Letter pubs.acs.org/Langmuir
Surface Occupancy Plays a Major Role in Cholesterol’s Condensing Effect Martin R. Krause, Serhan Turkyilmaz, and Steven L. Regen* Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, United States S Supporting Information *
ABSTRACT: The condensing power of cholesterol and 5α-cholestane has been examined in liposomal membranes made from 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC). Quantitative nearest-neighbor recognition (NNR) analysis and fluorescence measurements using phase-sensitive probe Laurdan have demonstrated that 5α-cholestane exhibits a substantially weaker condensing effect. This fact, in and of itself, provides compelling evidence that cholesterol’s condensing effect is critically dependent on having its steroid nucleus at the membrane surface.
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INTRODUCTION The ability of cholesterol to condense fluid-phase phospholipids has been known for almost 90 years.1−14 Despite numerous investigations, a molecular-level understanding of this phenomenon has remained elusive. In this Letter, we provide compelling evidence that surface occupancy (i.e., the taking up of space at the membrane surface by cholesterol’s steroid nucleus) plays a major role in its condensing action. This finding, together with previous evidence for a template effect by cholesterol, provides the most detailed insight to date into one of the oldest mysteries surrounding biological membraneshow cholesterol condenses phospholipids. We have previously introduced a variation of the nearestneighbor recognition (NNR) technique that allows one to quantify the condensing power of steroids in lipid bilayers.15 In essence, this method measures the influence that a steroid has on the association of an exchangeable phospholipid, A, with an exchangeable steroid analogue, B (Chart 1). More specifically, a
chemical equilibrium that is established between homodimers of A and B (i.e., AA and BB, respectively) and the corresponding heterodimer (AB) via thiolate−disulfide exchange affords a measure of the compactness of a host membrane.16 For example, the equilibrium constant, K (where K = [AB]2/([AA][BB]), in liposomes made from 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol closely tracks the conversion of the membrane from the liquid-disordered to the liquid-ordered state (i.e., higher percentages of the liquid-ordered phase are reflected by higher values of K)15. If one takes statistical considerations into account, then this equilibrium constant can also be converted into a nearest-neighbor interaction free energy between A and B, where ωAB = −1/2RT ln(K/4).17 Of particular significance is the fact that such measurements are highly sensitive and can detect differences in free energy down to tens of calories per mole.18 By the use of this method, we have previously demonstrated that the condensing power of cholesterol is virtually identical to that of dihydrocholesterol, a commonly used surrogate (Chart 2).19 The fact that a kinked isomer of dihydrocholesterol (i.e., coprostanol) has a substantially weaker condensing power has also provided strong support for a template effect by the steroid nucleus whereby the flexible acyl chains of a neighboring phospholipid are able to complement, perfectly, the steroid’s planar nucleus to produce a large number of close hydrophobic contacts and tight packing.19 In a related study, we have compared cholesterol’s condensing power to that of an isomer, which has its hydroxyl group translocated from the C-3 to the C-25 position (i.e., 25OH′, Chart 2).20 In a sense, this sterol yields an upside-down view of the condensing power of cholesterol because the pendant chain is anchored to the membrane’s surface and the
Chart 1
Received: June 14, 2013 Revised: July 25, 2013 Published: July 31, 2013 © 2013 American Chemical Society
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reduced pressure. Purification by flash column chromatography (silica, CHCl3/CH3OH, 10/1 v/v, Rf = 0.24) afforded N-[1-(carboxyethyldithio)-2-ethyl]cholesteryl carbamate (91 mg, 0.152 mmol, 66%) as a white solid having the same 1H NMR spectrum as previously reported.15 To a solution that was composed of N-[1-(carboxyethyldithio)-2ethyl]cholesteryl carbamate (91 mg, 0.152 mmol), N-hydroxysuccinimide (19.4 mg, 0.168 mmol), DMAP (1.9 mg, 0.015 mmol), and 5 mL of CHCl3 was added DCC (47.4 mg, 0.23 mmol). After stirring for 5 h at room temperature, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (107 mg, 0.155 mmol) and DIPEA (81 μL, 0.463 mmol) were added. After additional stirring overnight at room temperature, the solution was diluted with CHCl3 (50 mL) and washed sequentially with 1 mM HCl and brine (50 mL). The organic layer was then separated, dried over MgSO4, and concentrated under reduced pressure. Purification by preparative TLC (silica, CHCl3/CH3OH/ H2O, 8/2/0.2 v/v/v, Rf = 0.25) afforded AB (42 mg, 0.033 mmol, 21%) after filtration through a 0.45 μm filter (Whatman, regenerated celllulose) as a glassy solid having the same 1H NMR spectrum as previously reported.15 Nearest-Neighbor Recognition and Fluorescence Measurements. The procedures used for nearest-neighbor and fluorescence measurements were similar to those previously reported.19
Chart 2
steroid nucleus is free to penetrate the hydrocarbon interior. The fact that 25-OH′ was found to exhibit a relatively weak condensing effect was interpreted as being due to weaker condensing action within the hydrocarbon interior of the membrane. A caveat for this interpretation, however, is that the double bond of the 25-OH′molecule has the potential to act as a second headgroup that could result in an unnatural “looped” conformation20,21 The greater compressibility of 25-OH′ at the air/water interface relative to that of cholesterol lends credibility to such a possibility.21 The primary aim of the work reported herein was to establish firmly that the surface occupancy of the steroid nucleus does indeed play a major role in cholesterol’s condensing effect. With this purpose in mind, we have compared the condensing power of cholesterol to that of a steroid analogue that is devoid of any functionality that can serve as a headgroup (i.e., 5αcholestane (Chart 2)). Thus, by replacing the hydroxyl group of dihydrocholesterol with a hydrogen atom this steroid is now free to adopt any orientation and any depth within the hydrocarbon interior of the membrane to maximize hydrophobic interactions with the acyl chains of neighboring phospholipids. This is in sharp contrast to cholesterol, which has its steroid nucleus anchored to the membrane surface through its hydroxyl group.
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RESULTS AND DISCUSSION Before making quantitative NNR measurements using host membranes derived from DPPC, we first compared the condensing power of cholesterol and 5α-cholestane using phase-sensitive probe Laurdan.19,23 As discussed elsewhere, one can use Laurdan to detect the gel to liquid-crystalline phase transition of phospholipids by following its generalized polarization (GP) value as a function of temperature.23 Here, GP = (I440 − I490)/(I440 + I490), and I440 and I490 are the emission intensities at these wavelengths (λex = 350 nm). These values of GP, which reflect the polarity surrounding the Laurdan moiety, are also sensitive to changes in the phase of the membrane. In Figure 1 is shown a plot of GP as a function of temperature for liposomal membranes made from DPPC and
EXPERIMENTAL METHODS
Synthesis of Exchangeable Homodimers AA and BB. Synthesis methods that were used to prepare AA and BB were similar to those previously reported.15,22 Synthesis of Exchangeable Heterodimer AB. Heterodimer AB was synthesized using an improved procedure as outlined in Scheme 1.15 Thus, to a mixture of 3-(2-aminoethyldithio)propanoic acid hydrochloride salt (50 mg, 0.23 mmol) and triethylamine (100 μL) in anhydrous CHCl3 (5 mL) was added N-(cholesteryl-carbonyloxy)succinimide (194 mg, 0.38 mmol) in one portion.18 The resulting solution was stirred overnight at room temperature, diluted with CHCl3 (50 mL), and washed with water (50 mL, pH 3, HCl). The organic phase was then dried over MgSO4 and concentrated under
Figure 1. Plot of general polarization vs temperature in liposomes made from DPPC/DPPG/cholesterol/5α-cholestane with the following molar percentages: (+) 57.5/2.5/40/0, (●) 57.5/2.5/30/10, (▲) 57.5/2.5/20/20, (⧫) 57.5/2.5/2.5/37.5, and (×) 95/2.5/2.5/0. Data for (+) and (×) have previously been reported.19
Scheme 1
40 mol % steroid using varying ratios of cholesterol/5αcholestane. Also included in this figure is a plot made for DPPC containing only 2.5% cholesterol. In each case, a small quantity (2.5 mol %) of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-racglycerol) (DPPG) has been added to match the net negative charge that is introduced by the exchangeable phospholipid, A, for related membranes used in NNR experiments. When only 2.5% cholesterol is present, the gel-to-fluid phase transition of 10304
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DPPC is evident at ca. 41 °C.19 In sharp contrast, for liposomes containing 40 mol % cholesterol, the GP values decrease modestly and almost linearly with increasing temperature. Over the temperature range examined, such membranes are known to maintain the liquid-ordered phase. A similar trend is observed for membranes containing 30% cholesterol plus 10% 5α-cholestane, except that the GP values are lower, especially at temperatures above ca. 41 °C. This finding indicates that these bilayers are less compact and allow for a greater penetration of water. For analogous membranes containing 20% cholesterol plus 20% 5α-cholestane, a further decrease in GP values is apparent at the highest temperatures measured. When the liposomes are rich in 5α-cholestane (i.e., for bilayers containing 2.5 mol % cholesterol and 37.5 mol % 5α-cholestane), a dramatic change in the fluorescence profile can be seen. In this case, the slope of the plot is greatly increased, implying that there has been a substantial softening of the membrane. Taken together, these results indicate that 5α-cholestane is a much weaker condensing agent than cholesterol. Using experimental procedures similar to those previously reported, we then made NNR measurements in liposomes containing 2.5 mol % A and 2.5 mol % B plus varying ratios of cholesterol/5α-cholestane.19 As shown in Figure 2, the
Figure 3. Stylized illustration showing hydrated CH2 groups of phospholipids in the liquid-disordered (cholesterol-poor) state being transformed into the liquid-ordered (cholesterol-rich) state with the partial release of water and the partial straightening of their acyl chains.
formation of lipid bilayers (i.e., it is the hydrophobic effect). Thus, the template effect maximizes the hydrophobic effect by maximizing the number of close hydrophobic contacts between the steroid nucleus and the acyl chains of a neighboring phospholipid. As a consequence, the number of water molecules that are released from the bilayer is also maximized.
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CONCLUSIONS Fluorescence measurements that have been made using Laurdan, and NNR measurements that have been made using exchangeable lipids A with B have revealed a dramatic difference in the ability of cholesterol and 5α-cholestane to condense fluid bilayers of DPPC.24 Thus, whereas cholesterol has a strong condensing effect, 5α-cholestane has a close to negligible condensing power. Because cholesterol is anchored to the membrane surface through its hydroxyl group and 5αcholestane must be confined to the hydrocarbon interior of the membrane by virtue of its total absence of polar functionality, we conclude that the taking up of space at the membrane surface by cholesterol’s steroid nucleus (i.e., surface occupancy) plays a major role in its condensing action. When combined with our previous evidence for a template effect, a simple picture emerges that accounts for one of the oldest mysteries surrounding biological membraneshow cholesterol condenses phospholipids. Thus, cholesterol’s steroid nucleus maximizes the hydrophobic effect by serving as a planar hydrophobic template that maximizes the hydrophobic contact between neighboring lipids and, consequently, the release of water from the surface of the membrane.
Figure 2. Bar graph of K for liposomes made from DPPC/cholesterol/ 5α-cholestane/AB having the following mole percentages: (i) 57.5/ 37.5/0/2.5, (ii) 57.5/27.5/10/2.5, (iii) 57.5/17.5/20/2.5, and (iv) 57.5/0.0/37.5/2.5. Error bars represent one standard deviation. All NNR experiments were carried out in triplicate at 45 °C.
compactness of these membranes, which is reflected by the values of K, closely matches what is indicated from our fluorescence experiments (i.e., a moderate decrease in bilayer compactness accompanies a moderate replacement of cholesterol by 5α-cholestane). For bilayers that are rich in 5αcholestane (i.e., 37.5 mol %), however, a substantial decrease in compactness occurs. On the basis of these findings, it is clear that 5α-cholestane has close to a negligible condensing effect. When combined with our previous evidence for a template effect, the present findings provide clear insight into cholesterol’s condensing behavior. Thus, in the liquiddisordered state, a phospholipid’s headgroup occupies only about half of its total surface area; the remaining area is occupied by exposed and partially hydrated CH2 groups (Figure 3). By occupying space at the membrane surface, the steroid nucleus replaces some of these wet CH2 groups and allows them to undergo partial dehydration with partial straightening of the acyl chains. The fact that a much stronger condensing effect occurs when the steroid nucleus lies at the membrane surface implies that the main driving force behind cholesterol’s condensing effect is the same one that drives the
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures, tables of NNR and fluorescence data, fluorescence spectra, and dynamic light scattering data. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the National Science Foundation (CHE-1145500). 10305
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(23) Parassi, T.; DiStefano, M.; Loiero, M.; Ravagnan, G.; Gratton, E. Influence of Cholesterol on Phospholipid Bilayers Phase Domains As Detected by Laurdan Fluorescence. Biophys. J. 1994, 66, 120−132. (24) In this study, we have used DPPC as a host membrane for comparing the relative condensing power of cholesterol with 5αcholestane. Although, in principle, different tightly packed host membranes could yield different absolute values of K, the relative differences in condensing power should remain the same. Moreover, such differences are expected to be extremely small. In this regard, it should be noted that the value of K that was previously measured in host membranes made from 1,2-distearoyl-sn-glycero-3-phosphocholine in the liquid-ordered state differed by less than 10% as compared to that measured in DPPC.15
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