Lipid headgroups mediate organization and dynamics in bilayers

We report on the fluorescence lifetime and anisotropy decay dynamics of the tethered chromophore NBD in unilamellar vesicles comprised of phosphoglycerol and phosphocholine lipids with C(12) and C(18) saturated acyl chains, with or without cholesterol and/or sphingomyelin. For the phosphocholine vesicles, we use the chromophore 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-PC), and for the phosphoglycerol vesicles, we use the chromophore 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (NBD-PG). The addition of cholesterol and/or sphingomyelin to the PC vesicles restricts the chromophore environment, in agreement with the known rigidizing effect of cholesterol on PC membranes. The PG systems do not exhibit an analogous effect with the addition of cholesterol and/or sphingomyelin. The motional freedom of the NBD chromophore is, in general, more restricted in the PC bilayers than it is in the PG bilayers, and we understand this behavior in the context of the role of the lipid headgroups in mediating bilayer organization.     

Effect of submicellar concentrations of conjugated and unconjugated bile salts on the lipid bilayer membrane

The interaction of submicellar concentrations of various physiologically important unconjugated [sodium deoxycholate (NaDC), sodium cholate (NaC)] and conjugated [sodium glycodeoxycholate (NaGDC), sodium glycocholate (NaGC), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC)] bile salts with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles in solid gel (SG) and liquid crystalline (LC) phases was investigated using the excited-state prototropism of 1-naphthol. Steady-state and time-resolved fluorescence of the two excited-state prototropic forms of 1-naphthol indicate that submicellar bile salt concentration induces hydration of the lipid bilayer membrane into the core region. This hydration effect is a general phenomenon of the bile salts studied. The bilayer hydration efficiency of the bile salt follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC for both DPPC and DMPC vesicles in their SG and LC phases.     

Facilitated phosphatidylcholine flip-flop across erythrocyte membranes using low molecular weight synthetic translocases.

The transmembrane distribution of phospholipids plays an important regulatory role in human erythrocytes. Membrane-bound translocase enzymes maintain an asymmetric phospholipid distribution across the membrane monolayers by promoting transmembrane diffusion or flip-flop. Mechanistic understanding of the flip-flop process is weak at the molecular level. Recently, we discovered that amide and sulfonamide derivatives of tris(aminoethyl)amine facilitate phospholipid flip-flop across vesicle membranes; that is, they act as low molecular weight, synthetic translocases. In this report, NMR evidence is provided that suggests that the synthetic translocases work by forming a hydrogen-bonded complex with the phosphocholine headgroup which decreases headgroup polarity and promotes diffusion across the lipophilic interior of the membrane. Also cell morphology and fluorescence probe methods are used to show that these synthetic translocases facilitate phosphatidylcholine flip-flop across erythrocyte membranes. Addition of a small amount of dilauroylphosphatidylcholine to erythrocytes produces echinocyte morphology which takes days to revert back to the original discocyte shape. The rate of return is significantly accelerated by the presence of the synthetic translocases. The synthetic translocases facilitate inward-translocation (flip) of the fluorescent phosphatidylcholine probe, 1-palmitoyl-2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoyl)-sn-glycero-3-phosphocholine (PC-NBD).     

Micellization of bile salts in aqueous medium: a fluorescence study

Owing to the physiological importance of the micellization process of bile salts, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activities. The present study suggests fluorescence probing, using 1,6-diphenylhexatriene (DPH), as a simple, convenient, sensitive and economic method for monitoring the micellization process of bile salts in aqueous medium. Three independent parameters: fluorescence intensity, anisotropy and lifetime of DPH have been employed successfully for determining the CMC of two bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC), in aqueous medium. The CMC values reported by all the above three parameters of DPH are found to be same and it is 16 mM for NaC and 6 mM for NaDC at 25 degrees C in unbuffered solution. The effect of temperature and ionic strength on the micellization process has also been investigated employing DPH as a fluorescent probe. Increasing temperature leads to the formation of fluffier micelles with less rigid interior for both NaC and NaDC. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt.     

Quantitating the dynamics of NBD hexanoic acid in homogeneous solution and in solutions containing unilamellar vesicles

We report here on the motional and fluorescence lifetime dynamics of the chromophore NBDHA (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) in neat solvents and in aqueous solutions containing unilamellar vesicles of varying composition. We measure the transient response of this chromophore by time-correlated single-photon counting, using one- and two-photon excitation to resolve the Cartesian components of the rotational diffusion constant, D. Our experimental data for NBDHA in selected solvents of varying viscosity demonstrate that one- and two-photon excitation probe different components of the rotational diffusion constant and that this chromophore reorients as a prolate rotor with an aspect ratio of approximately 2. For NBDHA in aqueous solutions containing unilamellar vesicles of varying composition, we recover the same reorientation behavior regardless of vesicle composition. Fluorescence lifetime and steady-state fluorescence data show the chromophore to reside in a polar environment that is different from neat water. We understand these data in the context of the chromophore residing in close proximity to the unilamellar vesicle polar headgroups in all cases.     

Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching

Lateral diffusion of membrane constituents plays an important role in membrane organization and represents a central theme in current models describing the structure and function of biological membranes. Fluorescence recovery after photobleaching (FRAP) is a widely used approach that provides information regarding dynamic properties and spatial distribution of membrane constituents. On the basis of the unique concentration-dependent fluorescence emission properties of a fluorescently labeled cholesterol analogue modified at the tail region, 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (25-NBD-cholesterol), we have previously shown that it exhibits local organization even at very low concentrations in membranes. In this paper, we address aspects regarding the molecular size and dynamics of such an organized assembly of 25-NBD-cholesterol by monitoring its lateral diffusion characteristics using FRAP. To obtain a comprehensive understanding of the organization and dynamics of 25-NBD-cholesterol in the membrane, we compare its diffusion properties to that of a fluorescent phospholipid analogue 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-(1,3-benzoxadiazol-4-yl)) (NBD-PE). Our results indicate significant differences in the membrane dynamics of these NBD-labeled lipids. Importantly, on the basis of a novel wavelength-selective FRAP approach, our results show that the organization of 25-NBD-cholesterol is heterogeneous, with the presence of fast- and slow-diffusing species which could correspond to predominant populations of monomers and dimers of 25-NBD-cholesterol. The potential application of the wavelength-selective FRAP approach to monitor the organization and dynamics of molecules in membranes therefore represents an exciting possibility.     

Structure/activity study of tris(2-aminoethyl)amine-derived translocases for phosphatidylcholine

Sulfonamide and amide derivatives of tris(aminoethyl)amine (TREN) are known to facilitate phospholipid translocation across vesicle and erythrocyte membranes; that is, they act as synthetic translocases. In this report, a number of new TREN-based translocases are evaluated for their abilities to bind phosphatidylcholine and translocate a fluorescent phosphatidylcholine probe. Association constants were determined from (1)H NMR titration experiments, and translocation half-lives were determined via 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite quenching assays. A rough correlation exists between translocase/phosphatidylcholine association constants and translocation half-lives. The tris-sulfonamide translocases are superior to the tris-amide versions because they associate more strongly with the phospholipid headgroup. The stronger association is due to the increased acidity of the sulfonamide NHs as well as a molecular geometry (as shown by X-ray crystallography) that is able to form tridentate complexes with one of the phosphate oxygens. Two fluorescent translocase analogues were synthesized and used to characterize membrane partitioning properties. The results indicate that the facilitated translocation of phospholipids by TREN-derived translocases is due to the formation of hydrogen-bonded complexes with the phospholipid headgroups. In the case of zwitterionic phosphatidylcholine, it is the neutral form of the translocases that rapidly associates with the phosphate portion of the phosphocholine headgroup. Complexation masks the headgroup polarity and promotes diffusion of the phospholipid-translocase complex across the lipophilic interior of the membrane.     

Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholine with substrate

Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.     

Drastic changes in the fluorescence properties of NBD probes with the polarity of the medium: involvement of a TICT state?

The spectroscopic and photophysical properties of two 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) derivatives with different substituents on the nitrogen group are reported in 18 solvents. The solvatochromic shifts were analysed by correlating with polarity scales. The results, together with the help of modified neglect of diatomic overlap (MNDO) calculations, enable the polarity of the ground and first singlet excited states to be determined. Experiments based on variations in temperature and viscosity establish that the two probes undergo different de-excitation pathways. The possibilities of internal rotation leading to a twisted intramolecular charge transfer (TICT) state in the case of diethylamino-NBD are discussed. A study in binary solvent mixtures outlines specific solvent—solute interactions. Appropriate restrictions are emphasized on the utilization of NBD probes in biological fields.     

Nitroxide-fluorophore double probes: a potential tool for studying membrane heterogeneity by ESR and fluorescence

A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide-fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to the water-lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on cells show preferential localization of both probes in the plasma membrane, with a relatively slow redistribution to other membranes of the cell. We believe that such double probes would be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.     

微流控芯片电泳分离血清中小而密低密度脂蛋白的研究

应用微流控芯片电泳,以40 mmol/L Tricine(pH9.8)作为电泳缓冲体系,十二烷基硫酸钠(SDS)作为添加剂(0.1 mmol/L SDS样品溶液,0.02 mmol/L SDS分离缓冲液),分离血清小而密低密度脂蛋白(sdLDL)。研究荧光染料硝基苯并噁二唑-C6-酰基鞘胺醇(NBD C6-ceramide)与脂蛋白结合的特异性、饱和性以及血清保存和检测时间对脂蛋白电泳行为的影响;探讨SDS有效降低蛋白吸附,提高血清脂蛋白分辨率的作用。冠心病(CHD)组sdLDL检出率(75%)显著高于对照组(6%,P<0.01)。该法具有简易、快速、高效等优点,可望成为CHD危险性评估的常规分析手段。     

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin_1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin_1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.     

Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A2 activity

In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot–fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A_2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C₆-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A_2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A₂ as low as 0.0075 U mL^?1 and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A₂ inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).     

Fluid biomembranes supported on nanoporous aerogel/xerogel substrates

Planar supported lipid bilayers have attracted immense interest for their properties as model cell membranes and for potential applications in biosensors and lab-on-a-chip devices. We report the formation of fluid planar biomembranes on hydrophilic silica aerogels and xerogels. Scanning electron microscopy results showed the presence of interconnected silica beads of approximately 10-25 nm in diameter and nanoscale open pores of comparable size for the aerogel and grain size of approximately 36-104 nm with approximately 9-24 nm diameter pores for the xerogel. When the aerogel/xerogel was prehydrated and then allowed to incubate in l-alpha-phosphatidylcholine (egg yolk PC) unilamellar vesicle (approximately 30 nm diameter) solution, lipid bilayers were formed due to the favorable interaction of vesicles with the hydroxyl-abundant silica surface. Lateral mobility of labeled lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine was retained in the membranes. A diffusion coefficient of 0.61 +/- 0.22 microm(2)/s was determined from fluorescence recovery after photobleaching analysis for membranes on aerogels, compared to 2.46 +/- 0.35 microm(2)/s on flat glass. Quartz crystal microbalance-dissipation was utilized to monitor the kinetics of the irreversible adsorption and fusion of vesicles into bilayers on xerogel thin films.     

Small-angle neutron scattering study of sodium cholate and sodium deoxycholate interacting micelles in aqueous medium

Small angle neutron scattering (SANS) measurements of D₂O solutions (0.1 M) of sodium cholate (NaC) and sodium deoxycholate (NaDC) were carried out atT= 298 K. Under compositions very much above the critical micelle concentration (CMC), the bile salt micelle size growths were monitored by adopting Hayter-Penfold type analysis of the scattering data. NaC and NaDC solutions show presence of correlation peaks atQ = 0.12 and 0.1 ?_-1 respectively. Monodisperse ellipsoids of the micelles produce best fits. For NaC and NaDC systems, aggregation number (9.0, 16.0), fraction of the free counterions per micelle (0.79, 0.62), semi-minor (8.0 ?) and semi-major axes (18.4, 31.7 ?) values for the micelles were deduced. Extent of micellar growth was studied using ESR correlation time measurements on a suitable probe incorporating NaC and NaDC micelles. The growth parameter (axial ratio) values were found to be 2.3 and 4.0 for NaC and NaDC systems respectively. The values agree with those of SANS.     

Mixed Micellization of Dimeric (Gemini) Surfactants and Conventional Surfactants

The aqueous solutions of mixtures of various conventional surfactants and dimeric anionic and cationic surfactants have been investigated by electrical conductivity, spectrofluorometry, and time-resolved fluorescence quenching to determine the critical micelle concentrations and the micelle aggregation numbers in these mixtures. The following systems have been investigated: 12-2-12/DTAB, 12-2-12/C(12)E(6), 12-2-12/C(12)E(8), 12-3-12/C(12)E(8), Dim3/C(12)E(8), and Dim4/C(12)E(8) (12-2-12 and 12-3-12=dimethylene-1,2- and trimethylene-1,3-bis(dodecyldimethylammonium bromide), respectively; C(12)E(6) and C(12)E(8)=hexa- and octaethyleneglycol monododecylethers, respectively; Dim3 and Dim4=anionic dimeric surfactants of the disodium sulfonate type, Scheme 1; DTAB=dodecyltrimethylammonium bromide). For the sake of comparison the conventional surfactant mixtures DTAB/C(12)E(8) and SDS/C(12)E(8) (SDS=sodium dodecylsulfate) have also been investigated (reference systems). Synergism in micelle formation (presence of a minimum in the cmc vs composition plot) has been observed for the Dim4/C(12)E(8) mixture but not for other dimeric surfactant/nonionic surfactant mixtures investigated. The aggregation numbers of the mixed reference systems DTAB/C(12)E(8) and SDS/C(12)E(8) vary monotonously with composition from the value of the aggregation number of the pure C(12)E(8) to that of the pure ionic component. In contrast, the aggregation number of the dimeric surfactant/C(12)E(8) mixtures goes through a minimum at a low value of the dimeric surfactant mole fraction. This minimum does not appear to be correlated to the existence of synergism in micelle formation. The initial decrease of the aggregation number of the nonionic surfactant upon addition of ionic surfactant, up to a mole fraction of ionic surfactant of about 0.2 (in equivalent per total equivalent), depends little on the nature the surfactant, whether conventional or dimeric. The results also show that the microviscosity of the systems containing dimeric surfactants is larger than that of the reference systems. Copyright 2001 Academic Press.     

Temperature dependence of the interaction of cholate and deoxycholate with fluid model membranes and their solubilization into mixed micelles

The interaction of bile salts with model membranes composed of soybean phosphatidylcholine (SPC) and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using high sensitivity isothermal titration calorimetry (ITC). The partitioning and incorporation of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) from an aqueous phase (pure water or 0.1 M NaCl) into fluid bilayer vesicles was studied as a function of temperature and ionic strength. The thermodynamic parameters of partitioning were determined with a model taking electrostatic interactions into account. In addition, the solubilization of SPC and POPC vesicles with NaC and NaDC as a function of temperature was also studied by ITC and the phase diagrams for the vesicle to micelle transition at two different temperatures were established. Unsaturated phospholipids require higher amounts of detergent to be transformed into micelles compared to saturated phospholipids. In addition, the width of the coexistence region of mixed micelles and mixed vesicles is larger for phosphatidylcholines with unsaturated chains. A comparison of NaDC with NaC shows the higher solubilization effectiveness of NaDC in agreement with its lower cmc. Furthermore, increasing the ionic strength decreases the amount of bile salt necessary for the formation of mixed micelles, which is also expected from the decrease of the cmc with ionic strength due to the shielding of the charges of the bile salts.     

稳态荧光探针法测定三聚季铵盐表面活性剂的胶束聚集数

以芘为荧光探针, 十六烷基氯化吡啶(CPC)为猝灭剂, 以芘的饱和水溶液为溶剂配制表面活性剂溶液, 根据芘的荧光强度之比I1/I3随表面活性剂水溶液浓度的变化, 测定了三聚季铵盐表面活性剂CTTTA的cmc值, 测定值与表面张力法测定的cmc值一致；当猝灭剂CPC的浓度取0.1~0.3 mmol·L-1范围时, 用稳态荧光探针法测定了CTTTA的胶束聚集数. 实验数据表明, 表面活性剂溶液浓度为6~10倍cmc时, 胶束聚集数N随表面活性剂浓度增大而线性增大, 并用外推法得到CTTTA的临界胶束聚集数.     

Investigation of nonspecific effects of different dyes in the screening of labeled carbohydrates against immobilized proteins

[structure: see text] Carbohydrates play an important role in life processes, and combinatorial chemistry can provide useful sources of thousands of synthetic carbohydrates as potential ligands for biological receptors. To accelerate the detection of positive hits arising from specific interactions between a carbohydrate and a protein, the use of fluorescent dyes can serve as a reliable detecting tool. A study of labeled carbohydrates to lectins conjugated to a solid-support shows that succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X) dye provides by far the lowest level of nonspecific interaction with immobilized protein. This observation is in stark contrast with the commonly used labeling reagents constituted of charged and aromatic groups, for instance, FITC and TAMRA dyes.