were made by evaporating a chloroform solution containing 0.30 μmol [c1-Phos-Chol] or [c3Phos-Chol] and 11.4 μmol of PDSPC (See Table SI-1) under a stream of argon. After drying overnight under reduced pressure (0.5 Torr), 2.0 mL of a 10 mM Tris-HCl buffer (10 mM Tris,


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150 mM NaCl, 2 mM NaN3, 1 mM EDTA, pH=7.4 at 25 °C) was added to each of the films. The mixture was heated in a water bath (60 °C) and vortexed every 5 min for 30 seconds for a total of 40 min. Then the dispersions were subjected to six freeze/thaw cycles (liquid nitrogen/60 °C water bath) and extruded 20 times through a 200 nm pore diameter polycarbonate membrane (Nuclepore, Whatman Inc.) using argon at the pressure of ~100 psi. Then, a 60 μL aliquot of 1.68 mM monesin in Tris-HCl buffer was added to the dispersion to aid in pH equilibration across the membrane during NNR reactions. The LUV dispersions were then heated to 45 °C, 50 °C, 55 °C, 60 °C or 65 °C (oxygen was removed by purging with argon). The thiolate-disulfide interchange reactions were initiated by adding threo-dithiothreitol (DTT, 20 μL of a 20 mM solution in Tris-HCl buffer, 1.3 equivalent relative to disulfide content) and sufficient amounts of 0.1 M NaOH (25 μL) were added to bring the pH to 7.4. Aliquots (250 μL) were withdrawn as a function of time and quenched by adding 25 μL of 8.30 mM acetic acid to the test tubes containing theses aliquots followed by brief vortexing. Then aliquots were quickly frozen by liquid nitrogen and stored at -20 °C prior to HPLC analysis. For HPLC analysis of the thawed aliquots 1.0 mL of CHCl3/MeOH (2:1 v/v) and Aldrithiol-2 (40 μL of a 10 mM solution in chloroform) were added. The test tubes were then vortexed and centrifuged. The aqueous phases were removed using a pipette. The organic phases were concentrated under reduced pressure using a Savant SVC-100 SpeedVac concentrator (~45 min at ~0.4 torr). The dried lipids were dissolved in a mixture of 20 μL of CHCl3 and the 80 μL of HPLC mobile phase eluent [EtOH/ H2O/ Hexane/ n-Bu4NOAc (1 M) 760/120/100/10 v/v/v/v]. These samples were analyzed by HPLC using a C18 reverse phase column with the mobile phase shown above using a flow rate of 0.9 mL/min. The column was maintained at 31 °C and the components were monitored at 203 nm. The peaks were manually integrated. Values of K were calculated from the peak areas obtained from HPLC traces using appropriate calibration curves.


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Monte Carlo Simulations. Monte Carlo (MC) simulations were performed as previously described.29 The lipid membrane was represented by a triangular lattice, of 100×100 sites, with skew-periodic boundary conditions.31 Each site on the lattice is occupied by a phospholipid (PDSPC, c1-Phos, or c3-Phos) or a Chol molecule. The interactions between nearest neighbors on the lattice are specified by the interaction parameter ω (for identical lipids ω = 0). To obtain equilibrium configurations and to calculate average properties, MC simulations were performed as previously described using standard methods.29,31-36 The lipids are exchanged by randomly selecting partners on the lattice, using a non-nearest-neighbor Kawasaki step.37 The Metropolis criterion is used to accept or reject the moves, with a probability that depends exponentially on the interaction free energy change.38 A random number is used for the decision.39 The simulations included a pre-equilibration period of 2.5×105 MC cycles followed by a period of 106 acquisition cycles. These periods were found to be sufficient to reach equilibrium.29 The interaction parameters ω used in the simulations were chosen to obtain the best match to the equilibrium constant for dimer exchange K experimentally determined by NNR. To this end, the value of ω was varied in the simulations until the calculated K matched its experimental value. RESULTS AND DISCUSSION In this study, we sought to determine whether the “repulsive” interaction between unsaturated phospholipids and cholesterol is dominated by entropy or by enthalpy. To this end, we examined the temperature dependence of the interaction of Chol with c1-Phos and with c3Phos, as measured by the unlike nearest-neighbor interaction parameter ω. The exchangeable phospholipids c1-Phos and c3-Phos are mimics of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine

(POPC

or

16:0,18:1PC)

and

1-palmitoyl-2-linolenoyl-sn-glycero-3-

phosphocholine (16:0,18:3PC), which are major components of eukaryotic membranes. The phospholipids c1-Phos and c3-Phos contain cis-cyclopropyl moieties to lock in permanent


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kinks. This avoids potential complications resulting from cis-trans isomerization of double bonds, which can occur under NNR conditions.40-43 The procedures that were used to carry out the NNR reactions in membranes of large unilamellar vesicles (LUVs) and those used to synthesize

the

host

lipid,

1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphocholine

(PDSPC) and the exchangeable dimers of the possible combinations of c1-Phos and Chol or c3Phos and Chol have previously been described.21,28 Experimentally, we carried out a series of NNR measurements and determined equilibrium constants, K, at 45°C, 50°C, 55°C, 60°C, and 65°C. These constants can then be related to the Gibbs energy of interaction by ω = −½ RT ln(K/4). The bilayers used in these NNR experiments were large unilamellar vesicles (LUVs) that were prepared by extrusion (diameter, 200 nm). These bilayers consisted of 95 mol% of a disordered host lipid, 1-palmitoyl-2-dihydrosterculoyl-

sn-glycero-3-phosphocholine (PDSPC), plus 2.5 mol% of Chol and c1-Phos or c3-Phos. Our principal results are shown in Table 1 and Figure 3. As is readily apparent, the dependence of K on temperature is weak for both c1-Phos and c3-Phos interacting with Chol.


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Biochemistry

Table 1. Values of K and ω as a function of temperaturea Lipid

c1-Phos

c3-Phos

a

Temperature (°C)

K

ω

45

2.37 ± 0.31

166 ± 41

50

2.24 ± 0.23

186 ± 33

55

2.35 ± 0.33

173 ± 45

60

2.43 ± 0.16

165 ± 21

65

2.33 ± 0.16

182 ± 23

45

1.02± 0.15

432 ± 48

50

1.12± 0.10

409 ± 27

55

1.16 ± 0.05

404 ± 13

60

1.17 ± 0.09

408 ± 24

65

1.17 ± 0.06

412 ± 16

The QC approximation, ω = −½ RT ln(K/4), was used to calculate values of ω. To determine the thermodynamic origin of these lipid—lipid interactions, we performed

Monte Carlo (MC) simulations in a triangular lattice of 100×100 sites, where each site represents a phospholipid or cholesterol. The simulations were first performed for the same mixtures used in the NNR experiments, namely PDSPC:ck-Phos:Chol 95:2.5:2.5, where ck-Phos is either c1Phos or c3-Phos. The QC approximation is accurate for small values of ω, but not for large values.26,44 The deviation of the QC approximation from the exact values obtained from MC simulations begins to occur for ω > 300 cal/mol (Figure S1).44 Therefore, rather than using the QC approximation to obtain ω from the equilibrium constant K, we calculated K directly in the MC simulations from the statistics of the different pairs of nearest neighbors in the lattice. The interaction parameters ω used in these simulations were chosen to obtain the best match to the dimer exchange constant K determined by NNR analysis. To this end, ω was varied and K was


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calculated in the simulations until a value of ω was found such that K matched the experimental value. The results of the simulations are shown in Figure 3 together with the experimental data.

Figure 3. Temperature dependence of the equilibrium constant K determined in NNR experiments (solid symbols, averages and standard deviations) in PDSPC/ck-Phos/Chol 95.5:2.5:2.5 for mixtures containing c1-Phos (black) or c3-Phos (red), and calculated in MC simulations (open symbols) with different values of ω (in cal/mol), as indicated. Triangles show simulations with the ω value (in cal/mol) that produced the best match to experiment. Shaded areas represent the variations in K corresponding to simulations with small variations in ω (open squares and circles). The experimental values of K (Figure 3, solid circles) are compared with those obtained in the MC simulation for the best matches (open triangles) and for small variations (±10–15 cal/mol) from the match (shaded area between the open squares and circles) in mixtures that mimic those used experimentally (PDSPC:ck-Phos:Chol 95:2.5:2.5). It is clear that using a constant value of ω for each mixture in the simulations is sufficient to obtain agreement with experiment (ω=165 cal/mol for c1-Phos/Chol and ω=395 cal/mol for c3Phos/Chol.) The experimental data for c1-Phos/Chol is noisier, but in the case of c3Phos/Chol the calculated value of K (with constant ω=+395 cal/mol) matches exactly the experimental value of K (Figure 3, right; except at 45 C, but that is the value with the largest o

experimental error). Thus, based on the simulations, and given the experimental error of K, we conclude that the interaction parameters ω for both of these lipid mixtures are essentially

independent of temperature.


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Biochemistry

The change of K with temperature is given by K= 4 exp(−2ω/RT) in the QC approximation. Thus, a constant, positive, small but non-zero value of ω will lead to a slight increase of K with temperature. A statistical analysis of the experimental values of K shows that the variation of lnK with 1/T (van’t Hoff plot) is not significant for c1-Phos/Chol. What this means is that the value of ω calculated from the van’t Hoff plot cannot be distinguished from zero within the experimental error. This is consistent with the very small value (ω=165 cal/mol) found in the Monte Carlo simulations to provide a match to the experiments (Figure 3, left). The variation of lnK with 1/T is significant for c3-Phos/Chol but this temperature dependence is very small (Figure S2). What this means is that ω is essentially constant with temperature (in the temperature interval examined) but clearly different from zero. This is consistent with the value of ω=395 cal/mol determined in the Monte Carlo simulations to provide a match to the experimental values of K. The MC simulations calculate K directly from the lipid distributions in the lattice and they are not affected by the QC approximation. The unfavorable interaction of Chol with c1-Phos is very small, with ω = +165 cal/mol, so the QC approximation remains valid throughout the temperature range examined (Figure S1). The same is not true, however, regarding the interaction of Chol with c3-Phos, for which ω ≈ 400 cal/mol. Hence the importance of performing the MC simulations, to ensure that the lack of variation of ω calculated through the QC approximation is correct. In general, we can write the Gibbs energy change for the formation of a heterodimer from two homodimers as ω = ΔhAB − T ΔsAB, where ΔhAB and ΔsAB are the enthalpic and entropic components of the interaction parameter. In the present case, we find that ω is independent of T, as shown in Figure 3. Therefore, we must have ΔsAB ≈ 0 and ω ≈ ΔhAB. That is, the net interactions between Chol and c1-Phos or c3-Phos are determined exclusively by enthalpy. In the case of c3-Phos the interaction with Chol is strongly repulsive, with a "push" that corresponds to ω=+395 cal/mol. In the case of c1-Phos, the "push" is much weaker, with


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ω=+165 cal/mol. The MC simulations provide a very intuitive illustration of these two situations. Figure 4 shows snapshots of lattices of 60:40 mixtures of c1-Phos /Chol (A) and c3-Phos /Chol (B). The snapshots of the simulations show the striking difference between the two cases (PC is black, Chol is red). To further place these results into context, recall that the enthalpy of a hydrogen bond is about 4 kcal/mol, and the thermal energy at physiological temperatures is

RT≈600 cal/mol. Thus, ω = +165 cal/mol, is much smaller than RT. This is the case for the interaction of c1-Phos with Chol, which mix very well (Figure 4, left). The mixing, however, is not ideal as can be seen from a comparison with a simulation of an ideal mixing case (ω = 0, Figure S3). Note also that ideal mixing does not mean uniform mixing. The situation is very different for the interaction of c3-Phos with Chol. Here ω≈400 cal/mol, which is not much smaller than RT. Thus we expect to observe phase separation and we do (Figure 4, right). These mixtures are mimics of of POPC/Chol 60:40 and 16:0,18:3PC/Chol 60:40. The differences between the two show the dramatically different behavior of these lipids in biological membranes. All previous reports that have indicated that disordered, polyunsaturated phospholipids “repel” cholesterol are fully consistent with our own experimental results and our conclusions that low-melting lipids push cholesterol out of the Ld phase and into Lo regions of a bilayer.13-19 They are also consistent with our findings that the greater the number of permanent kinks or unsaturations (cis double bonds or cyclopropyl rings) in a low-melting phospholipid, the greater its push on cholesterol.


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Biochemistry

Figure 4. Snapshots of Monte Carlo simulations of mixtures of Chol with (A) c1-Phos and (B) c3-Phos. The mixtures contain 40% Chol (red) and 60 Phos (black).

To the best of our knowledge, the present results provide the first experimental determination of the thermodynamic origin of repulsive interactions in lipid bilayers. These results imply that unfavorable interaction enthalpies between cholesterol and the acyl chains of neighboring unsaturated phospholipids provide a major driving force for pushing cholesterol out of the Ld phase and into the Lo phase. These interactions are likely to play a major role in determining the organization of cell membranes. ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on the ACS Publication website at DOI:xxx. Tables of raw data of the values of K at different temperatures; Dependence K on ω from the QC approximation and from MC simulations (Figure S1); statistical analysis of the van’t Hoff plot of lnK as a function of 1/T (Figure S2); snapshot of a MC simulation of an ideal mixture of two lipids (Figure S3).


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AUTHOR INFORMATION Corresponding Authors [email protected]; [email protected] ORCID Paulo F. Almeida: 0000-0003-4591-938X Steven L. Regen: 0000-0001-6192-7916

ACKNOWLEDGMENT We are grateful to Lehigh University for support of this research. This work was supported in part by a grant from the National Science Foundation (CHE-1464769).

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Net Interactions That Push Cholesterol Away From ... - ACS Publications

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