We recently reported the equilibrium maximum solubility of cholesterol in a lipid bilayer, chi*chol, to be 0.66 in four different phosphatidylcholines, and 0.51 in a phosphatidylethanolamine (Huang, J.,J.T. Buboltz, and G. W. Feigenson. 1999. Biochim. Biophys. Acta. in press). Here we present a model of cholesterol-phospholipid mixing that explains these observed values of chi*chol. Monte Carlo simulations show that pairwise-additivity of nearest-neighbor interactions is inadequate to describe all the chi*chol values. Instead, if cholesterol multibody interactions are assigned highly unfavorable energy, then jumps occur in cholesterol chemical potential that lead to its precipitation from the bilayer. Cholesterol precipitation is most likely to occur near three discrete values of cholesterol mole fraction, 0.50, 0.57, and 0.67, which correspond to cholesterol/phospholipid mole ratios of 1/1, 4/3, and 2/1, respectively. At these solubility limits, where cholesterol chemical potential jumps, the cholesterol-phospholipid bilayer mixture forms highly regular lipid distributions in order to minimize cholesterol-cholesterol contacts. This treatment shows that dramatic structural and thermodynamic changes can occur at particular cholesterol mole fractions without any stoichiometric complex formation. The physical origin of the unfavorable cholesterol multibody interaction is explained by an "umbrella model": in a bilayer, nonpolar cholesterol relies on polar phospholipid headgroup coverage to avoid the unfavorable free energy of cholesterol contact with water. Thus, at high cholesterol mole fraction, this unfavorable free energy, not any favorable cholesterol-phospholipid interaction, dominates the mixing behavior. This physical origin also explains the "cholesterol condensing effect" and the increase in acyl chain order parameter in cholesterol-phospholipid mixtures.

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.

The condensing effect of cholesterol in dioleoylphosphatidylcholine (DOPC) lipid bilayers was systematically investigated via atomistic molecular dynamics (MD) simulation. Fourteen independent 200 ns simulations, spanning the entire range of cholesterol mole fraction (xc) in DOPC bilayers (i.e. from xc = 0 to 0.66), were performed at 323 K. The molecular areas occupied by DOPC and cholesterol at different distances from the bilayer center were analyzed using a slicing method based on the VDW radii of atoms. Curiously, while the average area per lipid and the cholesterol tilt angle, in respect to the bilayer normal, both show monotonic decreases as xc increases, the average bilayer height shows a significant decrease for xc > 0.35, following an initial increase. The calculated partial-specific areas of lipids clearly show the condensing effect of cholesterol. The VDW areal analysis showed that the condensing effect is limited only to the cholesterol sterol ring region, where the acyl chains of DOPC are severely compressed by cholesterol. As xc increases, the headgroups of DOPC gradually expand along the bilayer-aqueous interface to occupy more lateral area. Thus, it confirmed a key prediction of the Umbrella model. At high cholesterol mole fractions, the calculated area per DOPC and area per cholesterol using some existing methods showed an inconsistent result: Both increase, while the overall area per lipid decreases. The inconsistency stems from the problematic assumption that cholesterol and DOPC have cylindrical shape and the same height. Our results showed that the total area of a PC/cholesterol bilayer is primarily determined by the molecular packing in the cholesterol sterol ring region. An alternative analysis of area per molecule within this region is proposed, which takes into account the cholesterol tilt angle and the practical incompressibility of cholesterol sterol rings. The new calculation shows that the majority of the area lost due to the cholesterol condensing effect is taken from PC molecules.

Cholesterol plays a vital role in determining the physiochemical properties of cell membranes. However, the detailed nature of cholesterol-lipid interactions is a subject of ongoing debate. Existing conceptual models, including the Condensed Complex Model, the Superlattice Model, and the Umbrella Model, identify different molecular mechanisms as the key to cholesterol-lipid interactions. In this work, the compositional dependence of the chemical potential of cholesterol in cholesterol/phosphatidylcholine mixtures was systematically measured at high resolution at 37°C by using an improved cholesterol oxidase (COD) activity assay. The chemical potential of cholesterol was found to be much higher in di18:1-PC bilayers than in di16:0-PC bilayers, indicating a more favorable interaction between cholesterol and saturated chains. More significantly, in 16:0,18:1-PC and di18:1-PC bilayers, the COD initial-reaction rate displays a series of distinct jumps near the cholesterol mole fractions ( biomembrane ͉ chemical activity ͉ free energy ͉ liposome ͉ rapid solvent exchange method C holesterol plays a vital role in determining the physiochemical properties of cell membranes. The presence of cholesterol in a lipid membrane can drastically increase lipid acyl chain order, induce cholesterol regular distributions (superlattices) or lipid raft domains, and modulate the activities of surface-acting enzymes (1-4). Despite significant technical advances in membrane research in recent years, the detailed nature of cholesterol-lipid interactions is a subject of ongoing debate. Existing conceptual models, including the Condensed Complex Model, the Superlattice Model, and the Umbrella Model, identify different molecular mechanisms as the key to cholesterol-lipid interactions in biomembranes. Clearly, there is an urgent need to establish a general cholesterol-lipid interaction theory that can explain how cholesterol supports or modulates important functions in cell membranes and perhaps can predict the behavior and functional role of cholesterol in complex membranes. Condensed Complex Model.This model was initially proposed based on a study of lipid monolayers at the air-water interface (5). The model hypothesizes the existence of low free-energy stoichiometric cholesterol-lipid complexes that occupy smaller molecular lateral areas (5, 6). At a stoichiometric composition, a sharp jump in cholesterol chemical potential ( C ) has been predicted (6, 7), as shown in Fig. 1a. Because the proposed condensed complex has a compact low-energy structure, the model explicitly predicted that cholesterol can form condensed complexes with lipids with which it can mix favorably, such as phosphatidylcholine (PC) with long saturated chains or sphingomyelins. It has also been suggested that cholesterol superlattices as well as lipid rafts are examples of the proposed condensed complexes (6,8). According to this model, the ability to form cholesterol-lipid condensed complexes represents an essential feature of cholesterol-lipid interactions.Superlatti...

Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols) in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM) in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm −) were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm − mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm − causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.

Abstract.Wide angle x-ray scattering (WAXS) from oriented lipid multilayers was used to study the effect of adding cholesterol (Chol) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Small quantities (X < 0.10 mole fraction) of both molecules disrupt the tight packing of tilted chains of pure gel-phase DPPC, forming a more disordered, untilted phase. The addition of larger quantities of DOPC causes the sample to phase-separate into a gel phase, characterized by a narrow WAXS peak, and liquid disordered phase, characterized by wide, diffuse WAXS scattering. In contrast, two WAXS peaks indicative of two coexisting phases were not observed in Chol/DPPC mixtures (X Chol = 0.07 to 0.40). Instead, Chol caused a gradual increase in the width of the WAXS peak, consistent with a gradual change from a more gel-like to a more liquid-like state rather than passing through a region of two phase coexistence. Our WAXS data include a huge amount of information. A new method of analysis suggests that WAXS data may provide definitive results relating to the disagreements between previously published phase diagrams for Chol/DPPC.

Time-resolved fluorescence and Fourier transform infrared spectroscopies were used to investigate the lateral organization of lipids in compositionally uniform and fully equilibrated 1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPC/CHOL) liposomes prepared by a recently devised low-temperature trapping method. Independent fluorescence decay lifetime and rotational dynamics parameters of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) in these liposomes were recovered from the time-resolved fluorescence measurements as a function of cholesterol molar fraction (X(CHOL)) at 23 degrees C. The results indicate significantly greater lifetime heterogeneity, shorter average lifetime, rotational correlation time, and lower order parameter of the DPH moiety at X(CHOL) approximately 0.40 and 0.50 as compared to the adjacent cholesterol concentrations. Less prominent changes were also detected at, for example, X(CHOL) approximately 0.20 and 0.33. These X(CHOL)'s coincide with the "critical" X(CHOL)'s predicted by the previously proposed superlattice (SL) model, thus indicating that POPC and cholesterol molecules tend to form SL domains where the components tend to be regularly distributed. The data also support another prediction of the SL model, namely that lateral packing defects coexist with the ordered SL domains. It appears that unfavorable interaction of the DPH-moiety of DPH-PC with cholesterol results in a preferential partition of DPH-PC to the defect regions. Fourier transform infrared analysis of the native lipid O=P=O, C=O, and C-H vibrational bands of POPC/CHOL liposomes in the absence of DPH-PC revealed an increase in the conformational order of the acyl chains and a decrease in the conformational order (or increased hydration) of the interfacial and headgroup regions at or close to the predicted critical X(CHOL)'s. This provides additional but probe-independent evidence for SL domain formations in the POPC/CHOL bilayers. We propose that the defect regions surrounding the putative SL domains could play an important role in modulating the activity of various membrane-associated enzymes, e.g., those regulating the lipid compositions of cell membranes.