Interaction of cytochrome c with 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine: evidence for acyl chain reversal

The conformational dynamics of the oxidatively modified phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) were assessed by observing by fluorescence energy transfer the association of the water-soluble cationic protein cytochrome c with micelles composed of this lipid. In keeping with reversal of the azelaoyl chain so as to expose its carboxyl function on the micelle surface, cytochrome c bound avidly to the micelles. In contrast, the aldehyde group bearing 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) interacted only weakly. While the physiological significance of the above interaction is uncertain, our results demonstrate that oxidative damage alters the physical properties of lipid bilayers, involving enrichment of the polar moieties of oxidatively modified lipid chains within the membrane surface.     

Surface planar bilayers of phospholipids used in protein membrane reconstitution: an atomic force microscopy study

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.     

A needle-and-thread approach to bilayer transport: permeation of a molecular umbrella-oligonucleotide conjugate across a phospholipid membrane

A di-walled molecular umbrella, composed of two choloyl groups, one spermidine moiety, and a 5-thiol(2-nitrobenzoyl) "handle", was covalently attached to a 16-mer oligonucleotide (S-dT16) through a disulfide bond. Incubation of this conjugate (1) with vesicles made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (95/5, mol/mol) (200 nm diameter, extrusion) that contained entrapped glutathione (GSH) at 37 degrees C resulted in the liberation of the oligonucleotide and the umbrella-bound 5-mercapto(2-nitrobenzoyl) moiety (USH) via thiolate-disulfide interchange. The appearance of USH, together with the absence of leakage of entrapped GSH and a vesicular capture of the oligonucleotide that matches the extent of USH formation, provides compelling evidence for delivery of S-dT16 into the aqueous compartment of these vesicles. In a sense, the molecular umbrella functions like a "needle" in providing a pathway for the oligonucleotide (the "thread") to cross the membrane.     

Viewing membrane-bound molecular umbrellas by parallax analyses

Fluorescence quenching measurements have been made for a series of di-walled and tetra-walled molecular umbrellas having moderate (i.e., hydroxyl-) and strong (i.e., sulfate-) facial hydrophilicity, using Cascade Blue as the fluorophore. Through the use of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotempocholine, 1-palmitoyl-2-stearoyl-(5-DOXYL)-sn-glycero-3-phosphocholine, and 1-palmitoyl-2-stearoyl-(12-DOXYL)-sn-glycero-3-phosphocholine as fluorescence quenchers, evidence has been obtained for a membrane-bound state in which the umbrella molecules lie on the surface of the lipid bilayer. In the case of the sulfated molecular umbrellas, evidence has also been obtained for a subpopulation in which the fluorophore lies deeper within the membrane. Probable structures for the shallow-lying and deep-lying molecular umbrellas are discussed.     

Membrane association and contact formation by a synthetic analogue of polymyxin B and its fluorescent derivatives

sP-B is a synthetic analogue of the natural lipopeptide antibiotic polymyxin B (PxB) that maintains the ability of the parent compound to form vesicle-vesicle contacts and induce lipid exchange. Exchange is selective, and only monoanionic phospholipids such as 1-palmitoyl-2-oleoyl-glycero-sn-3-phosphoglycerol (POPG) are transferred, whereas dianionic phospholipids such as 1-palmitoyl-2-oleoyl-glycero-sn-3-phosphate (POPA) are not, as shown by fluorescence experiments based on the excimer/monomer ratio of pyrene-labeled phospholipids. Synthetic fluorescent analogues of sP-B are used to investigate the peptide position and orientation in the intermembrane contacts: sP-Bw, an analogue that contains D-tryptophan (D-Trp) instead of the naturally occurring D-phenylalanine, and sP-Bpy, incorporating a pyrene group at the N-terminus. Tryptophan fluorescence, anisotropy, and quenching measurements performed with sP-Bw indicate that the peptide binds and inserts in anionic vesicles of POPG and POPA. However, significant differences are seen depending on the lipid composition, as also demonstrated by fluorescence resonance energy transfer (FRET) experiments from Trp to 7-nitro-2-1,3-benzoxadiazol (NBD) groups at the interface. Intermolecular FRET using sP-Bw as the donor and sP-Bpy as the acceptor indicates self-association of the peptide, possibly forming dimers, when bound to POPG vesicles at concentrations that induce the vesicle-vesicle contacts.     

Improved synthesis of the epoxy isoprostane phospholipid PEIPC and its reactivity with amines

An improved synthesis of the naturally occurring hydroxy ketone 1-palmitoyl-2-(5,6)-epoxyisoprostane E 2- sn-glycero-3-phosphocholine (PEIPC) 1, a compound that plays a role in endothelial activation in atherosclerosis, has been carried out using a PMB ether as the key protecting group. Opening of an intermediate with pentylamine shows that the allylic epoxide is the position of attack by nucleophiles.     

Analysis of 1,2(2,3)- and 1,3-positional isomers of diacylglycerols from vegetable oils by reversed-phase high-performance liquid chromatography

Separation of 1,2(2,3)- and 1,3-positional isomers of diacylglycerols (DAG) from vegetable oils by reversed-phase high-performance liquid chromatography (RP-HPLC) is investigated. The method is based on isocratic elution using 100% acetonitrile and UV detection at 205 nm. The following elution order of DAG molecular species is identified: 1,3-dilinolein < 1,2-dilinolein < 1,3-dimyristin < 1-oleoyl-3-linoleoyl-glycerol < 1,2-dimyristoyl-rac-glycerol < 1(2)-oleoyl-2(3)-linoleoyl-glycerol < 1-linolenoyl-3-stearoyl-glycerol < 1(2)-linolenoyl-2(3)-stearoyl-glycerol < 1,3-diolein < 1-palmitoyl-3-oleoyl-glycerol < 1,2-dioleoyl-sn-glycerol < 1(2)-palmitoyl-2(3)-oleoyl-glycerol < 1-linoleoyl-3-stearoyl-glycerol < 1,3-dipalmitin < 1(2)-linoleoyl-2(3)-stearoyl-glycerol < 1-oleoyl-3-stearoyl-glycerol < 1,2-dipalmitoyl-rac-glycerol < 1-palmitoyl-3-stearoyl-sn-glycerol < 1,3-distearin < 1,2-distearoyl-rac-glycerol. Linearity is observed over three orders of magnitude. Limits of detection and quantitation range 0.2-0.7 microg/mL for 1,3-dilinolein to 0.6-1.9 microg/mL for 1,2-dioleoyl-sn-glycerol, respectively. Precision and accuracy of the method are also demonstrated. The method is developed to separate mixtures of DAG molecular species produced from edible oils.     

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。     

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。     

Effect of liposome composition on β-blocker interactions studied by capillary electrokinetic chromatography

Liposome capillary electrokinetic chromatography was used to investigate the interactions between three β-blockers of different hydrophobicity and various liposome solutions. The studied β-blockers comprised alprenolol, propranolol, and carvedilol. The composition of the liposome solutions, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phos-phoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l -serine, and cholesterol in various molar ratios, was designed by a response surface methodology-central composite design approach. Subsequently, after conducting the liposome capillary electrokinetic chromatography experiments and determining the retention factors from the electrophoretic mobilities of the compounds, and further calculating the distribution coefficients, an analysis of variance was performed. After extracting the statistical models, optimal operational conditions were obtained based on the developed models. To further investigate the interactions between the β-blockers and the liposomes, nanoplasmonic sensing experiments were carried out on two different liposome systems. The overall results demonstrate the strong influence of cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l -serine on the distribution coefficients.     

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.     

Probing the hydration of lipid bilayers using a solvent isotope effect on phospholipid mixing

Nearest-neighbor recognition experiments, which have been carried out using exchangeable dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,and 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine, indicate that replacement of H2O by D2O can significantly influence phospholipid mixing, but only in bilayers that are saturated and devoid of cholesterol. These findings, together with those of previous electron spin resonance spin-labeling studies,indicate that mammalian membranes, which are rich in cholesterol and unsaturated phospholipids, are ideal hydrophobic barriers.     

Synthesis of a polymerizable metal-ion-chelating lipid for fluid bilayers.

Hydrated lipid structures, such as liposomes, that display tethered metal-ion-chelating groups have proven useful in peptide and protein binding, as well as 2D protein crystallization through molecular recognition of accessible histidine sites in proteins and peptides. Polymerizable metal-ion-chelating lipids bearing a reactive diacetylene group have been described. These interesting compounds can be polymerized in the solid-analogous phase. Here we describe the design of the first polymerizable metal-ion-chelating lipid that can be used in the fluid, i.e., liquid analogous, phase of lipid bilayers. The synthesis of 1-palmitoyl-2-[8-[(E,E)-2',4'-hexadienoyloxy]octanoyl]-sn-glycero-3-N-[11-[N',N'-bis[carboxymethyl]imino]-3,6,9-trioxaundecanoyl] phosphatidylethanolamine (1) is described. The chelator moiety, iminodiacetate (IDA), was linked to the polymerizable phosphatidylethanolamine (PE) with a terminal 2,4-hexadienoyl (sorbyl) group through an oligo(ethylene glycol)-based spacer. Lipid 1-Cu complex is designed to be combined with the corresponding polymerizable matrix lipids (bis-SorbPC) to form functionalized liposomes that can be stabilized by various polymerization methods.     

Kinetic evidence for the existence and mechanism of formation of a barrel stave structure from pore-forming dendrimers

A dendritic approach to the construction of a homologous series of pore-forming amphiphiles has been developed, based on the use of spermidine, spermine, lysine, and cholic acid. A kinetic analysis of Na+ transport across bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by three dendritic amphiphiles has provided the strongest evidence to date for a barrel stave structure.     

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.     

Scrutiny of annexin A1 mediated membrane-membrane interaction by means of a thickness shear mode resonator and computer simulations

The dissipational quartz crystal microbalance (D-QCM) technology was applied to monitor the adsorption of vesicles to membrane-bound annexin A1 by simultaneously reading out the shifts in resonance frequency and dissipation. Solid-supported membranes (SSMs) composed of a chemisorbed octanethiol monolayer and a physisorbed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine monolayer were immobilized on the gold electrode of a 5 MHz quartz plate. Adsorption and desorption of annexin A1 to the SSM was followed by means of the QCM technique. After nonbound annexin A1 was removed from solution, the second membrane binding was monitored by the D-QCM technique, which allowed distinguishing between adsorbed and ruptured vesicles. The results show that vesicles stay always intact independent of the amount of bound annexin and the vesicle and buffer composition. It was shown that the vesicle adsorption process to membrane-bound annexin A1 is fully irreversible and is mediated by two-dimensional annexin clusters. For N-terminally truncated annexin A1, a decrease in the amount of bound vesicles was observed, which might be the result of fewer binding sites presented by the annexin A1 core. Supported by computer simulations, the results demonstrate that the vesicle adsorption process is electrostatically driven, but compared to those of sole electrostatic binding, the rate constants of adsorption are 1-2 orders of magnitude smaller, indicating the presence of a potential barrier.     

Complexes of nucleolipid liposomes with single-stranded and double-stranded nucleic acids

We report on the association of anionic liposomes from POP-Ade:POPC (1-palmitoyl-2-oleoyl-phosphatidyladenosine and 1-palmitoyl-2-oleoyl-phosphatidylcholine, respectively) with single- and double-strand nucleic acids, mediated by Ca(2+) bridging. The structural and dynamical features of such complexes are compared with those displayed when the nucleolipid is replaced by POPG (1-palmitoyl-2-oleoyl-sn-phosphatidyl-glycerol), characterized by the same apolar skeleton and negative charge as POP-Ade, but lacking the nucleic polar head. For single-stranded nucleic acids, we demonstrate that specific interactions drive the formation of complexes with nucleolipid liposomes, while no association is present for POPG-based samples. For double-stranded nucleic acids, Ca(2+) bridging promotes association with both liposomal formulations, but the corresponding complexes have different structural features, in terms of size, overall charge and internal liquid-crystalline structure.     

Probing the kinetics of membrane-mediated helix folding

The kinetics of peptide-membrane association have been studied previously using stopped-flow tryptophan fluorescence; however, such experiments do not directly report the coil-to-helix transition process, which is a hallmark of peptide-membrane interaction. Herein, we report a new method for directly assessing the kinetics of the helix formation accompanied by the peptide-membrane association. This method is based on the technique of fluorescence resonance energy transfer (FRET) and an amino acid FRET pair, p-cyano-L-phenylalanine and tryptophan. To demonstrate the utility of this method, we have studied the membrane-mediated helix folding dynamics of a mutant of magainin 2, an antibiotic peptide found in the skin of the African clawed frog, Xenopus laevis. Our results indicate that the coil-to-helix transition occurs during the binding of the peptide to the lipid vesicle (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], 3:1, wt/wt) but prior to the full insertion of the peptide into the hydrophobic region of the lipid bilayers.     

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.     

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.