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...

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.

Recently, a number of ternary phase diagrams of lipid mixtures have been constructed using various experimental techniques with a common goal of understanding the nature of lipid domains. An accurate experimental phase diagram can provide rich thermodynamic information, and can also be used to extract molecular interactions using computer simulation. In this study, the liquid-ordered and liquid-disordered (l o -l d ) phase boundary of DSPC/DOPC/Cholesterol ternary mixtures is simulated in a lattice model using pairwise interactions. The block composition distribution (BCD) technique was used to accurately locate the compositions of coexisting phases and thermodynamics tie-lines in the 2-phase region, and the Binder ratio method was used to determine the phase boundary in the critical region. In simulations performed along a thermodynamic tie-line, the BCD method correctly samples the compositions as well as the relative amounts of coexisting phases, in excellent agreement with the Lever Rule. A "best-fit" phase boundary was obtained that has a top boundary closely resembling the experimental boundary. However, the width of the simulated 2-phase region is significantly wider than the experimental one. The results show that pairwise interactions alone are not sufficient to describe the complexity of molecular interactions in the ternary lipid mixtures; more complex forms of interactions, possibly multibody interaction or domain interfacial energy, should be included in the simulation.

Human apolipoprotein E (ApoE) exists as three common isoforms that differ by single amino acid substitutions. ApoE4 (Arg112/Arg158) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD)-a significant cause of mortality and disability worldwide. At the protein level, previous studies suggest that the conformational landscape of ApoE4 is different to that of the wild-type ApoE3 (Cys112/Arg158). To date, however, effective ligands capable of selectively targeting the ''ApoE4-conformation'' remain elusive. In the present study we sought to use phage display technology to isolate several single domain antibody fragments (i.e. V NAR ), initially derived from the wobbegong shark, that are reactive against human ApoE isoforms. To this end we have expressed, purified, and characterized recombinant ApoE3 and ApoE4 for use as bait in phage display. After 3-5 rounds of panning enrichment against ApoE isoforms was observed. From these rounds of panning several unique clones were identified and subsequently subcloned, expressed, and purified to near homogeneity. We are currently in the process of more fully characterising the binding propensities of these clones.

No abstract