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Physical studies of cholesterol-phospholipid interactions
Physical studies of cholesterol-phospholipid interactions Todd PW McMullen and Ronald N McElhaney Our understanding of the role of cholesterol in biological membranes requires a detailed knowledge of the forces which mediate cholesterol-lipid interactions and of the lateral organization of cholesterol in natural and model membranes. The recent literature amply illustrates the strength of a multidisciplinary approach in determining the effects of cholesterol on the host bilayer lipids. To confidently apply our current knowledge of cholesterol-lipid interactions and cholesterol organization to natural membranes, however, a systematic analysis of the effects of cholesterol on a much broader range of host lipid bilayers will be required.
Address Department of Biochemistry. University of Alberta, Edmonton. Alberta, Canada T6G 2H7 Current Opinion in Colloid & Interface SCience 1996. 1 :83-90
e Current Science Ltd ISSN 1359-0294 Abbreviations DMPC dimyristoylphosphatidylcholine DPPC dipalmitoylphosphatidylcholine LQ liquid crystalline PC phosphatidylcholine SPM sphingomyelin
Introduction Cholesterol or closely rel ated sterols are major lipid components of the plasma membranes of most eukaryotic cells, and are 'also found in lower concentrations in many intracellular membranes. Cholesterol has numerous different functions in eukaryotic cells, one of its primary roles being to modulate the physical properties of the plasma membrane phospholipid bilayer (see [1-3] for reviews). Thus a large number of studies of cholesterol/phospholipid bilayer model membranes have been carried out, using a wide range of physicochemical techniques [2-5]. Briefly, cholesterol incorporation into bilaycrs has four effects: first, it broadens and eventually eliminates the cooperative gel to liqu id-crystalline phase transition of phospholipid bilayers; second, it decreases (increases) the area per molecule of the liquid-crystalline (gel) st ate monolayers; third, it increases (decreases) the orientational order of the hydrocarbon chains of liquid-crystalline (gel) bilayers; and fourth, it decreases (increases) the passive permeability of phospholipid bilayers above (below) their gel to liquidcrystalline ph ase transition temperatures. At relatively low cholesterol concentrations (up to 22 mol%), there exist two thermodynamically distinct domains, a cholesterol-poor domain with properties similar to those of the pure phospholipid bilayer, and a cholesterol-rich domain. The cholesterol-rich domain, known as the liquid-ordered phase or fJ-phase, is the only phase which exists at cholesterol levels <22 mol% in phospholipid bilayers. It
is characterized by phospholipid intra- and intermolecular motional rates which are similar to those of a fluid phospholipid bilayer, but w hich contain hydrocarbon cha in orientationa I order and phospholipid area compressibility values th at more closely resemble the gel ph ase of a pure phospholipid. Although we currently know a considerable amount about the overall effect of cholesterol on phospholipid bilayer phase transitions, our understanding , of phospholipidcholesterol interactions at the molecular level is incomplete. The largest contribution to cholesterol-phospholipid interactions appears to be from van der Waals forces and hydrophobic forces, although hydrogen bonding to the polar headgroup and interfacial reg ions of the host lipid bilayer m ay be of considerable importance, especially in the sphingolipids and anionic phospholipids. Moreover, depending on temperature and cholesterol concentration, as well as on the headgroup and hydrocarbon ch ain composition of the host bilayer, cholesterol may exist in regularly distributed arrays or in laterally segregated cholesterol-rich or cholesterol-poor domains of variable number and size. Some of the important questions driving recent investigations include the following: Does cholesterol exhibit phospholipid-specific interactions? Does cholesterol exist as monomers or as aggregates in phospholipid bilayers, and, if the former, are these monomers dispersed randoml y or in ordered arrays within the plane of the bil ayer? Is the structure of the cholesterol-rich liquid-ordered phase, whi ch is usually considered to be homogeneous, actually dependent upon cholesterol concentration and temperature? While these questions may persist for some time, recent work clearly demonstrates that even simple binary cholesterol/phospholipid mixtures may exhibit very complex thermotropic phase behavior. Moreover, both the thermotropic phase behavior and the molecular organization of sterol-containing lipid bilayers is acutely sensitive to differences in the chemical structure of both the sterol and the phospholipid with which it is interacting.
Sterol-phospholipid Interactions Cholesterol has a dramatically different structure than that of the phospholipids and sphingolipids that compose eukaryotic membranes. Cholesterol consists of a planar tetracyclic ring system with an alkyl side-chain extending towards the bilayer center and a hydroxyl group located at the polar/non-polar interface, as shown in Figure 1 [2,3,5,6-]. Most structural and stereochemical alterations to the cholesterol molecule result in some loss of its ability to produce its characteristic effects on phospholipid bilayers [2,7-9]. However, the relat ionship between the chemical structure of sterols and the magnitude and direction of their various effects on the physical properties of model membrane systems is not always clear or
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consistent [2,9,10,11-]. This may be due in part either to experimental or interpretational errors on the part of different investigators examining different sterols in a variety of model and biological membranes, or to real differences in host phospholipid bilayer physical properties. For example, McMullen er 01. [9) have determined that the effects of cholesterol and of androstenol (a sterol which lacks the alkyl side-chain) on phosphatidylcholine (PC) bilayers are quite different, but that the type and magnitude of these differences are dependent on the chain length of the host PC bilayer. Specifically, the relative importance of the alkyl side-chain of cholesterol was found to depend on the thickness of the host phospholipid bilayer. It is clear, however, that none of the sterol analogues examined was able to quantitatively duplicate cholesterol-like effects on any of the host bilayers tested. Even the position of the double bond in the sterol ring system, initially believed to be of little importance, was found to be important in mediating cholesterol-like effects in certain model membranes [7,11-]. These results indicate that sterol miscibility and organization in the host PC monolayer or bilayer are sensitive to even small changes in the structure of the cholesterol molecule. Thus it is not surprising that sterol -related molecules, such as the oxysterols or vitamin D 3, do not always have cholesterol-like effects on the physicochemical properties of PC bilayers into which they are incorporated [12,13].
Figure 1
Cholesterol-phospholipid interactions Although the techniques chosen to investigate the phase behavior and organization of cholesterol-containing bilayers or monolayers are large in number, the vast majority of the past and present work on cholesterol-phospholipid interactions involves one class of phospholipids, the PCs. Recently, one of the most important factors governing the phase behavior of cholesterol/PC mixtures, namely, the hydrophobic mismatch effect, has been revealed by Mclvlullen et 01. (14). Using high-sensitivity differential scanning calorimetry (HS-DSC), these workers demonstrated that cholesterol progressively decreases the host bilayer transition temperature (Tm) of the cholesterol-rich domains of PC bilayers with hydrocarbon chains longer than 17:0, while increasing the chain-melting T m of PCs with chain lengths shorter than 17:0 (specific phosphatidylcholines are designated by the notation n:O PC, where n is the number of carbon atoms per hydrocarbon chain and zero indicates the absence of double bonds). As the hydrophobic thickness of the PC bilayer decreases by approximately one-third upon chain melting (due to introduction of gauche conformers along the acyl chain), the relative stabilities of the gel and liquid-crystalline (LQ) phases should be least affected when the length of another molecule (i.e. cholesterol) incorporated into the bilayer matches the mean hydrophobic thickness of the bilayer (a hydrophobic length midway between the gel and LQ states) [15]. Molecules with a greater hydrophobic length than the mean would tend todifferentially stabilize
lateral view of a cholesterollDMPC dimer. The a face of cholesterol faces towards the DMPC molecule (into the page). The oxygen atoms are shaded. P indicates the phosphorous group, N indicates nitrogen. Note that the hydrogen bond denoled by the dolled line (cholesterol hydroxyl group to the sn·2 carbonyl oxygen) is purely speculative. (Adapted from [6°])
the gel phase, thus increasing the T m- and molecules with a smaller hydrophobic moiety than the mean would tend to de-stabilize the gel phase. In fact, these authors argued that the reason that cholesterol did not alter the T m of 17:0 PC bilayers is that the mean hydrophobic length of the host lipid molecule and the length of the cholesterol molecule are equal. Indeed, the Fourier transform infrared (FTIR) spectroscopic data of Chia er 01. [16] confirm that bilayers of longer-chain PCs exhibited considerably more conformational disorder in the gel state than do shorter-chain PC bilayers with a comparable level of cholesterol.
Physical studies of cholesterol-phospholipid interactions McMullen and McElhaney
A number of additional studies support the existence of the hydrophobic mismatch effect in l-stearoyl-z-oleoylphosphatidylcholine (SOPC) as well as in dipalmitoylphosphatidylcholine (DPPC) bilayers containing various alkyl side-chain analogues of cholesterol [10,17,18]. In each case the magnitude of sterol/phospholipid hydrophobic mismatch, whether due to variations in the length of the alkyl side-chain of the cholesterol analogue or in the length of the acyl chains of the phospholipid, is the primary consideration in determining the nature of the sterol-induced changes in the thermotropic phase behavior of the host phospholipid bilayer [18]. In addition to the above studies, recent spectroscopic evidence supports the importance of non-polar forces in determining the organization of sterol-containing phospholipid bilayers. Guerneve and Auger (19-], using a combination of magic angle spinning and ramped-amplitude cross-polarization techniques with natural abundance carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy, obtained individual resonances for different carbons along the entire dimyristoylphosphatidylcholine (DMPC) molecule for both pure phospholipid and cholesterol-containing bilayers in the LQ state. This study concluded that the effect of cholesterol is clearly dominated by sterol interactions with the acyl chains and glycerol backbone of the host PC bilayer. However, the effect of cholesterol is not uniform along the non-polar region of DMPC. DMPC glycerol carbons 1 to 3 exhibit increased orientational order and decreased mobility in the presence of cholesterol, whereas fatty acyl chain carbons exhibit both increased orientational order and increased fluctuations in the intermolecular acyl chain spacing (19-]. In addition, a deuterium wide-line NMR spectroscopic study of the effect of cholesterol on the hydrocarbon chains of 18:0 and 24:0 galactosylceramides (Galcers) in SOPC bilayers reported that the effect of cholesterol on the order parameter profiles of the hydrocarbon chains generally followed the pattern known for acyl chains of phospholipids of similar length [20]. Specifically, cholesterol incorporation significantly increased orientational order in the plateau region of both Galcer hydrocarbon chains (carbon atoms 3-10) but progressively less so toward the methyl terminus of these chains. The longer 24:0 chain, however, also exhibited a second plateau region from carbon atoms 14-23 in which the orientational order remained relatively low and constant, in contrast to the 18:0 chains, in which the orientational order decreases steeply toward the methyl terminus [20]. As an extension of earlier molecular model building, computational simulations are an approach used with increasing frequency to provide insight into the dynamics of cholesterol-phospholipid interactions. Robinson if 0/. [21], using a molecular dynamics simulation of cholesterolcontaining DMPC bilayers, determined that from carbons 3 to 14, cholesterol decreased the average number of gauche conformers while increasing the trans dihedrals
85
and hydrocarbon chain ordering in the acyl chains. This conclusion is qualitatively similar to that obtained by recent FTIR and NMR spectroscopic studies [16,20]. Moreover, the hydroxyl group of cholesterol is able to form a hydrogen bond, with either water, the carbonyl groups of the sn-l or sn-Z hydrocarbon chains, or possibly the sn-3 phosphate [21]. An energy minimization simulation of DMPC/ cholesterol bilayers (1:1 molar ratio) by Vanderkooi [6-] revealed that the minimal energy structure for DMPC-eholesterol interactions involves a hydrogen bond between the cholesterol hydroxyl group and the DMPC sn-Z carbonyl group, as well as the parallel alignment of the a face of cholesterol to the DMPC acyl chain (see Fig. 1). Again, as observed experimentally, the largest contribution to cholesterol-phospholipid interactions comes from the van der Waals forces between the cholesterol and DMPC molecules in the bilayer hydrophobic core [6-]. It is of interest that despite intense speculation and numerous investigations, there is no direct evidence in support of a hydrogen bond between the hydroxyl group of cholesterol and the carbonyl groups of the sn-l and/or sn-2 acyl chains in aqueous systems. Guerneve ef 0/. [19-] postulate that a hydrogen bond is present on the basis of the splitting of the cholesterol-containing DMPC bilayer sn-l and sn-Z carbonyl group resonances into two populations. In addition, both molecular modeling studies [6-,21] predict a similar hydrogen bond . However, no experimental data have yet been able to rule out the existence of a hydrogen bond between water and the hydroxyl group of cholesterol [2,3]. If the depth of cholesterol in the phospholipid bilayer can vary with temperature or cholesterol concentration, as has recently been suggested [21,22], the problem of identifying a particular hydrogen bond becomes even more difficult. Some evidence exists that the nature and stoichiometry of cholesrerol-PC interactions is very sensitive to the degree of unsaturarion of the hydrocarbon chains. Thus Keough and co-workers [23-25] have demonstrated that the thermotropic phase behavior of PCs containing one or two as-unsaturated fatty acyl chains can be quite different from that of cholesterol-containing DPPC bilayers. Moreover, we have unpublished work showing the dielaidoyl species of PC and phosphatidylethanolamine (PE) behave differently in the presence of cholesterol when compared with their distearoyl analogues. However, as at least some diunsaturated PCs exhibit complex, overlapping gel-ogel and gcl~LQ phase transitions in the absence of cholesterol [26], whereas DPPC does not, the differences observed in the calorimetric behavior of diunsarurated and disaturated PCs may reflect primarily the different nature of the phase transitions being monitored rather than fundamental differences in the nature of their interactions with cholesterol. In this regard, we have recently shown that the effects of cholesterol and its side-chain analogues on the thermotropic phase behavior of SOPC and DPPC bilayers are qualitatively similar when one discounts the differences in their hydrophobic thickness [10,17,18]. This
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Experimental self-assembly
result indicates that the presence of a single cis double bond in the middle of a hydrocarbon chain does not, per se, greatly alter the nature or stoichiometry of cholesterol-PC interactions. Cholesterol can, however, promote the formation of apparently homogeneous solutions of disaturated and diunsaturated PCs that would normally exhibit phase separations in the absence of sterol [27]. Lastly, the effect of cholesterol on different phospholipid headgroups has gathered little attention. Recently, only one phospholipid other than PC has been investigated. Borochov et 01. [28], using low-sensitivity differential scanning calorimetry (LS-DSC) and low-angle diffraction X-ray studies, determined that changes in phase transition enthalpy, T m and cooperativity of cholesterol-containing phosphatidylglycerol (PG) bilayers are significantly different from those observed in cholesterol/PC bilayers. Moreover, the miscibility of cholesterol in a PG bilayer is also less than that seen in PC bilayers and decreases with increasing chain length. These results support conclusions from earlier work in which the gel to LQ phase transition of bilayers composed of the phospholipids phosphatidic acid (PA), phosphatidylserine (PS) and PE each appear to exhibit unique 'changes in enthalpy, transition temperature and cooperativity with progressive increases in cholesterol content [28-30]. However, the effect of cholesterol on phospholipids other than PCs needs to examined in much more detail using modern, more sophisticated physical techniques.
Cholesterol-sphingomyelin interactions An interesting body of literature has developed regarding the interaction of cholesterol with sphingomyelin (SPM). Cholesterol is postulated to have a preferential affinity for SPM on the basis of the coordinated regulation of cholesterol and SPM levels in the membranes of eukaryotic cells; however, the molecular basis for this preferential interaction is unknown [2,3,31-]. Prior calorimetric investigations [30,32,33] of cholesterol/SPM bilayers revealed a qualitatively similar effect of cholesterol on the thermotropic phase behavior of SPM and DPPC bilayers, as. well as a similar progressive decrease in the overall transition enthalpy. However, the magnitude of these changes, as well as the absolute shifts in gel to LQ transition temperature, were quantitatively unlike those observed in PC bilayers. In monotectic binary phospholipid/SPM mixtures, cholesterol preferentially abolished the phase transition of SPM domains whether or not SPM was the high or lower melting component, indicating a. preferential interaction with the SPM species [30,34]. Similarly, prior monolayer pressure-area studies found that SPM/cholesterol mixtures exhibit decreased average molecular areas relative to comparable PC/cholesterol mixtures [35]. However, when Smaby er 01. [31-] investigated the monolayer behavior of SPM/cholesterol and PC/cholesterol mixtures which
were matched for hydrophobic length and structure, the condensing effect of cholesterol exhibited no quantitative differences between the SPM and PC monolayers at equimolar ratios. Moreover, when the sn-1 chain has a long and extended conformation, the structural requirements of the host PC or SPM monolayer sn-2 acyl chain for maximal cholesterol condensation are partially mitigated. Thus, in contrast to Lund-Katz et al. [35], Smaby etal. [31-] conclude that the basis for the preferential interaction of cholesterol with SPMs over PCs is due primarily to differences in the hydrocarbon chain length and structure [31-]. The same authors determined that acyl chain length and structure are also key parameters controlling the ability of GalCer to interact with cholesterol [36].
The accessibility of cholesterol to enzyme-catalyzed oxidation has also been used to probe the strength of cholesterol-PC and cholesterol-SPM interactions as well as to determine cholesterol-PC and cholesterol-SPM interaction stoichiometries [37]. These studies indicate that the accessibility of cholesterol to enzyme-catalyzed oxidation is greater in unsaturated than in comparable saturated monolayers of PCs or SPMs, and that cholesterol is also oxidized more readily in PC than in comparable SPM monolayers. To determine if cholesterol-SPM preferential interactions required enhanced van der Waals interactions or interfacial hydrogen bonding, Bittman et 01. [38] synthesized two stearoyl SPM analogues, both of which had a hydrogen in place of the glycerol carbon 3 hydroxyl, while at the glycerol carbon 2, one analogue had an ester group (2-0-SPM) and the other the natural SPM amide linkage (2-N-SPM). In monolayer studies the force-area isotherms for each of the pure compounds were similar and the cholesterol miscibility was approximately equal in each host monolayer. In addition, cholesterol was also able to condense the lateral packing of the various stearoyl SPM analogues to a similar degree. However, the oxidation of cholesterol by cholesterol oxidase was greatest in the 2-0-SPM analogue, indicating that the cholesterol present in the amide linked 2-N-SPM analogue monolayer is less accessible to the enzyme at the monolayer interface, presumably due to a hydrogen bond between the glycerol carbon 2 amide and the cholesterol hydroxyl group [38]. It also appears that the SPM glycerol carbon 3 hydroxyl group is not important in SPM-<:holesterol interactions.
In summary, although it appears that van der Waals and hydrophobic forces generally dominate cholesterol-PC interactions, interfacial or polar headgroup interactions may be quantitatively important in the interactions of cholesterol with SPMs and phospholipids other than PC (such as PG). Competing interfacial and hydrophobic interactions, which depend on hydrocarbon chain length, headgroup charge and hydration, may all contribute to the effect of cholesterol on host bilayer thermotropic phase behavior and organization.
Physical studies of cholesterol-phospholipid interactions McMullen and McElhaney
The lateral organization of cholesterol in PC and SPM monolayers and bilayers Beyond the microscopic interactions of cholesterol with phospholipid molecules, the macroscopic organization of cholesterol in the host phospholipid bilayer is of considerable physical and biological interest. Specifically, do cholesterol molecules exist as monomers, dimers or higher aggregates in phospholipid bilayers, and are these cholesterol molecules randomly dispersed within the bilayer or are they organized in regular arrays? One method that is used to probe the macroscopic organization of cholesterol in the bilayer involves monitoring the fluorescence intensity of fluorescent probes. Changes in fluorescence intensity are believed to coincide directly with changes in the lateral organization and spacing of the probes in the host cholesterol/phospholipid bilayer. Two different studies, using two different probes (dehydroergosterol [DHE] and diphenylhexatriene [DHP)) monitored reproducible changes in fluorescence intensity as the level of cholesterol increased from 2 to 54 mol% in LQ DMPC bilayers, thus providing compelling evidence for the ordered distribution of individual cholesterol molecules [39·,40]. The lateral distribution of cholesterol observed experimentally could be well modeled as a hexagonal superlattice with cholesterol molecules maximally separated [3tr,40]. However, as the temperature increases significantly above the gel~LQ T rn- the lateral organization of the bilayer appears to become more random [40]. Using a different probe, Laurdan (2dimethylamino-6-lauroylnaphthalene), Parasassi et al. [41] also determined that ordered microdomains are formed with particular local order and physical properties at certain critical cholesterol concentrations. Only three critical chol~sterol concentrations, however, were identified in this latter study, and one critical concentration differed from that observed in other studies. This may be due in part 'to the fact that the Laurdan probe primarily monitors th interfacial region rather than the hydrocarbon core of the lipid bilayer. Lastly, monitoring the steady-state anisotropy of the fluorescent lipid Irons-parinaric acid, Mateo el al. [42] revealed the presence of two liquid-crystalline phases, an a phase (liquid-disordered phase) and a ~ phase (liquid-ordered phase) in both DMPC and 1palmitoyl-2-0Ieoyl-phosphatidylcholine bilayers. The level of cholesterol required to form the ~ phase as well as the cholesterol levels in which the a and ~ phases coexist vary significantly for the' different PCs, however. This study again illustrates the importance of the host bilayer phase state and structure on cholesterol-phospholipid interactions and cholesterol organization. In addition to these fluorescence studies, Slone [43] has also attempted to directly monitor the lateral domain formation of cholesterol-containing PC and SPM bilayers using 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl-]amino)-23,24bisnor (NBD)-eholesterol and NBD-PC probes and epifluorescence microscopy. Both reporter molecules exhibit
87
similar partitioning into cholesterollPC bilayers and were treated as impurities within the pure phospholipid or mixed cholesterol/phospholipid monolayers [44]. Using these probes, Slotte [43] found that the liquid-condensed domains of cholesterol/DPPC and cholesterol/N-P-SPM monolayers differed in their number, size and properties. It should be noted that another study by Slone [11·] indicated that small changes to the cholesterol molecule can significantly alter the thermotropic behavior and organization of sterol-containing phospholipid bilayers. Thus, the interpretation of data derived from the use of these probes is somewhat problematic [7,11·,30]. There is also considerable indirect evidence for the presence of thermodynamically and laterally distinct cholesterolrich and cholesterol-poor domains in cholesterol-containing phospholipid bilayers. In cholesterollPC systems with disaturated chain lengths of 10:0 to 20:0, the susceptibility of cholesterol to oxidation by cholesterol oxidase was significantly higher in the di-1O:0 and di-12:0, and in the di-18:0 and 20:0 PC monolayers [45]. Moreover, by correlating the observation of laterally heterogeneous fluorescent domains in the short and long chain PC monolayers, but not in the di-14:0 to di-17:0 rc rnonolayers, Mattjus et al. reasoned that cholesterol laterally phase separates in monolayers with hydrophobic thicknesses either significantly shorter or greater than that of cholesterol, thus permitting increased cholesterol oxidation [45]. Similarly, discontinuities in the rate of oxidation of cholesterol by cholesterol oxidase at 1:1 cholesterol/PC and 2:1 cholesterol/SPM molar ratios are believed to represent the formation of nearly pure cholesterol domains [37,43]. SPM is postulated to have cooperative effects which alter the two-dimensional packing of the monolayer which in turn permits the formation of nearly pure cholesterol domains at higher cholesterol concentrations than that seen in PC monolayers [37]. Unfortunately, because the above monolayer studies were all performed at the same temperature, the phase states of the various PC/SPM systems vary from predominantly gel to LQ state, possibly altering the miscibility of cholesterol in the lipid bilayer and complicating the interpretation of these results. Fluorescent studies in DMPC bilayers suggest that cholesterol tends to be maximally separated and to exhibit regular order, depending on the temperature and on the concentration of cholesterol. The organization of cholesterol-containing PC bilayers or cholesterol/SPM bilayers however, appears to differ significantly depending on changes in the relative contributions of hydrophobic and interfacial interactions. Thus it appears that the fluorescence studies that predict regular order in PC bilayers [39·,40] are probably limited to DMPC and DPPC, as significantly longer or shorter chain PCs, or SPMs, may interact with cholesterol with different stoichiometries because cholesterol exhibits lateral phase separation in such systems [37,45]. As the plasma membrane of eukaryotic cells contains many different phospholipids and
aa
Experimental self-assembly
glycolipids, the above studies only begin to explore the possibilities of the formation and distribution of different cholesterol domains [46].
Experimental and theoretical temperature/composition diagrams: past and present To promote a complete understanding of the effect of cholesterol on phospholipid thermotropic phase behavior over a wide range of concentrations and ternperatures, many investigators have atte mpted to develop temperature/composition phase diagrams of cholesterol/phospholipid model bilayers. One notable recent attempt is that of Vist and Davis [4], who employed HSDSC and zH-NMR spectroscopy to determine the phase boundaries of mixtures of cholesterol and chain-perdeuteratcd DPPC at cholesterol concentrations up to 25 mol%. They identified three phases: two pure phospholipid phases, the Lll (gel) and La (liquid-disordered) phases, which co-ex ist up to approximately 22 mol % cholesterol, and a cholesterol-containing phospholipid liquid-ordered (L o) or f3-phase. The f3-phase was postulated to form at a eutectic point of 7.5 mol% cholesterol, then to co-exist with fhe La and LfJ phases from 7.5 to 22 mol% cholesterol, becoming the sole ph ase above 22 mol% cholesterol. Above 22 mol% cholesterol the f3-phase was described as physically homogeneous with respect to temperature and cholesterol content. This phase diagram was thought to have universal form independent of the chemical structure of the PC molecule [47]. However, recently we reported 114,48-] that the appearance of a eutectic point at 7.5 mol% is an artifact of the different cholesterol-dependent shifts in the T rn- cooperativity and enthalpy of the cholesterol-rich and cholesterol-poor domains, which actually coexist at cholesterol concentrations as low as 1-2 mol%. Moreover, the phase boundaries and T m of the cholesterol-rich domain vary with the chain length of the PC bilayer [14,48-]. In addition, both Rein) er 01. [22] and Huang et 01. [49], using a combination of 13C_ and zH-NMR and FTIR spectroscopic experiments, revealed significant changes in the so-called homogeneous L o or f3-ph ase structure of PC bilayers with respect to temperature and PC acyl chain length. These changes are manifest as changes in the conformational order of the phospholipid acyl chains as well as in the relative position of cholesterol in the bilayer [22,48-,49]. Integrating these findings, we proposed [48-] a new and more complete phase diagr am which docs not include a eutectic point. It consists of the two pure phospholipid phases, La and LfJ, which exist upto 22 mol% cholesterol, and the two liquid-ordered or f3-phases, termed Lou and Loll, which co-exist from approximately 1 to 50 mol% but whose boundaries vary with temperature and depend on the chain length of the host PC bilayer [48-].
Applications The growing evidence for distinct cholesterol-rich or cholesterol-poor domains in model phospholipid bilayers
has provided investigators with some interesting insights into biological phenomena. For example, Pott and Dufourc [50] correlated ZH- and 3IP-NMR spectroscopic evidence for domain boundaries within the f3-phase to explain the rnclittin-induced disruption of cholesterol-containing DPPC bilayers. Therein, they propose a model that describes how the entry of the peptide into the bilayer is permitted by domain boundaries within the LQ phase, which then triggers membrane disruption. Phase separation in cholesterol-containing bilayers is also postulated to account for the domain-induced budding process in vesicles, or possibly to be a mechanism for the sorting of proteins into cholesterol-rich domains of membranes which exhibit increased hydrophobic thickness [48-,51,52]. Another interesting recent investigation revealed that quaternary ammonium based surfactants selectively disrupt cholesterol-poor bilayers [53]. Quaternary ammonium salts cannot cross the interface of the tightly packed non-polar region of cholesterol-containing lipid bilayers although proton .ionizable amine surfactants can shed their charge, thus permitting penetration into both cholesterol-rich and cholesterol-poor domains of the bilayer. These authors demonstrate the complexity and specificity of interfacial interactions and propose how to exploit this complexity by designing antibiotics with enhanced cytotoxicity towards bacteria and fungi compared to mammalian cells. Finally, small changes in cholesterol content may trigger large local changes in bilayer packing, as illustrated in fluorescence studies, which may explain, for example, why changes in cellular activities can occur when no overall change in bilayer fluidity is apparent [39-,40]. Cholesterol is also a prominent component of many liposomal formulations. Cholesterol is typically used to increase the retention of hydrophilic drugs while also inceasing bilayer rigidity and resistance to in vivo liposomal degradation [54]. Recently cholesterol has also been used to modulate liposomal pH-sensitivity [55] and Iiposomal fusion [56], and has been derivatized to produce cationic liposomes or act as lipid anchors for covalently-linked oligonucleotides [57,58]. Despite its widespread usc, the reported effects of cholesterol on liposomal behavior are often inconsistent [54,56]. It is now clear that the thermotropic phase behavior and organization of cholesterol-containing phosphlipid bilayers may vary dramatically with both vesicular contents and host phosphlipid cornpsition. Thus, continued use of cholesterol to optimize liposome encapsulation, stability and release of genetic materials or drugs will require careful consideration of cholesterol-lipid interactions.
Conclusion Although cholesterol/phospholipid systems have been studied for many years, it is clear that our understanding of the molecular basis of cholesterol-phospholipid interactions remains incomplete. However, the recent resurgence of interest in these biologically relevant model membrane systems, and the increasingly sophisticated biophysical
Physical studies of cholesterol-phospholipid interactions McMullen and McElhaney
and biochemical techniques being applied to them, have resulted in considerable progress in the understanding of a number of long-standing problems in this field. The complete resolution of the many outstanding issues in this area will continue to require the coordinated application of a wide variety of physical techniques to systematically study a broader range of host lipid bilayers existing in a comparable range of physical states.
Acknowledgements We would like to stress the importance of references t4 and t6 in relation 10 the review and its contents. Unfortunately these articles could not be bullctcd in the reference section as they were not published within the annual period of review,
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Davis PJ, Keough KMW: Differential scanning calorimetric studies of aqueous dispersions of mixtures of cholesterol with some mixed-acid and sln9le-acld phosphatldylchollne. Biochemistry 1983, 22:6334-6340.
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Keough KMW, Griffin B, Matthews PLJ: Phosphatlylchollnecholesterol Interactions: bilayers of heteroacld lipIds containing lin oleate lose calorimetric transitions at low cholesterol concentration. Biochim Biophys Acta 1989, 983:51-55.
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Kariel N, Davidson E, Keough KMW: Cholesterol does not remove the gel to liquid-crystalline phase transition of phosphatldylcholines containing two polyenolc acyl chains. Biochim Biophys Acta 1991, 1062:70-76.
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Silvius JR: Cholesterol modulation of lipid Intermixing In phospholipid and glycosphlngolipld mixtures_ Evaluation using fluorescent lipid probes and bromlnated lipid quenchers. Biochemistry 1992, 31:3398-3408.
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McMullen TPW, Lewis RNAH, McElhaneyRN: Comparative differential scanning calorimetric and FTIR and 31P-NMR spectroscopic studies of the effects of cholesterol and androstenol on the thermotropic phase behavior and organization of phosphatidylchollne bilayers. Biophys ) 1994, 66:741-752. McMullen TPW, Vilcheze C, McElhaneyRN, Bittman R: Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatldylcholine thermotropic phase behavior. Biophys ) 1995, 69:169-176.
Siotte JP: Effect of sterol strudure on molecular Interactions and lateral domain formation In monolayers containing dipalmitoylphosphatldylcholine_ Biochim Biophys Acta 1995, 1237:127-134. A systematicexamination of the effect of modificationson the hydroxyl group of cholesterol, as well as to the tetracyclic ring system double bond, on domainformationin DPPC monolayers. The author,in detailing data on sterol miscibility in DPPC monolayersand lateral sterol organization, demonstrates
11. •
Li a-T, Das NP: Comparison of the effects of cholesterol and oxysterols on phospholipid bilayer microheterogeneity: a study of fluorescence lifetime distributions. Arch Biochem Biophys 1994,315:473-478.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest
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Smaby JM, Brockman Hl, Brown RE: Cholesterol's Interfacial Interadions with sphlngomyellns and phosphatldylcholines: hydrocarbon chain strudure determines the magnitude of condensation. Biochemistry 1994, 33:9135-9142. This paper illustrates the importance of matching phase state and hydrocarbon chain structure when examining the condensing effect of cholesterol in monolayers composed of different lipid species. Moreover, the authors conclude that both headgroup and hydrocarbon structure is important in mediating cholesterol-SPM or cholesterol-PC interactions.
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Calhoun WI, Shipley GG: Sphingomyelin-lecithin bilayers and their Interadion with cholesterol. Biochemistry 1979, 18:1717-1721.
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Estep TN, Mountcastle DD, Barenholz Y, Biltonen Rl, Thompson TE: Thermal behavior of synthetic sphingomyelin-cholesterol dispersions. Biochemistry 1979, 18:2112-2117.
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Ali S, Smaby JM, Brockman HL, Brown RE: Cholesterol's Interfacial Interactions with galadosylceramldes. Biochemistry 1994, 33:~900-2906.
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Mattjus P, Siotte JP: Availability for enzyme-catalyzed oxidation of cholesterol In mixed monolayers containing both phosphatidylcholine and sphingomyelin. Chem Phys Lipids 1994, 71 :73-81.
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Bittman R, Kasireddy CR, Mattjus P, Siotte JP: Interadlon of cholesterol with sphingomyelin In monolayers and vesicles. Biochemistry 1994, 33:11776-11781.
Chong Pl-G: Evidence for regular distribution of sterols In liquid-crystalline phosphatidylchollne bilayers. Proc Nat! Acad Sci 1994, 91:10069-10073. This paper provides compelling evidencefor the regulardistribution of sterols in La DMPC bilayers by using a probe which closely resembles the cholesterol molecule. The data is carefully analyzed in order to illustrate how changes in fluorescence intensity relate to changes in the macroscopic organizationof the sterol-DMPC bilayer.
or N-palmitoylsphingomyelin. Biochim Biophys Acta 1995, 1235:419-427.
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Siotte JP, Malljus P: Visualization of lateral phases In cholesterol and phosphatidylcholine monolayers at the air/water Interface - a comparative study with two different reporter molecules. Biochim Biophys Acta 1995, 1254:22-29.
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Malljus P, Hedstrom G, Siotte JP: Monolayer Interadlon of cholesterol with phosphatidylchofines: effects of phospholipid acyl chain length. Chem Phys Lipids 1994, 74:195-203.
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McMullen TPW, McElhaney RN: New aspects of the Interaction of cholesterol with dipalmitoylphosphatidylcholine bilayers as revealed by high-sensitivity differential scanning calorimetry. Biochim Biophys Acta 1995, 1234:90-98. By correlating extensive HS-DSC data with prior spectroscopic analysis, we propose a new cholesterol-PC phase diagram which exhibits cholesterolrich and cholesterol-poor domain formation at very low cholesterol concentrations, and chain-length dependent cholesterol-rich Tm and phase boundaries. This analysisshows that bilayerscomposed of different phospholipids may exhibit significantly different phase behavior and organization.
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Huang T-H, Lee CWB, Das Gupta SK, Blume A, Griffin RG: A 13C and 2H nuclear magnetic resonance study of phosphatldylcholine/cholesterollnteradions: charaderlzation of liquid-gel phases. Biochemistry 1993, 32:13277-13287.
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Dobereiner H-G, Kas J, Noppl D, Sprenger I, Sackmann E: Budding and fission of vesicles. Biophys J 1993, 65:1396-1403.
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Bretscher MS, Munro S: Cholesterol and the golgl apparatus. Science 1993, 261 :1280-1281.
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Watanabe S, Regen SL: Quaternary ammonium-based surfactants that can recognize cholesterol-rich membranes and proton-Ionizable analogs that cannot JAm Chem Soc 1994, 116:5762-5765.
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Grit M, Crommelin DJA: Chemical stability of Iiposomes: Implications for their physical stability. Chem Phys Lipids 1993, 64:3-18.
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Hazemoto N, Harada M, Suzuki S, Kaiho F, Haga M, Kato Y: Effects of phosphotidylcholine and cholesterol on pH-sensitive Iiposomes. Chem Pharm Bull Tokyo 1993,41:1003-1006.
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Brock TG, Nagaprakash K, Margolis 01, Smolen JE: Modelling degranulation with IIposomes: effect of lipid composition on membrane fusIon. J Membrane BioI 1994, 141:139-148.
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Tang D, Van Der Meer BW, Simon Chen SoY: Evidence for a regular distribution of cholesterol In phospholipid bilayers from dlphenylhexatrlene fluorescence. Biophys J 1995, 68:1944-1955.
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Mateo CR, Acuna AU, Brochon J-C: liquid-crystalline phases of cholesterol/lipid bilayers as revealed by the florescence of trBns-parlnaric acid. Biophys J 1995, 68:978-987.
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Slotte JP: Lateral domain formation In mixed monolayers containing cholesterol and dlpalmitoylphosphatldylcholine
Address Department of Biochemistry. University of Alberta, Edmonton. Alberta, Canada T6G 2H7 Current Opinion in Colloid & Interface SCience 1996. 1 :83-90
e Current Science Ltd ISSN 1359-0294 Abbreviations DMPC dimyristoylphosphatidylcholine DPPC dipalmitoylphosphatidylcholine LQ liquid crystalline PC phosphatidylcholine SPM sphingomyelin
Introduction Cholesterol or closely rel ated sterols are major lipid components of the plasma membranes of most eukaryotic cells, and are 'also found in lower concentrations in many intracellular membranes. Cholesterol has numerous different functions in eukaryotic cells, one of its primary roles being to modulate the physical properties of the plasma membrane phospholipid bilayer (see [1-3] for reviews). Thus a large number of studies of cholesterol/phospholipid bilayer model membranes have been carried out, using a wide range of physicochemical techniques [2-5]. Briefly, cholesterol incorporation into bilaycrs has four effects: first, it broadens and eventually eliminates the cooperative gel to liqu id-crystalline phase transition of phospholipid bilayers; second, it decreases (increases) the area per molecule of the liquid-crystalline (gel) st ate monolayers; third, it increases (decreases) the orientational order of the hydrocarbon chains of liquid-crystalline (gel) bilayers; and fourth, it decreases (increases) the passive permeability of phospholipid bilayers above (below) their gel to liquidcrystalline ph ase transition temperatures. At relatively low cholesterol concentrations (up to 22 mol%), there exist two thermodynamically distinct domains, a cholesterol-poor domain with properties similar to those of the pure phospholipid bilayer, and a cholesterol-rich domain. The cholesterol-rich domain, known as the liquid-ordered phase or fJ-phase, is the only phase which exists at cholesterol levels <22 mol% in phospholipid bilayers. It
is characterized by phospholipid intra- and intermolecular motional rates which are similar to those of a fluid phospholipid bilayer, but w hich contain hydrocarbon cha in orientationa I order and phospholipid area compressibility values th at more closely resemble the gel ph ase of a pure phospholipid. Although we currently know a considerable amount about the overall effect of cholesterol on phospholipid bilayer phase transitions, our understanding , of phospholipidcholesterol interactions at the molecular level is incomplete. The largest contribution to cholesterol-phospholipid interactions appears to be from van der Waals forces and hydrophobic forces, although hydrogen bonding to the polar headgroup and interfacial reg ions of the host lipid bilayer m ay be of considerable importance, especially in the sphingolipids and anionic phospholipids. Moreover, depending on temperature and cholesterol concentration, as well as on the headgroup and hydrocarbon ch ain composition of the host bilayer, cholesterol may exist in regularly distributed arrays or in laterally segregated cholesterol-rich or cholesterol-poor domains of variable number and size. Some of the important questions driving recent investigations include the following: Does cholesterol exhibit phospholipid-specific interactions? Does cholesterol exist as monomers or as aggregates in phospholipid bilayers, and, if the former, are these monomers dispersed randoml y or in ordered arrays within the plane of the bil ayer? Is the structure of the cholesterol-rich liquid-ordered phase, whi ch is usually considered to be homogeneous, actually dependent upon cholesterol concentration and temperature? While these questions may persist for some time, recent work clearly demonstrates that even simple binary cholesterol/phospholipid mixtures may exhibit very complex thermotropic phase behavior. Moreover, both the thermotropic phase behavior and the molecular organization of sterol-containing lipid bilayers is acutely sensitive to differences in the chemical structure of both the sterol and the phospholipid with which it is interacting.
Sterol-phospholipid Interactions Cholesterol has a dramatically different structure than that of the phospholipids and sphingolipids that compose eukaryotic membranes. Cholesterol consists of a planar tetracyclic ring system with an alkyl side-chain extending towards the bilayer center and a hydroxyl group located at the polar/non-polar interface, as shown in Figure 1 [2,3,5,6-]. Most structural and stereochemical alterations to the cholesterol molecule result in some loss of its ability to produce its characteristic effects on phospholipid bilayers [2,7-9]. However, the relat ionship between the chemical structure of sterols and the magnitude and direction of their various effects on the physical properties of model membrane systems is not always clear or
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Experimental self'assembly
consistent [2,9,10,11-]. This may be due in part either to experimental or interpretational errors on the part of different investigators examining different sterols in a variety of model and biological membranes, or to real differences in host phospholipid bilayer physical properties. For example, McMullen er 01. [9) have determined that the effects of cholesterol and of androstenol (a sterol which lacks the alkyl side-chain) on phosphatidylcholine (PC) bilayers are quite different, but that the type and magnitude of these differences are dependent on the chain length of the host PC bilayer. Specifically, the relative importance of the alkyl side-chain of cholesterol was found to depend on the thickness of the host phospholipid bilayer. It is clear, however, that none of the sterol analogues examined was able to quantitatively duplicate cholesterol-like effects on any of the host bilayers tested. Even the position of the double bond in the sterol ring system, initially believed to be of little importance, was found to be important in mediating cholesterol-like effects in certain model membranes [7,11-]. These results indicate that sterol miscibility and organization in the host PC monolayer or bilayer are sensitive to even small changes in the structure of the cholesterol molecule. Thus it is not surprising that sterol -related molecules, such as the oxysterols or vitamin D 3, do not always have cholesterol-like effects on the physicochemical properties of PC bilayers into which they are incorporated [12,13].
Figure 1
Cholesterol-phospholipid interactions Although the techniques chosen to investigate the phase behavior and organization of cholesterol-containing bilayers or monolayers are large in number, the vast majority of the past and present work on cholesterol-phospholipid interactions involves one class of phospholipids, the PCs. Recently, one of the most important factors governing the phase behavior of cholesterol/PC mixtures, namely, the hydrophobic mismatch effect, has been revealed by Mclvlullen et 01. (14). Using high-sensitivity differential scanning calorimetry (HS-DSC), these workers demonstrated that cholesterol progressively decreases the host bilayer transition temperature (Tm) of the cholesterol-rich domains of PC bilayers with hydrocarbon chains longer than 17:0, while increasing the chain-melting T m of PCs with chain lengths shorter than 17:0 (specific phosphatidylcholines are designated by the notation n:O PC, where n is the number of carbon atoms per hydrocarbon chain and zero indicates the absence of double bonds). As the hydrophobic thickness of the PC bilayer decreases by approximately one-third upon chain melting (due to introduction of gauche conformers along the acyl chain), the relative stabilities of the gel and liquid-crystalline (LQ) phases should be least affected when the length of another molecule (i.e. cholesterol) incorporated into the bilayer matches the mean hydrophobic thickness of the bilayer (a hydrophobic length midway between the gel and LQ states) [15]. Molecules with a greater hydrophobic length than the mean would tend todifferentially stabilize
lateral view of a cholesterollDMPC dimer. The a face of cholesterol faces towards the DMPC molecule (into the page). The oxygen atoms are shaded. P indicates the phosphorous group, N indicates nitrogen. Note that the hydrogen bond denoled by the dolled line (cholesterol hydroxyl group to the sn·2 carbonyl oxygen) is purely speculative. (Adapted from [6°])
the gel phase, thus increasing the T m- and molecules with a smaller hydrophobic moiety than the mean would tend to de-stabilize the gel phase. In fact, these authors argued that the reason that cholesterol did not alter the T m of 17:0 PC bilayers is that the mean hydrophobic length of the host lipid molecule and the length of the cholesterol molecule are equal. Indeed, the Fourier transform infrared (FTIR) spectroscopic data of Chia er 01. [16] confirm that bilayers of longer-chain PCs exhibited considerably more conformational disorder in the gel state than do shorter-chain PC bilayers with a comparable level of cholesterol.
Physical studies of cholesterol-phospholipid interactions McMullen and McElhaney
A number of additional studies support the existence of the hydrophobic mismatch effect in l-stearoyl-z-oleoylphosphatidylcholine (SOPC) as well as in dipalmitoylphosphatidylcholine (DPPC) bilayers containing various alkyl side-chain analogues of cholesterol [10,17,18]. In each case the magnitude of sterol/phospholipid hydrophobic mismatch, whether due to variations in the length of the alkyl side-chain of the cholesterol analogue or in the length of the acyl chains of the phospholipid, is the primary consideration in determining the nature of the sterol-induced changes in the thermotropic phase behavior of the host phospholipid bilayer [18]. In addition to the above studies, recent spectroscopic evidence supports the importance of non-polar forces in determining the organization of sterol-containing phospholipid bilayers. Guerneve and Auger (19-], using a combination of magic angle spinning and ramped-amplitude cross-polarization techniques with natural abundance carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy, obtained individual resonances for different carbons along the entire dimyristoylphosphatidylcholine (DMPC) molecule for both pure phospholipid and cholesterol-containing bilayers in the LQ state. This study concluded that the effect of cholesterol is clearly dominated by sterol interactions with the acyl chains and glycerol backbone of the host PC bilayer. However, the effect of cholesterol is not uniform along the non-polar region of DMPC. DMPC glycerol carbons 1 to 3 exhibit increased orientational order and decreased mobility in the presence of cholesterol, whereas fatty acyl chain carbons exhibit both increased orientational order and increased fluctuations in the intermolecular acyl chain spacing (19-]. In addition, a deuterium wide-line NMR spectroscopic study of the effect of cholesterol on the hydrocarbon chains of 18:0 and 24:0 galactosylceramides (Galcers) in SOPC bilayers reported that the effect of cholesterol on the order parameter profiles of the hydrocarbon chains generally followed the pattern known for acyl chains of phospholipids of similar length [20]. Specifically, cholesterol incorporation significantly increased orientational order in the plateau region of both Galcer hydrocarbon chains (carbon atoms 3-10) but progressively less so toward the methyl terminus of these chains. The longer 24:0 chain, however, also exhibited a second plateau region from carbon atoms 14-23 in which the orientational order remained relatively low and constant, in contrast to the 18:0 chains, in which the orientational order decreases steeply toward the methyl terminus [20]. As an extension of earlier molecular model building, computational simulations are an approach used with increasing frequency to provide insight into the dynamics of cholesterol-phospholipid interactions. Robinson if 0/. [21], using a molecular dynamics simulation of cholesterolcontaining DMPC bilayers, determined that from carbons 3 to 14, cholesterol decreased the average number of gauche conformers while increasing the trans dihedrals
85
and hydrocarbon chain ordering in the acyl chains. This conclusion is qualitatively similar to that obtained by recent FTIR and NMR spectroscopic studies [16,20]. Moreover, the hydroxyl group of cholesterol is able to form a hydrogen bond, with either water, the carbonyl groups of the sn-l or sn-Z hydrocarbon chains, or possibly the sn-3 phosphate [21]. An energy minimization simulation of DMPC/ cholesterol bilayers (1:1 molar ratio) by Vanderkooi [6-] revealed that the minimal energy structure for DMPC-eholesterol interactions involves a hydrogen bond between the cholesterol hydroxyl group and the DMPC sn-Z carbonyl group, as well as the parallel alignment of the a face of cholesterol to the DMPC acyl chain (see Fig. 1). Again, as observed experimentally, the largest contribution to cholesterol-phospholipid interactions comes from the van der Waals forces between the cholesterol and DMPC molecules in the bilayer hydrophobic core [6-]. It is of interest that despite intense speculation and numerous investigations, there is no direct evidence in support of a hydrogen bond between the hydroxyl group of cholesterol and the carbonyl groups of the sn-l and/or sn-2 acyl chains in aqueous systems. Guerneve ef 0/. [19-] postulate that a hydrogen bond is present on the basis of the splitting of the cholesterol-containing DMPC bilayer sn-l and sn-Z carbonyl group resonances into two populations. In addition, both molecular modeling studies [6-,21] predict a similar hydrogen bond . However, no experimental data have yet been able to rule out the existence of a hydrogen bond between water and the hydroxyl group of cholesterol [2,3]. If the depth of cholesterol in the phospholipid bilayer can vary with temperature or cholesterol concentration, as has recently been suggested [21,22], the problem of identifying a particular hydrogen bond becomes even more difficult. Some evidence exists that the nature and stoichiometry of cholesrerol-PC interactions is very sensitive to the degree of unsaturarion of the hydrocarbon chains. Thus Keough and co-workers [23-25] have demonstrated that the thermotropic phase behavior of PCs containing one or two as-unsaturated fatty acyl chains can be quite different from that of cholesterol-containing DPPC bilayers. Moreover, we have unpublished work showing the dielaidoyl species of PC and phosphatidylethanolamine (PE) behave differently in the presence of cholesterol when compared with their distearoyl analogues. However, as at least some diunsaturated PCs exhibit complex, overlapping gel-ogel and gcl~LQ phase transitions in the absence of cholesterol [26], whereas DPPC does not, the differences observed in the calorimetric behavior of diunsarurated and disaturated PCs may reflect primarily the different nature of the phase transitions being monitored rather than fundamental differences in the nature of their interactions with cholesterol. In this regard, we have recently shown that the effects of cholesterol and its side-chain analogues on the thermotropic phase behavior of SOPC and DPPC bilayers are qualitatively similar when one discounts the differences in their hydrophobic thickness [10,17,18]. This
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Experimental self-assembly
result indicates that the presence of a single cis double bond in the middle of a hydrocarbon chain does not, per se, greatly alter the nature or stoichiometry of cholesterol-PC interactions. Cholesterol can, however, promote the formation of apparently homogeneous solutions of disaturated and diunsaturated PCs that would normally exhibit phase separations in the absence of sterol [27]. Lastly, the effect of cholesterol on different phospholipid headgroups has gathered little attention. Recently, only one phospholipid other than PC has been investigated. Borochov et 01. [28], using low-sensitivity differential scanning calorimetry (LS-DSC) and low-angle diffraction X-ray studies, determined that changes in phase transition enthalpy, T m and cooperativity of cholesterol-containing phosphatidylglycerol (PG) bilayers are significantly different from those observed in cholesterol/PC bilayers. Moreover, the miscibility of cholesterol in a PG bilayer is also less than that seen in PC bilayers and decreases with increasing chain length. These results support conclusions from earlier work in which the gel to LQ phase transition of bilayers composed of the phospholipids phosphatidic acid (PA), phosphatidylserine (PS) and PE each appear to exhibit unique 'changes in enthalpy, transition temperature and cooperativity with progressive increases in cholesterol content [28-30]. However, the effect of cholesterol on phospholipids other than PCs needs to examined in much more detail using modern, more sophisticated physical techniques.
Cholesterol-sphingomyelin interactions An interesting body of literature has developed regarding the interaction of cholesterol with sphingomyelin (SPM). Cholesterol is postulated to have a preferential affinity for SPM on the basis of the coordinated regulation of cholesterol and SPM levels in the membranes of eukaryotic cells; however, the molecular basis for this preferential interaction is unknown [2,3,31-]. Prior calorimetric investigations [30,32,33] of cholesterol/SPM bilayers revealed a qualitatively similar effect of cholesterol on the thermotropic phase behavior of SPM and DPPC bilayers, as. well as a similar progressive decrease in the overall transition enthalpy. However, the magnitude of these changes, as well as the absolute shifts in gel to LQ transition temperature, were quantitatively unlike those observed in PC bilayers. In monotectic binary phospholipid/SPM mixtures, cholesterol preferentially abolished the phase transition of SPM domains whether or not SPM was the high or lower melting component, indicating a. preferential interaction with the SPM species [30,34]. Similarly, prior monolayer pressure-area studies found that SPM/cholesterol mixtures exhibit decreased average molecular areas relative to comparable PC/cholesterol mixtures [35]. However, when Smaby er 01. [31-] investigated the monolayer behavior of SPM/cholesterol and PC/cholesterol mixtures which
were matched for hydrophobic length and structure, the condensing effect of cholesterol exhibited no quantitative differences between the SPM and PC monolayers at equimolar ratios. Moreover, when the sn-1 chain has a long and extended conformation, the structural requirements of the host PC or SPM monolayer sn-2 acyl chain for maximal cholesterol condensation are partially mitigated. Thus, in contrast to Lund-Katz et al. [35], Smaby etal. [31-] conclude that the basis for the preferential interaction of cholesterol with SPMs over PCs is due primarily to differences in the hydrocarbon chain length and structure [31-]. The same authors determined that acyl chain length and structure are also key parameters controlling the ability of GalCer to interact with cholesterol [36].
The accessibility of cholesterol to enzyme-catalyzed oxidation has also been used to probe the strength of cholesterol-PC and cholesterol-SPM interactions as well as to determine cholesterol-PC and cholesterol-SPM interaction stoichiometries [37]. These studies indicate that the accessibility of cholesterol to enzyme-catalyzed oxidation is greater in unsaturated than in comparable saturated monolayers of PCs or SPMs, and that cholesterol is also oxidized more readily in PC than in comparable SPM monolayers. To determine if cholesterol-SPM preferential interactions required enhanced van der Waals interactions or interfacial hydrogen bonding, Bittman et 01. [38] synthesized two stearoyl SPM analogues, both of which had a hydrogen in place of the glycerol carbon 3 hydroxyl, while at the glycerol carbon 2, one analogue had an ester group (2-0-SPM) and the other the natural SPM amide linkage (2-N-SPM). In monolayer studies the force-area isotherms for each of the pure compounds were similar and the cholesterol miscibility was approximately equal in each host monolayer. In addition, cholesterol was also able to condense the lateral packing of the various stearoyl SPM analogues to a similar degree. However, the oxidation of cholesterol by cholesterol oxidase was greatest in the 2-0-SPM analogue, indicating that the cholesterol present in the amide linked 2-N-SPM analogue monolayer is less accessible to the enzyme at the monolayer interface, presumably due to a hydrogen bond between the glycerol carbon 2 amide and the cholesterol hydroxyl group [38]. It also appears that the SPM glycerol carbon 3 hydroxyl group is not important in SPM-<:holesterol interactions.
In summary, although it appears that van der Waals and hydrophobic forces generally dominate cholesterol-PC interactions, interfacial or polar headgroup interactions may be quantitatively important in the interactions of cholesterol with SPMs and phospholipids other than PC (such as PG). Competing interfacial and hydrophobic interactions, which depend on hydrocarbon chain length, headgroup charge and hydration, may all contribute to the effect of cholesterol on host bilayer thermotropic phase behavior and organization.
Physical studies of cholesterol-phospholipid interactions McMullen and McElhaney
The lateral organization of cholesterol in PC and SPM monolayers and bilayers Beyond the microscopic interactions of cholesterol with phospholipid molecules, the macroscopic organization of cholesterol in the host phospholipid bilayer is of considerable physical and biological interest. Specifically, do cholesterol molecules exist as monomers, dimers or higher aggregates in phospholipid bilayers, and are these cholesterol molecules randomly dispersed within the bilayer or are they organized in regular arrays? One method that is used to probe the macroscopic organization of cholesterol in the bilayer involves monitoring the fluorescence intensity of fluorescent probes. Changes in fluorescence intensity are believed to coincide directly with changes in the lateral organization and spacing of the probes in the host cholesterol/phospholipid bilayer. Two different studies, using two different probes (dehydroergosterol [DHE] and diphenylhexatriene [DHP)) monitored reproducible changes in fluorescence intensity as the level of cholesterol increased from 2 to 54 mol% in LQ DMPC bilayers, thus providing compelling evidence for the ordered distribution of individual cholesterol molecules [39·,40]. The lateral distribution of cholesterol observed experimentally could be well modeled as a hexagonal superlattice with cholesterol molecules maximally separated [3tr,40]. However, as the temperature increases significantly above the gel~LQ T rn- the lateral organization of the bilayer appears to become more random [40]. Using a different probe, Laurdan (2dimethylamino-6-lauroylnaphthalene), Parasassi et al. [41] also determined that ordered microdomains are formed with particular local order and physical properties at certain critical cholesterol concentrations. Only three critical chol~sterol concentrations, however, were identified in this latter study, and one critical concentration differed from that observed in other studies. This may be due in part 'to the fact that the Laurdan probe primarily monitors th interfacial region rather than the hydrocarbon core of the lipid bilayer. Lastly, monitoring the steady-state anisotropy of the fluorescent lipid Irons-parinaric acid, Mateo el al. [42] revealed the presence of two liquid-crystalline phases, an a phase (liquid-disordered phase) and a ~ phase (liquid-ordered phase) in both DMPC and 1palmitoyl-2-0Ieoyl-phosphatidylcholine bilayers. The level of cholesterol required to form the ~ phase as well as the cholesterol levels in which the a and ~ phases coexist vary significantly for the' different PCs, however. This study again illustrates the importance of the host bilayer phase state and structure on cholesterol-phospholipid interactions and cholesterol organization. In addition to these fluorescence studies, Slone [43] has also attempted to directly monitor the lateral domain formation of cholesterol-containing PC and SPM bilayers using 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl-]amino)-23,24bisnor (NBD)-eholesterol and NBD-PC probes and epifluorescence microscopy. Both reporter molecules exhibit
87
similar partitioning into cholesterollPC bilayers and were treated as impurities within the pure phospholipid or mixed cholesterol/phospholipid monolayers [44]. Using these probes, Slotte [43] found that the liquid-condensed domains of cholesterol/DPPC and cholesterol/N-P-SPM monolayers differed in their number, size and properties. It should be noted that another study by Slone [11·] indicated that small changes to the cholesterol molecule can significantly alter the thermotropic behavior and organization of sterol-containing phospholipid bilayers. Thus, the interpretation of data derived from the use of these probes is somewhat problematic [7,11·,30]. There is also considerable indirect evidence for the presence of thermodynamically and laterally distinct cholesterolrich and cholesterol-poor domains in cholesterol-containing phospholipid bilayers. In cholesterollPC systems with disaturated chain lengths of 10:0 to 20:0, the susceptibility of cholesterol to oxidation by cholesterol oxidase was significantly higher in the di-1O:0 and di-12:0, and in the di-18:0 and 20:0 PC monolayers [45]. Moreover, by correlating the observation of laterally heterogeneous fluorescent domains in the short and long chain PC monolayers, but not in the di-14:0 to di-17:0 rc rnonolayers, Mattjus et al. reasoned that cholesterol laterally phase separates in monolayers with hydrophobic thicknesses either significantly shorter or greater than that of cholesterol, thus permitting increased cholesterol oxidation [45]. Similarly, discontinuities in the rate of oxidation of cholesterol by cholesterol oxidase at 1:1 cholesterol/PC and 2:1 cholesterol/SPM molar ratios are believed to represent the formation of nearly pure cholesterol domains [37,43]. SPM is postulated to have cooperative effects which alter the two-dimensional packing of the monolayer which in turn permits the formation of nearly pure cholesterol domains at higher cholesterol concentrations than that seen in PC monolayers [37]. Unfortunately, because the above monolayer studies were all performed at the same temperature, the phase states of the various PC/SPM systems vary from predominantly gel to LQ state, possibly altering the miscibility of cholesterol in the lipid bilayer and complicating the interpretation of these results. Fluorescent studies in DMPC bilayers suggest that cholesterol tends to be maximally separated and to exhibit regular order, depending on the temperature and on the concentration of cholesterol. The organization of cholesterol-containing PC bilayers or cholesterol/SPM bilayers however, appears to differ significantly depending on changes in the relative contributions of hydrophobic and interfacial interactions. Thus it appears that the fluorescence studies that predict regular order in PC bilayers [39·,40] are probably limited to DMPC and DPPC, as significantly longer or shorter chain PCs, or SPMs, may interact with cholesterol with different stoichiometries because cholesterol exhibits lateral phase separation in such systems [37,45]. As the plasma membrane of eukaryotic cells contains many different phospholipids and
aa
Experimental self-assembly
glycolipids, the above studies only begin to explore the possibilities of the formation and distribution of different cholesterol domains [46].
Experimental and theoretical temperature/composition diagrams: past and present To promote a complete understanding of the effect of cholesterol on phospholipid thermotropic phase behavior over a wide range of concentrations and ternperatures, many investigators have atte mpted to develop temperature/composition phase diagrams of cholesterol/phospholipid model bilayers. One notable recent attempt is that of Vist and Davis [4], who employed HSDSC and zH-NMR spectroscopy to determine the phase boundaries of mixtures of cholesterol and chain-perdeuteratcd DPPC at cholesterol concentrations up to 25 mol%. They identified three phases: two pure phospholipid phases, the Lll (gel) and La (liquid-disordered) phases, which co-ex ist up to approximately 22 mol % cholesterol, and a cholesterol-containing phospholipid liquid-ordered (L o) or f3-phase. The f3-phase was postulated to form at a eutectic point of 7.5 mol% cholesterol, then to co-exist with fhe La and LfJ phases from 7.5 to 22 mol% cholesterol, becoming the sole ph ase above 22 mol% cholesterol. Above 22 mol% cholesterol the f3-phase was described as physically homogeneous with respect to temperature and cholesterol content. This phase diagram was thought to have universal form independent of the chemical structure of the PC molecule [47]. However, recently we reported 114,48-] that the appearance of a eutectic point at 7.5 mol% is an artifact of the different cholesterol-dependent shifts in the T rn- cooperativity and enthalpy of the cholesterol-rich and cholesterol-poor domains, which actually coexist at cholesterol concentrations as low as 1-2 mol%. Moreover, the phase boundaries and T m of the cholesterol-rich domain vary with the chain length of the PC bilayer [14,48-]. In addition, both Rein) er 01. [22] and Huang et 01. [49], using a combination of 13C_ and zH-NMR and FTIR spectroscopic experiments, revealed significant changes in the so-called homogeneous L o or f3-ph ase structure of PC bilayers with respect to temperature and PC acyl chain length. These changes are manifest as changes in the conformational order of the phospholipid acyl chains as well as in the relative position of cholesterol in the bilayer [22,48-,49]. Integrating these findings, we proposed [48-] a new and more complete phase diagr am which docs not include a eutectic point. It consists of the two pure phospholipid phases, La and LfJ, which exist upto 22 mol% cholesterol, and the two liquid-ordered or f3-phases, termed Lou and Loll, which co-exist from approximately 1 to 50 mol% but whose boundaries vary with temperature and depend on the chain length of the host PC bilayer [48-].
Applications The growing evidence for distinct cholesterol-rich or cholesterol-poor domains in model phospholipid bilayers
has provided investigators with some interesting insights into biological phenomena. For example, Pott and Dufourc [50] correlated ZH- and 3IP-NMR spectroscopic evidence for domain boundaries within the f3-phase to explain the rnclittin-induced disruption of cholesterol-containing DPPC bilayers. Therein, they propose a model that describes how the entry of the peptide into the bilayer is permitted by domain boundaries within the LQ phase, which then triggers membrane disruption. Phase separation in cholesterol-containing bilayers is also postulated to account for the domain-induced budding process in vesicles, or possibly to be a mechanism for the sorting of proteins into cholesterol-rich domains of membranes which exhibit increased hydrophobic thickness [48-,51,52]. Another interesting recent investigation revealed that quaternary ammonium based surfactants selectively disrupt cholesterol-poor bilayers [53]. Quaternary ammonium salts cannot cross the interface of the tightly packed non-polar region of cholesterol-containing lipid bilayers although proton .ionizable amine surfactants can shed their charge, thus permitting penetration into both cholesterol-rich and cholesterol-poor domains of the bilayer. These authors demonstrate the complexity and specificity of interfacial interactions and propose how to exploit this complexity by designing antibiotics with enhanced cytotoxicity towards bacteria and fungi compared to mammalian cells. Finally, small changes in cholesterol content may trigger large local changes in bilayer packing, as illustrated in fluorescence studies, which may explain, for example, why changes in cellular activities can occur when no overall change in bilayer fluidity is apparent [39-,40]. Cholesterol is also a prominent component of many liposomal formulations. Cholesterol is typically used to increase the retention of hydrophilic drugs while also inceasing bilayer rigidity and resistance to in vivo liposomal degradation [54]. Recently cholesterol has also been used to modulate liposomal pH-sensitivity [55] and Iiposomal fusion [56], and has been derivatized to produce cationic liposomes or act as lipid anchors for covalently-linked oligonucleotides [57,58]. Despite its widespread usc, the reported effects of cholesterol on liposomal behavior are often inconsistent [54,56]. It is now clear that the thermotropic phase behavior and organization of cholesterol-containing phosphlipid bilayers may vary dramatically with both vesicular contents and host phosphlipid cornpsition. Thus, continued use of cholesterol to optimize liposome encapsulation, stability and release of genetic materials or drugs will require careful consideration of cholesterol-lipid interactions.
Conclusion Although cholesterol/phospholipid systems have been studied for many years, it is clear that our understanding of the molecular basis of cholesterol-phospholipid interactions remains incomplete. However, the recent resurgence of interest in these biologically relevant model membrane systems, and the increasingly sophisticated biophysical
Physical studies of cholesterol-phospholipid interactions McMullen and McElhaney
and biochemical techniques being applied to them, have resulted in considerable progress in the understanding of a number of long-standing problems in this field. The complete resolution of the many outstanding issues in this area will continue to require the coordinated application of a wide variety of physical techniques to systematically study a broader range of host lipid bilayers existing in a comparable range of physical states.
Acknowledgements We would like to stress the importance of references t4 and t6 in relation 10 the review and its contents. Unfortunately these articles could not be bullctcd in the reference section as they were not published within the annual period of review,
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