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。     

脂质体对细胞色素C结构的稳定作用

采用同步荧光光谱考察细胞色素C(CytC)在脂质体环境中分子内基团微环境的变化,推测分子的空间构象。结果表明:CytC结合到脂质体上引起分子内基团重新组装和排布,氨基酸残基所处的微环境发生明显变化,体现在荧光光谱上,酪氨酸和色氨酸的光活性增强,色氨酸在水溶液中的分子内电荷转移得到抑制,此过程不涉及化学键的断裂。在脂质体环境中,尿素引起的CytC解聚变性效应得到明显抑制,脂质体与尿素在促进CytC分子内氨基酸残基发光上有协同效应。     

磷脂膜色谱及其在药物跨膜转运评价中的应用

磷脂膜色谱是固态基质上的有序磷脂分子单层体系采用色谱学方法仿真药物与细胞膜相互作用过程,可用来评价药物的细胞膜渗透性和活性。硅胶表面上的磷脂单分子层模拟了单层细胞膜,因此药物的磷脂膜色谱保留行为可用于预测药物与细胞膜的相互作用。目前考察药物跨膜转运的模型主要有正辛醇/水系统、脂质体/水系统、反相色谱（ODS）以及磷脂膜色谱。与前述3种系统比较,磷脂膜色谱除了具有高效、简便等特点外,同时能模拟药物与生物膜之间疏水作用力以外的其他作用力,因此对磷脂膜色谱的研究也越来越深入。由于药物细胞膜渗透性对其有效性和安全性起着关键作用,因此磷脂膜色谱在新药研发早期阶段的介入可以有效地降低后期候选药物的淘汰率,提高新药的研发效率。该文就磷脂膜色谱的研究及在药物跨膜转运评价中的应用进行了综述。     

磷脂膜的相行为对氧化石墨烯与磷脂膜相互作用的影响

通过使用不同相变温度的磷脂分子并调节二者的比例构筑了不同相态的磷脂膜, 并利用表面增强红外光谱和激光共聚焦显微镜研究了磷脂膜的相行为对氧化石墨烯和磷脂膜相互作用的影响. 结果表明, 氧化石墨烯对磷脂膜中磷脂分子的抽提作用具有显著的相态选择性, 其选择性地抽提流动相的磷脂分子; 氧化石墨烯对流动相磷脂的抽提作用受到膜中凝胶相磷脂存在比例的影响, 只有在流动相磷脂分子占磷脂膜中磷脂分子的绝大部分时才能够发生抽提作用, 且只有流动相的磷脂分子被抽提.     

纳米雄黄与脂质体仿生膜的相互作用研究

本工作以卵磷脂与胆固醇组成的磷脂小单层脂质体（small unilamelarvesicles,suv）作为仿生膜的简单模型,采用表面等离子共振技术（SPR）、荧光偏振、拉曼（Raman）光谱、核磁共振（NMR）及原子力显微镜（AFM）研究纳米雄黄与SUV仿生膜的相互作用,证实了磷脂是纳米雄黄作用的关键靶分子．随纳米雄黄结合,SUV仿生膜的相对粘度聃值增大,膜的流动性减小．Raman光谱数据计算表明,作用后膜的纵向有序性参数s。。及横向有序性参数Slat值增大,说明纳米雄黄的结合使磷脂膜的脂酰基链全反式构型比例上升,膜的流动性减小．由Raman光谱和引PNMR结果推测,磷脂极性头部是纳米雄黄与磷脂的主要结合位点。AFM实时观测,纳米雄黄通过在膜表面打“孔”或“洞”的方式,损坏磷脂膜．     

聚半乳糖醛酸酶（PG2）中色氨酸残基的化学修饰

用化学修饰法以Ｎ－溴代琥珀酰亚胺作修饰剂，对聚半乳糖醛酸酶（ＰＧ２）中色氨酸（Ｔｒｐ）残基与酶活性的关系进行研究，发现１个ＰＧ２分子中有６个Ｔｒｐ残基，其中４个位于酶分子内部，２个位于酶分子表面，该发现通过表面荧光猝灭实验得到进一步证明，酶分子表面的２个Ｔｒｐ残基中，有１个为活性必需的氨基酸，它的修饰导致大部分酶活力丧失，底物保护实验进一步证明该活性必需的Ｔｒｐ可能位于酶的活性部位，酶被修饰后其圆     

肌红蛋白的同步荧光光谱

首次对肌红蛋白的同步荧光光谱进行了研究,并对肌红蛋白荧光峰予以归属。当△λ为20nm时,308nm处的荧光峰主要为酪氨酸残基的贡献,很小一部分是由色氨酸残基贡献的;△λ为40nm时,分别在322和596nm处观察到两个荧光峰,322nm的荧光峰为酪氨酸和色氨酸残基的共同贡献,596nm的荧光峰则归属为肌红蛋白分子中血红素的贡献。     

毒素蛋白ColicinE1与磷脂膜相互作用的荧光光谱研究临界摩尔比的测定

磷脂－蛋白相互作用的临界摩尔比是研究膜脂－蛋白相互作用的重要参数．本文利用荧光光谱技术首次测定了毒素蛋白ＣｏｌｉｃｉｎＥ１在不同条件下与不同磷脂膜相互作用的临界摩尔比并通过临界摩尔比的变化讨论了插膜蛋白与磷脂膜相互作用的规律，为进一步探讨毒素蛋白的插膜机制提供了重要的基础     

毒素蛋白Colicin E1与磷脂膜相互作用的荧光光谱研究——临界摩尔…

磷脂－蛋白相互作用的临界摩尔比是研究膜脂－蛋白相互作用的重要参数。本文利用荧光光谱技术首次测定了毒素蛋白Ｃｏｌｉｃｉｎ Ｅ１在不同条件下与不同磷脂膜相互作用的临界摩尔比并通过临界摩尔比的变化讨论了插膜蛋白与磷脂膜相互作用的规律，为进一步探讨毒素蛋白的插膜机制提供了重要的基础。     

家蝇幼虫抗菌肽MDL-1的荧光特性分析

本文研究了家蝇幼虫抗菌肽MDL-1的荧光光谱和淬灭剂对内源性荧光的影响。家蝇幼虫抗菌肽MDL-1在激发波长280 nm时,其荧光光谱为酪氨酸（Tyr）残基和色氨酸（Trp）残基共同提供。结果表明,KI不能淬灭抗菌肽MDL-1的Trp残基的荧光,而丙烯酰胺（Acr）能淬灭几乎所有的Trp残基的荧光（f-0．92）;这说明,Trp残基不是位于抗菌肽分子的表面,而是位于分子的内部。     

Phospholipase D-mediated aggregation, fusion, and precipitation of phospholipid vesicles

Large unilamellar vesicles with a diameter of 100 nm were prepared from the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at pH 8.0. After addition to these vesicles of the enzyme phospholipase D (PLD) from Streptomyces sp. AA586 at 40 degrees C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only approximately 68% in the absence of Ca2+. Furthermore, in the absence of Ca2+, the size of the vesicles did not change significantly with time upon PLD addition, as judged from turbidity, dynamic light scattering, and electron microscopy measurements. In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca2+ ions with the negatively charged phospholipids formed in the membranes. Vesicle fusion was monitored by using a novel fusion assay system involving vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. After vesicle fusion, chymotrypsinogen A transformed into a-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The alpha-chymotrypsin formed could be detected with benzoyl-L-Tyr-p-nitroanilide as a membrane permeable chymotrypsin substrate. The observed vesicle precipitation occurring after vesicle fusion in the presence of 1 mM Ca2+ was correlated with an increase of the main phase transition temperature, Tm, of POPA to values above 40 degrees C.     

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.     

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.     

Shrink-wrap vesicles

We describe a simple approach to the controlled removal of molecules from the membrane of large unilamellar vesicles made of fatty acids. Such vesicles shrink dramatically upon mixing with micelles composed of a mixture of fatty acid and a phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)), as fatty acid molecules leave the vesicle membrane and accumulate within the mixed micelles. Vesicle shrinkage was confirmed by dynamic light scattering, fluorescence recovery after photobleaching of labeled vesicles, and fluorescence resonance energy transfer between lipid dyes incorporated into the vesicle membrane. Most of the encapsulated impermeable solute is retained during shrinkage, becoming concentrated by a factor of at least 50-fold in the final small vesicles. This unprecedented combination of vesicle shrinkage with retention of contents allows for the preparation of small vesicles containing high solute concentrations, and may find applications in liposomal drug delivery.     

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.     

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.     

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.     

Lipid nanotube formation from streptavidin-membrane binding

A novel transformation of giant lipid vesicles to produce nanotubular structures was observed upon the binding of streptavidin to biotinylated membranes. Unlike membrane budding and tubulation processes caused by proteins involved with endocytosis and vesicle fusion, streptavidin is known to crystallize at near the isoelectric point (pI 5 to 6) into planar sheets against biotinylated films. We have found, however, that at neutral pH membranes of low bending rigidity (<10kT), such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), spontaneously produce tubular structures with widths ranging from micrometers to below the diffraction limit (<250 nm) and lengths spanning up to hundreds of micrometers. The nanotubes were typically held taut between surface-bound vesicles suggesting high membrane tension, yet the lipid nanotubes exhibited a fluidic nature that enabled the transport of entrained vesicles. Confocal microscopy confirmed the uniform coating of streptavidin over the vesicles and nanotubes. Giant vesicles composed of lipid membranes of higher bending energy exhibited only aggregation in the presence of streptavidin. Routes toward the development of these highly curved membrane structures are discussed in terms of general protein-membrane interactions.     

Fluorescence microscopic investigation on morphological changes of giant multilamellar vesicles induced by amphiphilic additives

Adding an artificial bolaamphiphile to a dispersion of giant multilamellar vesicles (GMVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) induced a cup-shaped deformation in GMVs accompanied by partial extrusion of the inner vesicle; thereafter, the deformed vesicles returned to their original shape. On the other hand, when the artificial bolaamphiphile together with a surfactant was added to the vesicular dispersion, these deformation and reformation dynamics were transmitted from the outer membranes in GMVs to the inner membranes until an intact inner vesicle was extruded out of the outer membrane. The microscopic aspects of these processes were investigated using amphiphiles tagged with individual fluorophores.