We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.

Insufficient supply to the developing brain of docosahexaenoic acid (22:6n3, DHA), or its omega-3 fatty acid precursors, results in replacement of DHA with docosapentaenoic acid (22:5n6, DPA), an omega-6 fatty acid that is lacking a double bond near the chain's methyl end. We investigated membranes of 1-stearoyl(d(35))-2-docosahexaenoyl-sn-glycero-3-phosphocholine and 1-stearoyl(d(35))-2-docosapentaenoyl-sn-glycero-3-phosphocholine by solid-state NMR, X-ray diffraction, and molecular dynamics simulations to determine if the loss of this double bond alters membrane physical properties. The low order parameters of polyunsaturated chains and the NMR relaxation data indicate that both DHA and DPA undergo rapid conformational transitions with correlation times of the order of nanoseconds at carbon atom C(2) and of picoseconds near the terminal methyl group. However, there are important differences between DHA- and DPA-containing lipids: the DHA chain with one additional double bond is more flexible at the methyl end and isomerizes with shorter correlation times. Furthermore, the stearic acid paired with the DHA in mixed-chain lipids has lower order, in particular in the middle of the chain near carbons C(10)(-)(12), indicating differences in the packing of hydrocarbon chains. Such differences are also reflected in the electron density profiles of the bilayers and in the simulation results. The DHA chain has a higher density near the lipid-water interface, whereas the density of the stearic acid chain is higher in the bilayer center. The loss of a single double bond from DHA to DPA results in a more even distribution of chain densities along the bilayer normal. We propose that the function of integral membrane proteins such as rhodopsin is sensitive to such a redistribution.

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 1C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 1C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.Membranes of most enveloped viruses form by budding out from the plasma membranes of their host cells a highly select subset of plasma membrane components. In general, the selected membrane proteins are coded by the viral genome, whereas lipids are recruited from host membranes; however, the lipid composition of the viral envelope differs from that of the host membrane 1,2 and from other budding viruses 3 . The envelope of influenza contains higher amounts of both cholesterol 1 and glycosphingolipids 4 -lipids known to partition into the liquid ordered (l o ) phase. The l o phase is characterized by extended hydrocarbon chains having a reduced gauche-trans isomerization compared with those of the liquid disordered (l d ) phase, but having a similar rotational and translational mobility 5 . Liquid ordered phases are thought to be at the core of lipid rafts 6 , which are defined as transient membrane microdomains that are enriched in sphingolipids and cholesterol (1) 7 -a hypothesis that has generated much debate 8,9 .The influenza virus has played a pivotal role in the development of the raft hypothesis, starting with early studies that inferred ordered domains using spin probes 10-12 and fluorescence 13,14 and that suggested that an ordered lipid domain is selected in toto as the envelope during budding from the plasma membrane 15 . These lipids are either selected at the time of budding or pre-selected as the 'pre-envelope' suggested by clusters of the viral envelope protein hemagglutinin seen in immunoelectron microscopy 16 .Although detection of virus-sized domains of lipids (B100-nm diameter) is below the limit of resolution of fluorescent microscopy, large micrometer-sized membrane domains are reliably detected [17][18][19] . Recently, proton magic angle spinning NMR ( 1 H MAS NMR) has been used to determine the phase diagram of the same lipid compositions used to study large membrane domains in lipid bilayers and othe...

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