Behavior of Bilayer Leaflets in Asymmetric Model Membranes: Atomistic Simulation Studies

Spatial organization within lipid bilayers is an important feature for a range of biological processes. Leaflet compositional asymmetry and lateral lipid organization are just two of the ways in which membrane structure appears to be more complex than initially postulated by the fluid mosaic model. This raises the question of how the phase behavior in one bilayer leaflet may affect the apposing leaflet and how one begins to construct asymmetric model systems to investigate these interleaflet interactions. Here we report on all-atom molecular dynamics simulations (a total of 4.1 μs) of symmetric and asymmetric bilayer systems composed of liquid-ordered (Lo) or liquid-disordered (Ld) leaflets, based on the nanodomain-forming POPC/DSPC/cholesterol system. We begin by analyzing an asymmetric bilayer with leaflets derived from simulations of symmetric Lo and Ld bilayers. In this system, we observe that the properties of the Lo and Ld leaflets are similar to those of the Lo and Ld leaflets in corresponding symmetric systems. However, it is not obvious that mixing the equilibrium structures of their symmetric counterparts is the most appropriate way to construct asymmetric bilayers nor that these structures will manifest interleaflet couplings that lead to domain registry/antiregistry. We therefore constructed and simulated four additional asymmetric bilayer systems by systematically adding or removing lipids in the Ld leaflet to mimic potential density fluctuations. We find that the number of lipids in the Ld leaflet affects its own properties, as well as those of the apposing Lo leaflet. Collectively, the simulations reveal the presence of weak acyl chain interdigitation across bilayer leaflets, suggesting that interdigitation alone does not contribute significantly to the interleaflet coupling in nonphase-separated bilayers of this chemical composition. However, the properties of both leaflets appear to be sensitive to changes in in-plane lipid packing, possibly providing a mechanism for interleaflet coupling by modulating local density and/or curvature fluctuations

Molecular Structures of Fluid Phosphatidylethanolamine Bilayers Obtained from Simulation-to-Experiment Comparisons and Experimental Scattering Density Profiles

Following our previous efforts in determining the structures of commonly used PC, PG, and PS bilayers, we continue our studies of fully hydrated, fluid phase PE bilayers. The newly designed parsing scheme for PE bilayers was based on extensive MD simulations, and is utilized in the SDP analysis of both X-ray and neutron (contrast varied) scattering measurements. Obtained experimental scattering form factors are directly compared to our simulation results, and can serve as a benchmark for future developed force fields. Among the evaluated structural parameters, namely, area per lipid <i>A</i>, overall bilayer thickness <i>D</i>_B, and hydrocarbon region thickness 2<i>D</i>_C, the PE bilayer response to changing temperature is similar to previously studied bilayers with different headgroups. On the other hand, the reduced hydration of PE headgroups, as well as the strong hydrogen bonding between PE headgroups, dramatically affects lateral packing within the bilayer. Despite sharing the same glycerol backbone, a markedly smaller area per lipid distinguishes PE from other bilayers (i.e., PC, PG, and PS) studied to date. Overall, our data are consistent with the notion that lipid headgroups govern bilayer packing, while hydrocarbon chains dominate the bilayer’s response to temperature changes

¹H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers

We measured the transbilayer diffusion of 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (<i>L</i>_β′) and fluid (<i>L</i>_α) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-<i>d</i>13 vesicles was monitored using ¹H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol^–1. In gel-phase DPPC vesicles, flip-flop was not observed over the course of 250 h. Our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. We reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement

Interactions between Ether Phospholipids and Cholesterol As Determined by Scattering and Molecular Dynamics Simulations

Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol’s molecular interactions with ether lipids as determined using a combination of small-angle neutron and X-ray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data. This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup’s phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid-mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules

Morphological Characterization of DMPC/CHAPSO Bicellar Mixtures: A Combined SANS and NMR Study

Spontaneously forming structures of a system composed of dimyristoyl phosphatidylcholine (DMPC) and 3-[(3-cholamidopropyl)­dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) were studied by small-angle neutron scattering (SANS), ³¹P NMR, and stimulated echo (STE) pulsed field gradient (PFG) ¹H NMR diffusion measurements. Charged lipid dimyristoyl phosphatidylglycerol (DMPG) was used to induce different surface charge densities. The structures adopted were investigated as a function of temperature and lipid concentration for samples with a constant molar ratio of long-chain to short-chain lipids (= 3). In the absence of DMPG, zwitterionic bicellar mixtures exhibited a phase transition from discoidal bicelles, or ribbons, to multilamellar vesicles either upon dilution or with increased temperature. CHAPSO-containing mixtures showed a higher thermal stability in morphology than DHPC-containing mixtures at the corresponding lipid concentrations. In the presence of DMPG, discoidal bicelles (or ribbons) were also found at low temperature and lower lipid concentration mixtures. At high temperature, perforated lamellae were observed in high-concentration mixtures (≥7.5 wt %) whereas uniform unilamellar vesicles and bicelles formed in low-concentration mixtures (≤2.5 wt %), respectively, when the mixtures were moderately and highly charged. From the results, spontaneous structural diagrams of the zwitterionic and charged systems were constructed

Tocopherol Activity Correlates with Its Location in a Membrane: A New Perspective on the Antioxidant Vitamin E

We show evidence of an antioxidant mechanism for vitamin E which correlates strongly with its physical location in a model lipid bilayer. These data address the overlooked problem of the physical distance between the vitamin’s reducing hydrogen and lipid acyl chain radicals. Our combined data from neutron diffraction, NMR, and UV spectroscopy experiments all suggest that reduction of reactive oxygen species and lipid radicals occurs specifically at the membrane’s hydrophobic–hydrophilic interface. The latter is possible when the acyl chain “snorkels” to the interface from the hydrocarbon matrix. Moreover, not all model lipids are equal in this regard, as indicated by the small differences in vitamin’s location. The present result is a clear example of the importance of lipid diversity in controlling the dynamic structural properties of biological membranes. Importantly, our results suggest that measurements of aToc oxidation kinetics, and its products, should be revisited by taking into consideration the physical properties of the membrane in which the vitamin resides

Morphology-Induced Defects Enhance Lipid Transfer Rates

Molecular transfer between nanoparticles has been considered to have important implications regarding nanoparticle stability. Recently, the interparticle spontaneous lipid transfer rate constant for discoidal bicelles was found to be very different from spherical, unilamellar vesicles (ULVs). Here, we investigate the mechanism responsible for this discrepancy. Analysis of the data indicates that lipid transfer is entropically favorable, but enthalpically unfavorable with an activation energy that is independent of bicelle size and long- to short-chain lipid molar ratio. Moreover, molecular dynamics simulations reveal a lower lipid dissociation energy cost in the vicinity of interfaces (“defects”) induced by the segregation of the long- and short-chain lipids in bicelles; these defects are not present in ULVs. Taken together, these results suggest that the enhanced lipid transfer observed in bicelles arises from interfacial defects as a result of the hydrophobic mismatch between the long- and short-chain lipid species. Finally, the observed lipid transfer rate is found to be independent of nanoparticle stability

Bilayer Thickness Mismatch Controls Domain Size in Model Membranes

The observation of lateral phase separation in lipid bilayers has received considerable attention, especially in connection to lipid raft phenomena in cells. It is widely accepted that rafts play a central role in cellular processes, notably signal transduction. While micrometer-sized domains are observed with some model membrane mixtures, rafts much smaller than 100 nmbeyond the reach of optical microscopyare now thought to exist, both in vitro and in vivo. We have used small-angle neutron scattering, a probe free technique, to measure the size of nanoscopic membrane domains in unilamellar vesicles with unprecedented accuracy. These experiments were performed using a four-component model system containing fixed proportions of cholesterol and the saturated phospholipid 1,2-distearoyl-<i>sn</i>-glycero-3-phosphocholine (DSPC), mixed with varying amounts of the unsaturated phospholipids 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC). We find that liquid domain size increases with the extent of acyl chain unsaturation (DOPC:POPC ratio). Furthermore, we find a direct correlation between domain size and the mismatch in bilayer thickness of the coexisting liquid-ordered and liquid-disordered phases, suggesting a dominant role for line tension in controlling domain size. While this result is expected from line tension theories, we provide the first experimental verification in free-floating bilayers. Importantly, we also find that changes in bilayer thickness, which accompany changes in the degree of lipid chain unsaturation, are entirely confined to the disordered phase. Together, these results suggest how the size of functional domains in homeothermic cells may be regulated through changes in lipid composition

The in vivo structure of biological membranes and evidence for lipid domains

<div><p>Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium <i>Bacillus subtilis</i> to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the <i>B</i>. <i>subtilis</i> cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments—performed under biologically relevant conditions—answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.</p></div

Comparison between the in-plane scans of DPPC-d62 bilayers with 32.5 mol% cholesterol using a) the conventional high energy resolution (small Δ<i>E</i>) setup and b) the low energy resolution (large Δ<i>E</i>) setup.

<p>The data are denoted by circles with the fit shown as a solid line. A disordered structure was observed in a), while the sharp features in b) are indicative of the presence of highly ordered lipid domains. A top view of the corresponding molecular structures are shown in the insets to the Figure using the same symbols as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066162#pone-0066162-g001" target="_blank">Figure 1</a> b); the quasi-Bragg reflections are indicated by vertical dashed lines and their associated Miller indices, [<i>hkl</i>]. Peaks resulting from the silicon substrates and the aluminum sample chamber (as described in the Materials and Methods Section) are highlighted in grey, but not accounted for in the fit.</p