Rhodopsin reconstituted into a planar-supported lipid bilayer retains photoactivity after cross-linking polymerization of lipid monomers

Transmembrane proteins (TMPs), particularly ion channels and receptors, play key roles in transport and signal transduction. Many of these proteins are pharmacologically important and therefore targets for drug discovery. TMPs can be reconstituted in planar-supported lipid bilayers (PSLBs), which has led to development of TMP-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects TMP structure and activity. In this study, we have examined the effects of UV polymerization of bis-Sorbylphosphatidylcholine (bis-SorbPC) on the photoactivation of reconstituted bovine rhodopsin (Rho), a model G-protein-coupled receptor. Plasmon-waveguide resonance spectroscopy (PWR) was used to compare the degree of Rho incorporation and activation in fluid and poly(lipid) PSLBs. The results show that reconstitution of Rho into a supported lipid bilayer composed only of bis-SorbPC, followed by photoinduced lipid cross-linking, does not measurably diminish protein function.     

Preparation and characterization of asymmetric planar supported bilayers composed of poly(bis-sorbylphosphatidylcholine) on n-octadecyltrichlorosilane SAMs

Planar supported lipid bilayers (PSLBs) have been widely studied as biomembrane models and biosensor scaffolds. For technological applications, a major limitation of PSLBs composed of fluid lipids is that the bilayer structure is readily disrupted when exposed to chemical, mechanical, and thermal stresses. A number of asymmetric supported bilayer structures, such as the hybrid bilayer membrane (HBM) and the tethered bilayer lipid membrane (tBLM), have been created as an alternative to symmetric PSLBs. In both HBMs and tBLMs, the inner monolayer is covalently attached to the substrate while the outer monolayer is typically composed of a fluid lipid. Here we address if cross-linking polymerization of the lipids in the outer monolayer of an asymmetric supported bilayer can achieve the high degree of stability observed previously for symmetric PSLBs in which both monolayers are cross-linked [E.E. Ross, L.J. Rozanski, T. Spratt, S.C. Liu, D.F. O'Brien, S.S. Saavedra, Langmuir 19 (2003) 1752]. To explore this issue, HBMs composed of an outer monolayer of a cross-linkable lipid, bis-sorbylphosphatidylcholine (bis-SorbPC), and an inner SAM were prepared and characterized. Several experimental conditions were varied: vesicle fusion time, polymerization method, and polymerization time and temperature. Under most conditions, bis-SorbPC cross-linking stabilized the HBM such that its bilayer structure was largely preserved after drying; however these films invariably contained sub-micron scale defects that exposed the hydrophobic core of the HBM. The defects appear to be caused by desorption of low molecular weight oligomers when the film is removed from water, rinsed, and dried. In contrast, poly(bis-SorbPC) PSLBs prepared under similar conditions by Ross et al. were nearly defect free. This comparison shows that formation of a cross-linked network in the outer leaflet of an asymmetric supported bilayer is insufficient to prevent lipid desorption; inter-leaflet covalent linking appears to be necessary to create supported poly(lipid) assemblies that are impervious to repeated drying and rehydration. The difference in stability is attributed to inter-leaflet cross-linking between monolayers which can form in symmetric bis-SorbPC PSLBs.     

Polymerized planar suspended lipid bilayers for single ion channel recordings: comparison of several dienoyl lipids

The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability of air-water transfers. BLM stability followed the order bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α-hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single-channel recordings of reconstituted ICs.     

Functional Dynamics of Deuterated β2‐Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy

G‐protein‐coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β₂‐adrenergic receptor (β₂AR) in lipid bilayers of reconstituted high‐density lipoprotein (rHDL) increased by approximately 5‐fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β₂AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.     

Engineered lipids that cross-link the inner and outer leaflets of lipid bilayers

The application of supported lipid bilayer systems as molecular sensors, diagnostic devices, and medical implants is limited by their lack of stability. In an effort to enhance the stability of supported lipid bilayers, three pairs of phosphatidylcholine lipids were designed to cross-link at the termini of their 2-position acyl chain upon the formation of lipid bilayers. The cross-linked lipids span the lipid bilayer, resembling naturally occurring bolaamphiphiles that stabilize archaebacterial membranes against high temperatures. The three reactions investigated here include the acyl chain cross-linking between thiol and bromine groups, thiol and acryloyl groups, and cyclopentadiene and acryloyl groups. All three reactive lipid pairs were found to cross-link in liposomal membranes, as determined by thin-layer chromatography, ion-spray mass spectrometry, and 1H NMR. The monolayer film properties of the reactive amphiphiles were characterized by surface pressure-area isotherms and showed that stable monolayers formed at the air-water interface with limiting molecular areas comparable to that of pure saturated phosphatidylcholine lipids. Langmuir-Blodgett bilayers of dimyristoylphosphatidylcholine incorporating 15 mol % of the reactive thiol and acryloyl lipids had diffusion coefficients comparable with pure dimyristoylphosphatidylcholine, while bilayers with more than 25 mol % of the reactive lipids were immobile, suggesting that interleaflet cross-linking of the lipids inhibited membrane diffusion. Our results show that the reactive lipids can cross-link within a lipid bilayer and are suitable for assembling supported lipid bilayers using Langmuir-Blodgett deposition. By using terminally reactive amphiphiles to build up supported lipid bilayers with cross-linked leaflets, bolaamphiphiles can be incorporated into asymmetric solid supported membranes to increase their stability in biosensor and medical implant applications.     

Two-Dimensional polymerization of supramolecular assemblies: Synthesis and bilayer polymerization of lipids containing α-methylene-substituted acyl chains

Polymerization of lipid assemblies may be usefully employed to alter the properties of the assemblies. The possible locations of the reactive group in the lipids include (1) the chain terminus, (2) the head group, and (3) near the lipid backbone. The third strategy yields polymerized assemblies which retain their head group functionality and lipid chain motion. We have designed and synthesized new members of this later category by the use of 2-methylene-substituted acyl chains. The main transition temperature (T_m) from gel to liquid crystalline phase of hydrated bilayers of 1-palmitoyl-2-(2-methylene)palmitoyl-sn-glycero-3-phosphocholine ( 1 ) and the disubstituted 1,2-bis(2-methylenepalmitoyl)-sn-glycero-3-phosphocholine ( 2 ) were 33.6 and 25.3°C, respectively. The T_m of the mono-substituted 1-oleoyl-2-(2-methylene)palmitoyl-sn-glycero-3-phosphocholine ( 3 ) bilayers was detected in a range from ?15 to ?10°C by x-ray diffraction. Hydrated bilayers of each individual lipid were successfully polymerized with a water-soluble initiator, azobis(2-amidinopropane) dihydrochloride (AAPD). These results indicate the lipid 2-methylene groups are accessible to the water interface. Thermal polymerization of the mono-substituted lipids in aqueous suspensions with AAPD, yielded oligomers. However the bis-2-methylene PC ( 2 ) was successfully polymerized to yield stabilized crosslinked bilayers. © 1994 John Wiley & Sons, Inc.     

Fluid supported lipid bilayers containing monosialoganglioside GM1: a QCM-D and FRAP study

In an effort to use model fluid membranes for immunological studies, we compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic volumes of extruded small unilamellar vesicles (E-SUVs) containing physiologically relevant concentrations (0.5-5 mol%) of monosialoganglioside GM1 (GM1) from exclusive egg yolk L-alpha-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evaporated SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs containing 5 mol% GM1 reached a approximately 12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are approximately 18% heavier than 100 mol% egg PC and approximately 11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal immunoglobulin G anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equilibrium were roughly proportional to the concentrations of anti-GM1 administered in the solution. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio approximately 1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approximately 16% lower diffusion coefficient of 1.32+/-0.06 microm2/s, compared to 1.58+/-0.04 microm2/s for egg PC membranes without GM1 (p<0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for molecules of interest such as GM1 to exist on a more biologically relevant surface than those used in conventional methods such as ELISA. Our analysis of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.     

Material properties of matrix lipids determine the conformation and intermolecular reactivity of diacetylenic phosphatidylcholine in the lipid bilayer

Photopolymerizable phospholipid DC(8,9)PC (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) exhibits unique assembly characteristics in the lipid bilayer. Because of the presence of the diacetylene groups, DC(8,9)PC undergoes polymerization upon UV (254 nm) exposure and assumes chromogenic properties. DC(8,9)PC photopolymerization in gel-phase matrix lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monitored by UV-vis absorption spectroscopy occurred within 2 min after UV treatment, whereas no spectral shifts were observed when DC(8,9)PC was incorporated into liquid-phase matrix 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Liquid chromatography-tandem mass spectrometry analysis showed a decrease in the amount of DC(8,9)PC monomer in both DPPC and POPC environments without any change in the matrix lipids in UV-treated samples. Molecular dynamics (MD) simulations of DPPC/DC(8,9)PC and POPC/DC(8,9)PC bilayers indicate that the DC(8,9)PC molecules adjust to the thickness of the matrix lipid bilayer. Furthermore, the motions of DC(8,9)PC in the gel-phase bilayer are more restricted than in the fluid bilayer. The restricted motional flexibility of DC(8,9)PC (in the gel phase) enables the reactive diacetylenes in individual molecules to align and undergo polymerization, whereas the unrestricted motions in the fluid bilayer restrict polymerization because of the lack of appropriate alignment of the DC(8,9)PC fatty acyl chains. Fluorescence microscopy data indicates the homogeneous distribution of lipid probe 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (N-Rh-PE) in POPC/DC(8,9)PC monolayers but domain formation in DPPC/DC(8,9)PC monolayers. These results show that the DC(8,9)PC molecules cluster and assume the preferred conformation in the gel-phase matrix for the UV-triggered polymerization reaction.     

Structure of a gel phase lipid bilayer prepared by the Langmuir-Blodgett/Langmuir-Schaefer method characterized by sum-frequency vibrational spectroscopy

The structure of a planar supported lipid bilayer (PSLB) prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method was investigated by sum-frequency vibrational spectroscopy (SFVS). By using asymmetric lipid bilayers composed of selectively deuterated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids, the orientation of the fatty acid chains and phosphocholine headgroups has been determined independently for both leaflets of the bilayer. The alkyl chains of the lipids were found to be orientated approximately 13 degrees +/- 4 degrees from the surface normal for both leaflets. The lipid chains in both leaflets also contain some gauche content, which is consistent with previous NMR and FTIR studies of similar lipid systems. More importantly, the relative number of gauche defects does not seem to be influenced by the deposition method, LB versus LS. The headgroup orientation for the lipid film in contact with the silica support was determined to be 69 degrees +/- 3 degrees , whereas that in contact with the aqueous phase was 66 degrees +/- 4 degrees from the surface normal. The SFVS results indicate that the structure of the DSPC lipid film in contact with the solid support and the film adjacent to the aqueous phase are nearly identical in structure. These results suggesting the LB/LS deposition method do indeed produce symmetric lipid bilayers. These studies further add to the growing information on the efficacy of PSLBs as suitable models for biological membrane studies.     

Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy.

Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers. We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6omega3) at the sn-2 position. Measurements have been performed on fluid (L(alpha))-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments, and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.     

Structure and thermotropic phase behavior of fluorinated phospholipid bilayers: a combined attenuated total reflection FTIR spectroscopy and imaging ellipsometry study

Lipid bilayers consisting of lipids with terminally perfluoroalkylated chains have remarkable properties. They exhibit increased stability and phase-separated nanoscale patterns in mixtures with nonfluorinated lipids. In order to understand the bilayer properties that are responsible for this behavior, we have analyzed the structure of solid-supported bilayers composed of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and of a DPPC analogue with 6 terminal perfluorinated methylene units (F6-DPPC). Polarized attenuated total reflection Fourier-transform infrared spectroscopy indicates that for F6-DPPC, the tilt of the lipid acyl chains to the bilayer normal is increased to 39 degrees as compared to 21 degrees for native DPPC, for both lipids in the gel phase. This substantial increase of the tilt angle is responsible for a decrease of the bilayer thickness from 5.4 nm for DPPC to 4.5 nm for F6-DPPC, as revealed by temperature-controlled imaging ellipsometry on microstructured lipid bilayers and solution atomic force microscopy. During the main phase transition from the gel to the fluid phase, both the relative bilayer thickness change and the relative area change are substantially smaller for F6-DPPC than for DPPC. In light of these structural and thermotropic data, we propose a model in which the higher acyl-chain tilt angle in F6-DPPC is the result of a conformational rearrangement to minimize unfavorable fluorocarbon-hydrocarbon interactions in the center of the bilayer due to chain staggering.     

基于超高效液相色谱-四极杆-静电场轨道离子阱串联质谱的冷链贮藏过程中滩羊肉的脂质组学研究

该文建立了超高效液相色谱-四极杆-静电场轨道离子阱串联质谱(UHPLC-Q-Orbitrap MS/MS)结合脂质组学分析滩羊肉在冷链贮藏过程中脂质变化规律和脂质分子碎裂机理的方法。样品经异丙醇提取后,采用质谱全扫描模式和二级扫描模式对目标物质进行定性。共鉴定出48个变化显著性脂质,包括8个脂肪酰基肉碱、23个磷脂酰胆碱(PC)、3个溶血性磷脂酰胆碱(LPC)、13个磷脂酰乙醇胺(PE)、1个溶血性磷脂酰乙醇胺(LPE)。含量差异表现为部分PC、PE和脂肪酰基肉碱在前12 d短暂性升高,12 d后开始降低,而LPE和LPC在整个冷链贮藏期间表现为上升趋势。PC、PE和脂肪酰基肉碱短暂性的积累易导致大量的脂质氧化反应,进一步阐明最佳冷链时间为12 d。该方法适用于复杂基质中脂质分子的分离与定量,为肉及肉类产品在冷链贮藏过程中的脂质变化规律研究及质量控制提供了依据。     

The alphaM1 transmembrane segment of the nicotinic acetylcholine receptor interacts strongly with model membranes

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) plays a role in the regulation of the activity of this important ligand-gated ion channel. The lipid composition of the host membrane affects conformational equilibria of the nAChR and several classes of inhibitors, most notably anaesthetics, interact directly or indirectly with the four transmembrane M-segments, M1-M4, of the nAChR subunits. It has proven difficult to gain insight into structure-function relationships of the M-segments in the context of the entire receptor and the biomembrane environment. However, model membrane systems are well suited to obtain detailed information about protein-lipid interactions. In this solid-state NMR study, we characterized interactions between a synthetic alphaM1 segment of the T. californica nAChR and model membranes of different phosphatidylcholine (PC) lipids. The results indicate that alphaM1 interacts strongly with PC bilayers: the peptide orders the lipid acyl chains and induces the formation of small vesicles, possibly through modification of the lateral pressure profile in the bilayer. The multilamellar vesicle morphology was stabilized by the presence of cholesterol, implying that either the rigidity or the bilayer thickness is a relevant parameter for alphaM1-membrane interactions, which also has been suggested for the entire nAChR. Our results suggest that the model systems are to a certain extent sensitive to peptide-bilayer hydrophobic matching requirements, but that the lipid response to hydrophobic mismatch alone is not the explanation. The effect of alphaM1 on different PC bilayers may indicate that the peptide is conformationally flexible, which in turn would support a membrane-mediated modulation of the conformation of transmembrane segments of the nAChR.     

Nitrous oxide vibrational energy relaxation is a probe of interfacial water in lipid bilayers

Ultrafast infrared spectroscopy of N 2O is shown to be a sensitive probe of hydrophobic and aqueous sites in lipid bilayers. Distinct rates of VER of the nu 3 antisymmetric stretching mode of N 2O can be distinguished for N 2O solvated in the acyl tail, interfacial water, and bulk water regions of hydrated dioleoylphosphatidylcholine (DOPC) bilayers. The lifetime of the interfacial N 2O population is hydration-dependent. This effect is attributed to changes in the density of intermolecular states resonant with the nu 3 band ( approximately 2230 cm (-1)) resulting from oriented interfacial water molecules near the lipid phosphate. Thus, the N 2O VER rate becomes a novel and experimentally convenient tool for reporting on the structure and dynamics of interfacial water in lipids and, potentially, in other biological systems.     

Molecular dynamics simulations of bilayers containing mixtures of sphingomyelin with cholesterol and phosphatidylcholine with cholesterol

We performed six molecular dynamics simulations: three on hydrated bilayers containing pure phospholipids and three on hydrated bilayers containing mixtures of these phospholipids with cholesterol. The phospholipids in our simulations were SSM (sphingomyelin containing a saturated 18:0 acyl chain), OSM (sphingomyelin with an unsaturated 18:1 acyl chain), and POPC (palmitoyloleoylphosphatidylcholine containing one saturated and one unsaturated chain). Data from our simulations were used to study systematically the effect of cholesterol on phospholipids that differed in their headgroup and tail composition. In addition to the structural analysis, we performed an energetic analysis and observed that energies of interaction between cholesterol and neighboring SM molecules are similar to the energies of interaction between cholesterol and POPC. We also observed that the interaction energy between cholesterol and neighboring lipids cannot be used for the determination of which lipids are involved in the creation of a complex.     

Stabilized Lipid Membrane Based Biosensors with Incorporated Enzyme for Repetitive Uses

This work reports a technique for the stabilization of lipid membrane based biosensors with incorporated enzyme that retains its activity for repetitive uses. Microporous filters composed of glass fibers were used as supports for the stabilization of these sensors. The lipid film is formed on the filter by polymerization using UV (ultraviolet) radiation prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′‐azobis‐(2‐methylpropionitrile) was the initiator. The enzyme (acetylcholinesterase) is incorporated within this mixture prior to polymerization. The polymerization process takes place by using UV irradiation instead of heating at 60 °C the lipid mixture because this temperature might denature the enzyme. This method for preparation of stabilized lipid membranes was investigated using Raman spectroscopy. The results have indicated that the kinetics of polymerization are completed within 4 hours. The retain in activity of the enzyme was studied using electrochemical experiments which have shown that this mild technique of polymerization can now be used to incorporate a protein in these lipid membranes without loss of their activity. This will allow the practical use of the techniques for chemical sensing based on lipid membranes based biosensors and commercialization of these devices.     

Influence of highly polyunsaturated lipid acyl chains of biomembranes on the NMR order parameters

We investigate the effect of specific conformations of double-bond segments in highly polyunsaturated acyl chains on the deuterium (2)H NMR order parameters of a fully hydrated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC, 18:0/22:6 PC) lipid bilayer. The system is analyzed by performing a molecular dynamics simulation study at ambient conditions in the fluid lamellar phase. By separately calculating the different partial contributions to the total order parameter profiles measurable experimentally, we are able to get insights into the molecular origin of earlier experimental and theoretical observations. The effect of the position of the different conformations of double-bond segments along the polyunsaturated acyl chain is also examined. As in experiments performed in a series of lipid bilayers with an increasing number of cis double bonds per lipid molecule [Holte, L. L., et al. Biophys. J. 1995, 68, 2396], we find that unsaturations influence mainly the order of the bottom half of the saturated chain. Specific conformations of the polyunsaturated chain close to the lipid headgroups have a distinct effect on the order of the bottom half of the saturated chain and on the top half of the polyunsaturated chain. Our results indicate that for SDPC the conformation of the region of the polyunsaturated chain located between the first three cis double bonds is responsible for the major effects on the orientational order of both the saturated and the polyunsaturated chains.     

G-Protein-Coupled Receptor–Membrane Interactions Depend on the Receptor Activation State

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and serve as primary targets of approximately one-third of currently marketed drugs. In particular, adenosine A₁ receptor (A₁AR) is an important therapeutic target for treating cardiac ischemia–reperfusion injuries, neuropathic pain, and renal diseases. As a prototypical GPCR, the A₁AR is located within a phospholipid membrane bilayer and transmits cellular signals by changing between different conformational states. It is important to elucidate the lipid–protein interactions in order to understand the functional mechanism of GPCRs. Here, all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method were performed on both the inactive (antagonist bound) and active (agonist and G-protein bound) A₁AR, which was embedded in a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer. In the GaMD simulations, the membrane lipids played a key role in stabilizing different conformational states of the A₁AR. Our simulations further identified important regions of the receptor that interacted distinctly with the lipids in highly correlated manner. Activation of the A₁AR led to differential dynamics in the upper and lower leaflets of the lipid bilayer. In summary, GaMD enhanced simulations have revealed strongly coupled dynamics of the GPCR and lipids that depend on the receptor activation state. © 2019 Wiley Periodicals, Inc.     

Inclusion-mediated lipid organization in supported membranes on a patterned substrate

We theoretically investigate the effects of inclusions on the domain formation in mixed lipid bilayers supported on a geometrically patterned substrate. It is found that the inclusions may distribute quite differently with varying volume fraction and size of inclusions. The distribution of inclusions will effectively change the spontaneous curvature of the inclusion-rich lipid domains, and consequently can sort the lipid domains in the supported bilayers. By varying the volume fraction and size of inclusions, we obtain a rich variety of laterally organized lipid bilayers and reveal some interesting transitions between these structures. The present model provides a possible strategy to control the domain formation in the supported membranes, and may yield some theoretical insight into the design of biosensors by the reorganization of lipids and inclusions.     

Substrate-supported lipid nanotube arrays

This Communication describes the self-assembly of phospholipids into lipid nanotubes inside nanoporous anodic aluminum oxide substrate. Orientations of the lipid molecules in such lipid nanoscale structures were verified by high-resolution spin labeling EPR at 95 GHz. The static order parameter of lipids in such nanotube arrays was determined from low-temperature EPR spectra and was found to be exceptionally high, Sstatic approximately 0.9. We propose that substrate-supported lipid nanotube arrays have potential for building robust biochips and biosensors in which rigid nanoporous substrates protect the bilayer surface from contamination. The total bilayer surface in the lipid nanotube arrays is much greater than that in the planar substrate-supported membranes. The lipid nanotube arrays seem to be suitable for developing patterned lipid deposition and could be potentially used for patterning of membrane-associated molecules.