Miscibility behavior and nanostructure of monolayers of the main phospholipids of Escherichia coli inner membrane

We report a thermodynamic study of the effect of calcium on the mixing properties at the air-water interface of two phospholipids that mimic the inner membrane of Escherichia coli: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. In this study, pure POPE and POPG monolayers and three mixed monolayers, χ(POPE) = 0.25, 0.5, and 0.75, were analyzed. We show that for χ(POPE) = 0.75, the values of the Gibbs energy of mixing were negative, which implies attractive interactions. We used atomic force microscopy to study the structural properties of Langmuir-Blodgett monolayers that were transferred onto mica substrate at lateral surface pressures of 25 and 30 mN m(-1). The topographic images of pure POPE and POPG monolayers exhibited two domains of differing size and morphology, showing a step height difference within the range expected for liquid-condensed and liquid-expanded phases. The images captured for χ(POPE) = 0.25 were featureless, and for χ(POPE) = 0.5 small microdomains were observed. The composition that mimics quantitatively the proportions found in the inner membrane of E. coli , χ(POPE) = 0.75, showed large liquid condensed domains in the liquid expanded phase. The extension of each domain was quantitatively analyzed. Because calcium is used in the formation of supported bilayers of negatively charged phospholipids, the possible influence of the nanostructure of the apical on the distal monolayer is discussed.     

Specific adsorption of cytochrome C on cardiolipin-glycerophospholipid monolayers and bilayers

In this study, we examined the adsorption of cytochrome c (cyt c) on monolayers and liposomes formed from (i) pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), or cardiolipin (CL) and on (ii) the more thermodynamically stable binary mixtures of POPE/CL (0.8:0.2 mol/mol) and POPC/CL (0.6:0.4 mol/mol). Constant surface pressure experiments showed that the maximum and minimum interactions occurred in the pure CL (anionic phospholipid) and the pure POPE (zwitterion) monolayers, respectively. Observation by atomic force microscopy (AFM) of the images of Langmuir-Blodgett (LB) films extracted at 30 mN m-1 suggests that the different interactions of cyt c with POPE/CL and the POPC/CL monolayers could be due to lateral phase separation occurring in the POPE/CL mixture. The competition between 8-anilino-1-naphthalene sulfonate (ANS) and cyt c for the same binding sites in liposomes that have identical nominal compositions with respect to those of the monolayers was used to obtain binding parameters. In agreement with the monolayer experiments, the most binding was observed in POPE/CL liposomes. All of our observations strongly support the existence of selective adsorption of cyt c on CL, which is modulated differently by different neutral phospholipids (POPE and POPC).     

Molecular umbrella-assisted transport of an oligonucleotide across cholesterol-rich phospholipid bilayers

A series of molecular umbrella conjugates, derived from cholic acid, deoxycholic acid, spermidine, lysine, and 5-mercapto-2-nitrobenzoic acid, have been synthesized and found capable of transporting an attached 16-mer oligonucleotide (S-dT16) across liposomal membranes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyldglycerol (POPG), and cholesterol [POPC/POPG/cholesterol (65/5/30; mol/mol/mol, v/v/v)] at 37 degrees C. Those molecular umbrellas containing four choloyl (or deoxycholoyl) groups resulted in significantly faster rates of transport as compared to those containing only two such moieties. A model that accounts for these membrane transport processes is proposed.     

Surface thermodynamic properties of monolayers versus reconstitution of a membrane protein in solid-supported bilayers

Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air-water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix.     

高效液相色谱-荧光检测法测定敬钊毒素-I的磷脂膜结合活性

敬钊毒素-I(JZTX-I)是一种能够抑制心肌钠通道失活的新型蜘蛛神经毒素,该文结合高效液相色谱与色氨酸荧光测定技术研究了JZTX-I的磷脂膜结合活性。脂质体共沉淀实验表明,JZTX-I具有不依赖于带负电荷磷脂组成的生物膜结合活性。当加入由酸性或中性磷脂构成的脂质体后,JZTX-I能够分别产生6.4和4.7 nm的蓝移以及7.4和8.0 nm的红移激发漂移,显示JZTX-I能够插入磷脂膜,同时该分子疏水表面的色氨酸残基处于一个运动受限的界面区域。荧光淬灭实验进一步证实,与脂质体结合能够减少该毒素分子表面色氨酸残基的溶剂暴露。该研究结果为阐明JZTX-I的离子通道门控调节机制提供了新的信息。     

高效液相色谱-荧光检测法测定敬钊毒素-I的磷脂膜结合活性

敬钊毒素-I(JZTX-I)是一种能够抑制心肌钠通道失活的新型蜘蛛神经毒素,该文结合高效液相色谱与色氨酸荧光测定技术研究了JZTX-I的磷脂膜结合活性。脂质体共沉淀实验表明,JZTX-I具有不依赖于带负电荷磷脂组成的生物膜结合活性。当加入由酸性或中性磷脂构成的脂质体后,JZTX-I能够分别产生6.4和4.7nm的蓝移以及7.4和8.0nm的红移激发漂移,显示JZTX-I能够插入磷脂膜,同时该分子疏水表面的色氨酸残基处于一个运动受限的界面区域。荧光淬灭实验进一步证实,与脂质体结合能够减少该毒素分子表面色氨酸残基的溶剂暴露。该研究结果为阐明JZTX-I的离子通道门控调节机制提供了新的信息。     

The reproducibility of phospholipid analyses by MALDI-MSMS

The identification of phosphocholine and phosphoethanolamine lipids by MALDI TOF/TOF, including characterisation of the headgroup and delineation of the acyl chain at each position of the glycerol backbone, has been explored using lipids representative of each type. The relative intensities of fragments involving the neutral loss of one or other of the acyl chains from ion adducts of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) were compared. For POPC and POPE, a statistical preference for the loss of the chain from the sn-1 position was observed in the presence of lithium. For OPPC this selectivity was reversed for one of the fragments. In the absence of lithium, fragmentation was favoured at the sn-2 position for all lipids. In all cases, spectra obtained in the presence of lithium yielded more intense product ion peaks. Although Collision Induced Dissociation (CID) could be used for complete lipid characterisation, LIFT? was found to be a better method due to the presence of a greater number of distinguishing product ion peaks and a better shot-to-shot reproducibility of peak intensities.     

钙离子和镁离子浓度变化对磷脂酰乙醇胺-磷脂酰甘油双分子层膜的影响

Ca²⁺ and Mg²⁺ ions are the main divalent cations in living cells and play vital roles in the structure and function of biological membranes. To date, the differences in the effects of these two ions on the Escherichia coli (E. coli) inner membrane at various concentrations remain unknown. Here, the effects of Ca²⁺ and Mg²⁺ ions on a mixed lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a 3 : 1 ratio (mol/mol), which mimics the E. coli inner membrane, were quantitatively differentiated at different concentrations by dynamic light scattering (DLS), zeta potential measurements and all-atom molecular dynamics (AA-MD) simulations. The DLS results demonstrated that the POPE/POPG liposomes were homogeneous and monodisperse in solutions with Ca²⁺ or Mg²⁺ ion concentrations of 0 and 1 mmol∙L^-1. As the Ca²⁺ or Mg²⁺ ion concentration was increased to 5-100 mmol∙L^-1, lipid aggregation or the fusion of unilamellar liposomes occurred in the ion solutions. The zeta potential measurements showed that both the Ca²⁺ and Mg²⁺ ions had overcharging effects on the negatively charged POPE/POPG liposomes. The AA-MD simulation results indicated that the Ca²⁺ ions irreversibly adsorbed on the membranes when the simulation time was longer than 100 ns, while the Mg²⁺ ions were observed to dynamically adsorb on and desorb from the membranes at various concentrations. These results are consistent with the DLS and zeta potential experiments. The average numbers of Ca²⁺ and Mg²⁺ ions in the first coordination shell of the oxygen atoms of the phosphate, carbonyl and hydroxyl groups of POPE and POPG (i.e., the first coordination numbers) in the pure membrane and membranes containing 5 and 100 mmol∙L^-1 ions were calculated from the radial distribution functions. The results indicated that the primary binding site of these two ions on POPE and POPG at the concentrations studied was the negatively charged phosphate group. Thus, these results might explain the overcharging effects of both the Ca²⁺ and Mg²⁺ ions on the POPE/POPG liposomes. Moreover, as the Ca²⁺ concentration increased, the area per lipid of the lipid bilayers decreased, and the membrane thickness increased, while the Mg²⁺ ions had negligible effects on these membrane parameters. In addition, these ions had different effects on the orientation of the lipid head groups. These simulation results may be used to provide the possible explanations for the differences between Ca²⁺ and Mg²⁺ ions in DLS and zeta potential measurements at the atomic level. The experimental results and MD simulations provide insight into various biological processes regulated by divalent cations, such as membrane fusion.     

A facile approach for assembling lipid bilayer membranes on template-stripped gold

Lipid vesicles are designed with functional chemical groups to promote vesicle fusion on template-stripped gold (TS Au) surfaces that does not spontaneously occur on unfunctionalized Au surfaces. Three types of vesicles were exposed to TS Au surfaces: (1) vesicles composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids; (2) vesicles composed of lipid mixtures of 2.5 mol % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio)propionate] (DSPE-PEG-PDP) and 97.5 mol % of POPC; and (3) vesicles composed of 2.5 mol % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG) and 97.5 mol % POPC. Atomic force microscopy (AFM) topography and force spectroscopy measurements acquired in a fluid environment confirmed tethered lipid bilayer membrane (tLBM) formation only for vesicles composed of 2.5 mol % DSPE-PEG-PDP/97.5 mol % POPC, thus indicating that the sulfur-containing PDP group is necessary to achieve tLBM formation on TS Au via Au-thiolate bonds. Analysis of force-distance curves for 2.5 mol % DSPE-PEG-PDP/97.5 mol % POPC tLBMs on TS Au yielded a breakthrough distance of 4.8 ± 0.4 nm, which is about 1.7 nm thicker than that of POPC lipid bilayer membrane formed on mica. Thus, the PEG group serves as a spacer layer between the tLBM and the TS Au surface. Fluorescence microscopy results indicate that these tLBMs also have greater mechanical stability than solid-supported lipid bilayer membranes made from the same vesicles on mica. The described process for assembling stable tLBMs on Au surfaces is compatible with microdispensing used in array fabrication.     

Persulfated molecular umbrellas as anti-HIV and anti-HSV agents

A series of persulfated molecular umbrellas have been synthesized from putrescine, spermidine, spermine, lysine, and cholic acid (1a, 2a, 3a, 4a, and 5a) and their anti-HIV and anti-HSV activities determined. Despite it size, the most active of these conjugates (5a) was able to cross phospholipid bilayers made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). The unique combination of anti-viral activity, an ability to cross hydrophobic barriers, a lack of cytotoxicity, and a simple three-step synthesis from biogenic starting material suggests that 5a and related conjugates may be exploitable as a novel class of anti-viral agents for systemic and topical applications.     

HIV fusion inhibitor peptide T-1249 is able to insert or adsorb to lipidic bilayers. Putative correlation with improved efficiency

T-1249 is a HIV fusion inhibitor peptide under clinical trials. Its interaction with biological membrane models (large unilamellar vesicles) was studied using fluorescence spectroscopy. A gp41 peptide that includes one of the hydrophobic terminals of T-1249 was also studied. Both peptides partition extensively to liquid-crystalline POPC (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine) (DeltaG = -7.0 kcal/mol and -8.7 kcal/mol, for T-1249 and terminal peptide, respectively) and are located at the interface of the membrane. T-1249 is essentially in a random coil conformation in this lipidic medium, although a small alpha-helix contribution is present. When other lipid compositions are used (DPPC, POPG + POPC, and POPC + cholesterol) (DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)), partition decreases, the most severe effect being the presence of cholesterol. Partition experiments and fluorescence resonance energy transfer analysis show that T-1249 adsorbs to cholesterol-rich membranes. The improved clinical efficiency of T-1249 relative to enfuvirtide (T20) may be related to its bigger partition coefficient and ability to adsorb to rigid lipidic areas on the cell surface, where most receptors are inserted. Moreover, adsorption to the sterol-rich viral membrane helps to increase the local concentration of the inhibitor peptide at the fusion site.     

Liposome electrokinetic capillary chromatography in the study of analyte-phospholipid membrane interactions. Application to pesticides and related compounds

Interactions between low-molar mass analytes and phospholipid membranes were studied by liposome electrokinetic capillary chromatography (LEKC). The analytes were pesticides, some degradation products, and compounds associated with the manufacture of pesticides. Negatively charged liposome dispersions with different zwitterionic lipids (PC) were applied to the determination of retention factors (k) of 15 charged and uncharged compounds. The liposome dispersions consisted of 80:20 mol% of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/POPS, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS. Retention factors were calculated from the effective electrophoretic mobilities of the analytes under LEKC and CZE conditions and from the effective electrophoretic mobilities of the liposomes, determined by CZE with a polyacrylamide-coated capillary. Determining the liposome mobilities in this way proved to be a good alternative to the conventional method employing a liposome marker compound. The log k values of the analytes for the different liposome dispersed phases were correlated with one another. In addition, correlation curves were determined between log k and calculated octanol-water partition coefficients. The results showed that the zwitterionic phospholipid in the liposome has a major impact on the interactions between the tested compounds and the lipid membranes.     

Effects of lactose permease on the phospholipid environment in which it is reconstituted: a fluorescence and atomic force microscopy study

The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.     

Membrane orientation of MSI-78 measured by sum frequency generation vibrational spectroscopy

Antimicrobial peptides (AMPs) selectively disrupt bacterial cell membranes to kill bacteria whereas they either do not or weakly interact with mammalian cells. The orientations of AMPs in lipid bilayers mimicking bacterial and mammalian cell membranes are related to their antimicrobial activity and selectivity. To understand the role of AMP-lipid interactions in the functional properties of AMPs better, we determined the membrane orientation of an AMP (MSI-78 or pexiganan) in various model membranes using sum frequency generation (SFG) vibrational spectroscopy. A solid-supported single 1,2-dipalmitoyl-an-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) bilayer or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) bilayer was used as a model bacterial cell membrane. A supported 1,2-dipalmitoyl-an-glycero-3-phosphocholine (DPPC) bilayer or a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer was used as a model mammalian cell membrane. Our SFG results indicate that the helical MSI-78 molecules are associated with the bilayer surface with ～70° deviation from the bilayer normal in the negatively charged gel-phase DPPG bilayer at 400 nM peptide concentration. However, when the concentration was increased to 600 nM, MSI-78 molecules changed their orientation to make a 25° tilt from the lipid bilayer normal whereas multiple orientations were observed for an even higher peptide concentration in agreement with toroidal-type pore formation as reported in a previous solid-state NMR study. In contrary, no interaction between MSI-78 and a zwitterionic DPPC bilayer was observed even at a much higher peptide concentration (～12,000 nM). These results demonstrate that SFG can provide insights into the antibacterial activity and selectivity of MSI-78. Interestingly, the peptide exhibits a concentration-dependent membrane orientation in the lamellar-phase POPG bilayer and was also found to induce toroidal-type pore formation. The deduced lipid flip-flop from SFG signals observed from lipids also supports MSI-78-induced toroidal-type pore formation.     

Unveiling a complex phase transition in monolayers of a phospholipid from the annular region of transmembrane proteins

The lateral packing properties of phospholipids that surround transmembrane proteins are fundamental in the biological activity of these proteins. In this work, Langmuir monolayers of one such lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), are studied with a combination of pressure-area isotherm analysis, Brewster angle microscopy, and atomic force microscopy of extracted films. The analysis reveals a sequence of phase transitions LE-LC-LC' occurring in a narrow packing range. The lateral pressures and area densities of these phases provided meanings for the packing requirements in the annular lipid region of typical transmembrane proteins.     

Calcium oxalate monohydrate precipitation at membrane lipid rafts

The precipitation of calcium oxalate monohydrate (COM) was monitored at a Langmuir monolayer containing lipid raft domains. The raft-forming monolayer consists of a 2:1:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/sphingomyelin/dihydrocholesterol, where the raft liquid ordered phase is enriched in sphingomyelin and the sterol. COM crystals, monitored by Brewster angle microscopy, appear at the phase boundary between the raft domains and the expanded phase.     

Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy

Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance ((2)H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state (2)H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed (2)H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.     

干燥过程中LEA-motif蛋白特征重复片段对POPE膜结构的保护研究简

通过分子动力学模拟的方法,模拟了1-棕榈酰基-2-油酰基磷脂酰乙醇胺(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,POPE)生物膜在添加胚胎发育晚期丰富蛋白(Late embryogenesis abundant proteins,LEA蛋白)特征重复片段(LEA-motif)前后两种体系在低温下的干燥过程,对比分析了干燥过程中两种体系在POPE生物膜结构、扩散系数、侧链有序性及分子间氢键数目的变化,从微观角度揭示了POPE生物膜因干燥失水导致的结构变化以及添加LEA-motif之后LEA-motif与POPE生物膜的相互作用. 结果表明,LEA-motif在脱水干燥过程中能够有效地稳定膜的结构,从而保护生物材料的生物活性.     

干燥过程中LEA-motif蛋白特征重复片段对POPE膜结构的保护研究

通过分子动力学模拟的方法, 模拟了1-棕榈酰基-2-油酰基磷脂酰乙醇胺(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE)生物膜在添加胚胎发育晚期丰富蛋白(Late embryogenesis abundant proteins, LEA蛋白)特征重复片段(LEA-motif)前后两种体系在低温下的干燥过程, 对比分析了干燥过程中两种体系在POPE生物膜结构、 扩散系数、 侧链有序性及分子间氢键数目的变化, 从微观角度揭示了POPE生物膜因干燥失水导致的结构变化以及添加LEA-motif之后LEA-motif与POPE生物膜的相互作用. 结果表明, LEA-motif在脱水干燥过程中能够有效地稳定膜的结构, 从而保护生物材料的生物活性.     

Cholesterol supports headgroup superlattice domain formation in fluid phospholipid/cholesterol bilayers

Fluorescence and Fourier transform infrared (FTIR) spectroscopic techniques were used to explore the effect of added cholesterol on the composition-dependent formation of putative phospholipid headgroup superlattices in fluid 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers. Steady-state fluorescence anisotropy measurements of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) revealed significant dips at several POPE-to-phospholipid mole fractions (X(PE)'s) when the cholesterol-to-lipid mole fraction (X(CHOL)) was fixed at 0.00, 0.35, 0.40, and 0.50. Most of the observed dips occur at or close to critical X(PE)'s predicted by the Headgroup Superlattice (SL) model, suggesting that phospholipid headgroups of different structures tend to adopt regular distributions even in the presence of cholesterol. Time-resolved fluorescence anisotropy measurements revealed that DPH-PC senses a disordered and highly mobile microenvironment in the POPE/POPC/CHOL bilayers at those critical X(PE)'s, indicating that this probe may partition to defect regions in the bilayers. The presence of coexisting packing defect regions and regularly distributed SL domains is a key feature predicted by the Headgroup SL model. Importantly, probe-free FTIR measurements of acyl chain C-H, interfacial carbonyl, and headgroup phosphate stretching peak frequencies revealed the presence of abrupt changes at X(PE)'s close to those observed in the fluorescence data. When X(PE) was varied from 0.60 to 0.72 and X(CHOL) from 0.34 to 0.46, a clear dip at the lipid composition coordinates (X(PE), X(CHOL)) approximately (0.68, 0.40) was observed in the three-dimensional surface plots of DPH-PC anisotropy as well as the carbonyl and phosphate stretching frequencies. The critical X(CHOL) at 0.40 agrees with the Cholesterol SL model, which assumes that cholesterol and phospholipid form SL domains at the lipid acyl chain level. In conclusion, this study provides evidence that cholesterol supports formation of phospholipid headgroup SLs in fluid state ternary lipid bilayers. The feasibility of the parallel existence of SLs at the lipid headgroup and acyl chain levels supports the relevance of the lipid SL model for the membranes of eukaryotic cells that typically contain significant amounts of cholesterol. We speculate that lipid SL formation may play a central role in the regulation of membrane lipid compositions, maintenance of organelle boundaries, and other crucial phenomena in those cells.