The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.     

Liposomal Co-Encapsulation of Two Novel Europium Complexes and Doxorubicin: Fluorescence Study

The present study was undertaken to design the novel liposomal drug formulation containing doxorubicin and europium coordination complexes. It was shown that co-encapsulation of the drugs facilitates the partitioning and permeation of lanthanides into the lipid bilayer. The obtained results suggest that new drug platform may have potential application in the design of novel antitumor agents.     

Europium Coordination Complexes as Potential Anticancer Drugs: Their Partitioning and Permeation Into Lipid Bilayers as Revealed by Pyrene Fluorescence Quenching

The present study was undertaken to evaluate the membrane-associating properties of a series of novel antitumor agents, Eu(III) coordination complexes (EC), using the pyrene fluorescence quenching as an analytical instrument. Analysis of EC-induced decrease in pyrene fluorescence intensity in terms of partition and solubility-diffusion models allowed us to evaluate the partition and permeation coefficients of the examined compounds into the lipid vesicles prepared from zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) and anionic lipid cardiolipin (CL). The drug-lipid interactions were found to have the complex nature determined by both EC structure and lipid bilayer composition. High values of the obtained partition and permeation coefficients create the background for the development of EC liposomal formulations.     

Investigation of the structure of multilayer lipid membranes by real-time neutron diffraction

This paper reports on the results of neutron diffraction experiments on the study of structural changes in multilayer lipid membranes as a function of the degree of hydration. The experiments have been performed on a time-of-flight diffractometer at the IBR-2 pulsed reactor (Joint Institute for Nuclear Research, Dubna, Russia) with a dipalmitoylphosphatidylcholine (DPPC) multilayer membrane oriented on a quartz plate. The structural parameters have been determined from the simultaneously measured several (up to five) diffraction orders of reflection, which has made it possible to calculate the profile of the DPPC bilayer structure. A high rate of the measurement of diffraction patterns has allowed one to trace the evolution the lamellar structure of the lipid bilayer in the course of its hydration and dehydration. The minimum time of accumulation of the necessary statistics is 3 min, which has made it possible to determine the characteristic times of transition processes in the membrane with a high accuracy.     

Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR

High-field W-band (95 GHz) electron paramagnetic resonance (EPR) study of partitioning of a small nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) in multilamellar liposomes composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is described. The high-resolution spectra with a high signal-to-noise ratio were combined with automated least-squares simulation analysis to derive accurate partitioning coefficients of TEMPO in the membrane lipid phase and to follow the membrane phase transitions. The isotropic magnetic parameters, g_iso and A_iso were used to characterize the average polarity the spin label is experiencing in the membrane. We also report an empirical correlation between g_iso and A_iso for a set of protic and aprotic solvents and use this correlation to assign domains formed by interdigitation of DPPC bilayer under a high ethanol concentration of 1.2 M.     

A molecular model for the line tension of lipid membranes

The line tension of a symmetric, lipid bilayer in its liquid-crystalline state is calculated on the basis of a molecular lipid model. The lipid model extends the opposing forces model by an expression for the conformational free energy of the hydrocarbon chains. We consider a membrane edge that consists of a perturbed bilayer covered by a section of a cylinder-like micelle. The structural rearrangement of the lipids implies an excess free energy which we minimize with respect to the cross-sectional shape of the membrane edge, including both the micellar and the bilayer region. The line tension is derived as a function of molecular lipid properties, like the lipid chain length or the head group interaction strength. We also relate it to the spontaneous curvature of the lipid layer. We find the line tension to become smaller for lipid layers that tend to curve more towards the hydrophobic core. Our predictions for the line tension and their relation to experimentally derived values are discussed. Received 2 January 2000     

Refined dynamic structure factor of a lipid bilayer on scales comparable to its thickness

The structural inhomogeneity of a lipid bilayer is an obstacle to applying the classical Canham–Helfrich model to describe its dynamics on nanometer length scales. In this paper, a refined expression for the free energy of a single-component lipid bilayer is used to describe the dynamics of lipid density fluctuations. In particular, the expression with a term involving the gradient of the area per lipid [8] is used for the free energy per lipid. A refined expression has been derived for the dynamic structure factor of a free lipid bilayer in the hydrodynamic region. It leads to differences in the interpretation and values of the bilayer parameters in comparison with the standard model.     

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

Hypothetical physical model for laser biostimulation

Coherent and polarized laser light has a special, and until now unexplained, effect on biological systems. This has been called biostimulation. A hypothetical model based on experimental facts to explain this effect is proposed. The substance of this model is that the polarized light reorders the polar heads of the lipid bilayer in the cell membrane, the lipid bilayer being near phase transition. This change in the quality of the cell membrane influences all the processes closely connected with it. These process modifications may yield an explanation for biostimulation.     

磷酸钾缓冲溶液与模型细胞膜相互作用的和频光谱

利用和频光谱技术详细研究了磷酸钾缓冲溶液与带负电荷的生物仿生膜(d₅₄-DMPG磷脂双层膜)相互作用的实时过程．通过监控CD₂、CD₃、磷脂分子头部的磷酸根以及羰基官能团的光谱信号随加入磷酸钾缓冲溶液的实时变化,获得了磷脂双层膜分子结构的动力学变化．结果表明K⁺能够结合到细胞膜上,并且很快地引起了CD₂、CD₃、磷脂头部磷酸根以及羰基官能团信号的变化．根据各官能团的和频信号响应,磷酸钾缓冲溶液很可能是通过在双层膜中形成环形气孔来与磷脂双层膜发生作用．该结果可以很好地解释磷酸钾缓冲溶液环境下的离子协助蛋白质的跨膜过程．     

Investigation of the dynamics of bacteriorhodopsin

Bacteriorhodopsin (bR) converted to the blue form by deionization has been reconstituted to the active purple membrane by addition of⁵⁷Fe ions. Mössbauer spectra measured in a wide temperature range reveal Fe³⁺ binding places with oxygen atoms in the neighbourhood. No evidence for a well defined functional binding place of the iron has been found. On a timescale faster 100 ns the purple membrane shows increasing flexibility above 200 K. In order to analyse the influence of the lipids, a bacteriorhodopsin sample where the lipid content has been increased artificially by the incorporation of DMPC as well as a sample consisting of lipid bilayer have been investigated.     

Examining Protein-Lipid Interactions in Model Systems with a New Squarylium Fluorescent Dye

The applicability of newly synthesized squarylium dye Sq to probing the changes in physical characteristics of lipid bilayer on the formation of protein-lipid complexes has been evaluated. Lipid vesicles composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and cholesterol (Chol) were employed as lipid component of model membrane systems while protein constituent was represented by lysozyme (Lz). Fluorescence intensity of Sq was found to decrease on Lz association with lipid bilayer. This effect was observed in all kinds of model systems suggesting that Sq is sensitive to modification of lipid bilayer physical properties on hydrophobic protein-lipid interactions. It was found that Sq spectral response to variations in Chol content depends on relative contributions of electrostatic and hydrophobic components of Lz-membrane binding.     

Permeation of nanocrystals across lipid membranes

Biological membranes are one of the major structural elements of cells, and play a key role as a selective barrier and substrate for many proteins that facilitate transport and signaling processes. Understanding the structural and mechanical properties of lipid membranes during permeation of nanomaterials is of prime importance in determining the toxicity of nanomaterials to living cells. It has been shown that the interaction between lipid membranes and nanomaterials and the disruption of lipid membranes are often determined by physicochemical properties of nanomaterials, such as size, shape and surface composition. In this work, molecular dynamic simulations were carried out using various sizes of nanocrystals as a probe to explore the transport of nanomaterials across dipalmitoylphosphatidylcholine (DPPC) bilayers and the changes in the structural and mechanical properties of DPPC bilayers during the permeation. A coarse-grained model was used to provide insight at large time and length scales. In this work, an external driving force helps the nanocrystals across the lipid bilayer. The minimum forces needed to penetrate the model membrane and the interaction of nanocrystals and lipid bilayers were investigated in simulations. The elastic and dynamic properties of lipid bilayers, including the local and bulk properties during the permeation of the nanocrystals, which are of considerable fundamental interest, were also studied. The findings described will lead to better understanding of nanomaterial–lipid membrane interactions and the mechanical and dynamic properties of lipid membranes under permeation.     

SERS技术在金属固体表面上人工双分子层膜(BLM)基的生物和化学传感器研究中的应用

ＳＥＲＳ技术在金属固体表面上人工双分子层膜（ＢＬＭ）基的生物和化学传感器研究中的应用方炎胡凤霞林书煌（首都师范大学综合技术研究所北京１０００３７）ＴｈｅＡｐｐｌｉｃａｔｉｏｎｏｆＳｕｒｆａｃｅ┐ＥｎｈａｎｃｅｄＲａｍａｎＳｃａｔｅｒｉｎｇ（ＳＥＲＳ）...     

Advances in solid-state NMR of membrane proteins

Solid-state nuclear magnetic resonance (SSNMR) is an NMR spectroscopy applied to condensed-phase systems, including membrane proteins. Membrane protein fold and function are dependent upon interactions with surrounding bilayer components. Structural and functional analyses are thus challenging, and new approaches are needed to better characterise these systems. SSNMR is uniquely suited to the examination of membrane proteins in native environments, and has the capabilities to elucidate complex protein mechanisms and structures. Notable research implementing SSNMR is aimed at developing new strategies and technology to efficiently target membrane proteins within synthetic and biological membranes. Significant advances have been made: observation of protein function in native environments, emergence of in situ methods to examine integral proteins within natural membranes, sensitivity enhancement techniques and cutting-edge structure determination methods. We present how these advances are applied to answer outstanding questions in structural biology. Experiments have shown consistent results for protein investigations in biological membranes and synthetic lipid compositions, indicating that SSNMR is an innovative and direct approach for the study of these systems.     

Novel benzanthrone aminoderivatives for membrane studies

The potential of novel benzanthrone aminoderivatives to trace the changes in physicochemical properties of lipid bilayer has been evaluated. Binding of the dyes to the lipid bilayers composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with anionic phospholipid cardiolipin (CL) and cholesterol (Chol) was followed by significant quantum yield increase with small blue shift of emission maximum. Analysis of partition coefficients of the dyes under study showed that all aminobenzanthrones possess high lipid-associating ability. The dyes A8 and AM2 proved to be sensitive to the variations in membrane chemical composition responding to the changes in bilayer hydration induced by CL and Chol.     

The effect of high-voltage electrostatic field (HVEF) on aged rice (Oryza sativa L.) seeds vigor and lipid peroxidation of seedlings

The study of the effect of high-voltage electrostatic field (HVEF) on plant seeds has been becoming one of the popular issues in electrostatic biology since the influence of HVEF on growing plants was observed early in 1980s and a few studies showed that HVEF could increase seeds activity. In this study the aged rice seeds were treated in HVEF, and their vitality and associated physiological variables such as membrane lipid peroxidation and the protective enzyme system, such as superoxide dismutase (SOD: EC 1.15.1.1), peroxidase (POD: EC 1.11.1.7) and catalase (CAT: EC 1.11.1.6) were also investigated. The results showed that the activity index of aged rice seeds was significantly increased, and the leakage conductivity was decreased with the appropriate HVEF treatment. The treatment also elevated the activity of SOD, POD and CAT, which improved the penetrating capability of membrane, and adjusted the level of lipid peroxidation and lowered the content of malondialdehyde (MDA). We concluded that HVEF could elevate aged rice seeds vigor and improve membrane system of aged rice seedlings, and the treatment for 55 min was better than that of 30 min.     

Energy of the interaction between membrane lipid domains calculated from splay and tilt deformations

Specific domains, called rafts, are formed in cell membranes. Similar lipid domains can be formed in model membranes as a result of phase separation with raft size may remaining small (～10–100 nm) for a long time. The characteristic lifetime of a nanoraft ensemble strongly depends on the nature of mutual raft interactions. The interaction energy between the boundaries of two rafts has been calculated under the assumption that the thickness of the raft bilayer is greater than that of the surrounding membrane, and elastic deformations appear in order to smooth the thickness mismatch at the boundary. When rafts approach each other, deformations from their boundaries overlap, making interaction energy profile sophisticated. It has been shown that raft merger occurs in two stages: rafts first merge in one monolayer of the lipid bilayer and then in another monolayer. Each merger stage requires overcoming of an energy barrier of about 0.08–0.12 k _BT per 1 nm of boundary length. These results allow us to explain the stability of the ensemble of finite sized rafts.     

Construction of a molecular model of a fragment of a lipid membrane in the gel and liquid crystalline states

A model of a lipid membrane in the liquid crystalline and gel states is constructed. The model of the membrane in the liquid crystalline state adequately reproduces the geometric parameters of the membrane, lipid packing, and the mobility parameters of molecules. The model of the membrane in the gel state of the bilayer reproduces the geometric parameters of the real membrane and lipid packing. New methods for calculation of the mobility parameters of phospholipid molecules in the plane of the bilayer are presented.     

On the Membrane Binding of the Potentiometric Probe Di-4-ANEPPS: A Fluorescence and Resonance Raman Study

Di-4-ANEPPS is a dye molecules which has been used to probe membrane electric potentials by its electrochromic response. The dye has an unusual blue shift in the absorption spectrum upon partitioning to lipid membranes. We studied its binding properties to membranes by absorption, fluorescence and resonance Raman spectroscopy. The latter technique provided evidence for the differential solubilization of the dye in the membrane, where the polar head of the molecule protrudes out of the lipid environment and is responsible for the spectral behavior.