ENERGY TRANSFER FROM CAROTENOIDS TO CHLOROPHYLL a IN BLACK LIPID MEMBRANES

Abstract— Black lipid membranes (BLM) were prepared from extracts of Chlorella and spinach chloroplasts. Excitation spectra of the 730 nm fluorescence of chlorophyll a in the BLM contained peaks identified as due to carotenoids and which therefore indicate sensitization of the chlorophyll fluorescence by them. The efficiency of this energy transfer was evaluated by comparison of the actual excitation spectra with those corresponding to 0 and 100 per cent transfer efficiency. Efficiencies were of the order of 40–50 per cent in BLM, but only 10 per cent in pigment solutions, when the mean distance between pigment molecules was 23 Å in both systems. The fluorescence quantum yield of chlorophyll a in such solutions was only 2 per cent of that found in BLM. Enhancement of energy transfer in BLM is considered to be mainly due to suppression of competing deactivation processes of excited carotenoid states, such as diffusional quenching by ground-state molecules and internal conversion. Favorable orientation of pigment molecules in the BLM constitutes a further enhancement factor.     

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.     

Unusual fluorescence spectral response of 1-(4-N,N-dimethylaminophenylethynyl)pyrene towards the thermotropic phase change in lipid bilayer membranes

The applicability of 1-(4-N,N-dimethylaminophenylethynyl)pyrene (DMAPEPy), a pyrene derivative showing intramolecular charge transfer, as a prospective probe for lipid bilayer membranes has been evaluated. High sensitivity of DMAPEPy to solvent polarity and viscosity makes it to act both as a polarity-sensitive probe and as a fluorescence anisotropy probe. The molecule shows high partition efficiency towards bilayer membranes in both solid gel as well as in the liquid crystalline phases. The emission spectrum, quenching experiment and lifetime data suggest bimodal distribution of DMAPEPy in the bilayer. Using the solvent polarity scales the polarity parameters of the two locations in lipid bilayer have been estimated. In the bilayer environment it exhibits remarkable spectral changes with temperature. The thermotropic phase change of the bilayer is sensitively monitored by fluorescence intensity as well as fluorescence anisotropy parameters. DMAPEPy is also capable of sensing the changes induced by membrane modifiers like cholesterol.     

Spectral properties and orientation of voltage-sensitive dyes in lipid membranes

Voltage-sensitive dyes are frequently used for probing variations in the electric potential across cell membranes. The dyes respond by changing their spectral properties: measured as shifts of wavelength of absorption or emission maxima or as changes of absorption or fluorescence intensity. Although such probes have been studied and used for decades, the mechanism behind their voltage sensitivity is still obscure. We ask whether the voltage response is due to electrochromism as a result of direct field interaction on the chromophore or to solvatochromism, which is the focus of this study, as result of changed environment or molecular alignment in the membrane. The spectral properties of three styryl dyes, di-4-ANEPPS, di-8-ANEPPS, and RH421, were investigated in solvents of varying polarity and in model membranes using spectroscopy. Using quantum mechanical calculations, the spectral dependence of monomer and dimer ANEPPS on solvent properties was modeled. Also, the kinetics of binding to lipid membranes and the binding geometry of the probe molecules were found relevant to address. The spectral properties of all three probes were found to be highly sensitive to the local environment, and the probes are oriented nearly parallel with the membrane normal. Slow binding kinetics and scattering in absorption spectra indicate, especially for di-8-ANEPPS, involvement of aggregation. On the basis of the experimental spectra and time-dependent density functional theory calculations, we find that aggregate formation may contribute to the blue-shifts seen for the dyes in decanol and when bound to membrane models. In conclusion, solvatochromic and other intermolecular interactions effects also need to be included when considering electrochromic response voltage-sensitive dyes.     

PHOTOSENSITIVE BILAYER MEMBRANES AS MODEL SYSTEMS FOR PHOTOBIOLOGICAL PROCESSES

Abstract— Photobiological processes such as photosynthesis, photomorphogenesis, photomovement, and photoreception are all associated with the membranous portions of cells. The unique properties of membrane surfaces are apparently required to achieve biologically relevant energy transduction and photocontrol phenomena and consequently the use of model membrane systems is suggested as an advantageous approach to elucidation of the important physical and chemical processes involved. Black lipid membrane (BLM) and liposome techniques are critically reviewed as preferred techniques for constructing and manipulating lipid bilayers. The lipid bilayer is considered to be the basic foundation for biological membrane models, and specific physical phenomena observed with the bilayers and their biological ramifications are analyzed. Light-stimulated polarization of the membrane and electron transfer across the bilayer are viewed as appropriate analogs of vision and photosynthesis, respectively. Bilayer-adsorbed dye experiments are the simplest systems explored that exhibit polarization and charge transfer across the membrane. Chloroplast extract BLM experiments are cited as an example of the light-stimulated transfer of electrons across the membrane under the influence of a preexisting redox gradient. Biliprotein (phycocyanin or phycoerythrin) on one side of the chloroplast extract membrane permits the direction of electron flow across the membrane so that a redox gradient is created in a manner truly analogous to photosynthesis. The potential for solar energy conversion from such membranes is explicitly considered utilizing a schematic photoelectrochemical cell. Model membranes containing bacterial rhodopsin and phytochrome represent examples of ionic gradients that result in biological energy transduction. Studies of membranes that exhibit transient photoeffects are considered potentially relevant for the elucidation of phototaxis. The analysis of many properties of photosensitive membranes is greatly aided by the use of appropriate theoretical models. It is apparent that there is a great potential for the application of photosensitive model membranes in many research areas involving complex photobiological phenomena and novel methods for solar energy conversion.     

Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.     

SPECTROSCOPIC AND ORIENTATIONAL PROPERTIES OF CHLOROPHYLL a AND CHLOROPHYLL b IN LIPID MEMBRANES

Abstract
Linear dichroism and fluorescence depolarization measurements on chlorophyll a and chlorophyll b molecules incorporated into macroscopically oriented multibilayers of the plant lipid digalactosyldiacylglycerol (DGDG) are presented. The results are combined with measurements of fluorescence anisotropy to obtain both the order parameters describing the orientational statistics and the directions of the absorption and emission transition moments in the frame of the molecules. The problem presented by the overlapping nature of the absorption and emission bands is overcome by determining the fluorescence depolarization at the two maxima of the emission spectrum.     

Ground- and excited-state proton transfer reaction of 3-hydroxyflavone in dimyristoylphosphatidylcholine liposome membrane

3-Hydroxyflavone (3HF), a molecule that exhibits excited-state intramolecular proton transfer, has been studied for its fluorescence characteristics in dimyristoylphosphatidylcholine (DMPC) liposome membrane. 3HF partitions to the lipid bilayer membrane with a reasonably large partition coefficient. On excitation at 417 nm, a weak emission from the ground-state anion species was observed at 483 nm, whereas excitation at absorption maxima (345 nm) gives the usual intense fluorescence of the phototautomeric emission at 530 nm. In this article, we report the observation of a ground-state proton transfer reaction of 3HF in DMPC liposome membrane.     

Retinoid chromophores as probes of membrane lipid order

There is a great need for development of independent methods to study the structure and function of membrane-associated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.     

PHOTOSENSITIZATION SYSTEMS OF COVALENTLY LINKED PHTHALOCYANINE COMPLEXES IN BOTH BILAYER LIPID MEMBRANES AND TIN OXIDE PHOTOVOLTAIC CELL

Abstract Zinc phthalolocyanine photosensitized donor-acceptor systems for light energy conversion and for the design of photoelectrochemical molecular devices are presented. Covalently linked phthalocyanine complexes were incorporated in bilayer lipid membranes (BLM) and deposited on SnO₂ transparent electrodes. Their photovoltages were measured and compared. It has been found that a more favorable orientation and closer proximity are attained in the diad compounds between the donor (phthalocyanine)-acceptor (anthraquinone) pair than in the reference compound for efficient light-induced charge separation and transfer. The triad compound is the best among all tested compounds. The decrease in the fluorescence yield and lifetime induced by quinones was examined and the apparent electron-transfer rate constants were calculated.     

Membrane electrochemistry

Electrochemistry and biomembranes are interface science in that both are concerned with the phenomena at, as well as across, the interfaces. Membrane electrochemistry may be defined as the application of electrochemistry to biomembrane studies. Additionally, transport processes within the membrane are involved in biomembranes. Since biomembranes are diverse and are usually not amenable to probing by electrochemicophysical techniques, model membrane systems have been developed for their investigation.

The introduction of experimental bilayer lipid membranes (BLM) technique and its modifications have been instrumental in the development and testing of membrane transport concepts (carriers vs channels) and electronic processes in membranes. Instead merely viewing a biomembrane as a physical barrier containing carriers or channels to carry out ionic processes, an ultrathin lipid or biological membrane can also be considered as a complete ‘electrochemical cell’ with one membrane/solution interface reducing (as a cathode) and the other membrane/solution interface oxidizing (as an anode). It is now possible to understand energy transduction (charge generation, separation, and redox reactions) in terms of ultrathin lipid membranes separating two aqueous solutions.

In this paper, we shall discuss the basic principles of electrochemistry as they are applied to membrane studies. Emphasis will be on experimental bilayer lipid membranes (BLM) which have been extensively investigated as models of biomembranes.     

自组装ITO/双层磷脂膜的制备及其光电行为研究

在ITO(Indium-tin-oxide)导电玻璃电极上制备上自组装双层磷脂膜和经C60修饰的双层磷脂膜,研究了这种自组装双层磷脂膜的光电行为,考察了偏压、溶液中的给体和受体的浓度对自组装膜光电流强度的影响,讨论了C60分子对光电子跨膜传递过程的促进作用。     

双层类脂膜及其在电化学生物传感器中的应用

详细评述了各种双层类脂膜包括传统的双层类脂膜（ＢＬＭ）、固体载体支撑的自组双层类脂膜（ｓ－ＢＬＭ）、固体载体支撑的混合双层类脂膜（ｅ－ＢＬＭ）的制备方法和特性,比较了其优缺点。介绍了双层类脂膜在电化学生物传感器中的应用,并展望了发展前景。     

含蒽醌基双分子膜的电子光谱

用荧光和紫外光谱研究了新合成的含蒽醌(2,6)生色基的单链双亲性分子(ANQU)在水溶液中形成的双分子膜聚集体结构。ANQU在稀水溶液中的吸收光谱比其在乙醇稀溶液中的谱峰有较大的红移；其凝胶态相对于液晶态的吸收谱亦有明显红移。结果表明,ANQU双分子膜中分子的堆积方式是J-聚集,蒽醌生色基以头对尾取向方式排列。变温荧光光谱观察双分子膜中蒽醌生色基的荧光光谱强度和峰位极敏感地受到双分子膜物理状态变化的影响。     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: detection by antibiotic membrane probes.

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylcholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmetrically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Molecular dynamics simulations of DiI-C18(3) in a DPPC lipid bilayer

We performed a 40 ns simulation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18(3)) in a 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) bilayer in order to facilitate interpretation of lipid dynamics and membrane structure from fluorescence lifetime, anisotropy, and fluorescence correlations spectroscopy (FCS). Incorporation of DiI of 1.6 to 3.2 mol% induced negligible changes in area per lipid but detectable increases in bilayer thickness, each of which are indicators of membrane structural perturbation. The DiI chromophore angle was 77 +/- 17 degrees with respect to the bilayer normal, consistent with rotational diffusion inferred from polarization studies. The DiI headgroup was located 0.63 nm below the lipid head group-water interface, a novel result in contrast to some popular cartoon representations of DiI but consistent with DiI's increase in quantum yield when incorporated into lipid bilayers. Importantly, the fast component of rotational anisotropy matched published experimental results demonstrating that sufficient free volume exists at the sub-interfacial region to support fast rotations. Simulations with non-charged DiI head groups exhibited DiI flip-flop, demonstrating that the positively-charged chromophore stabilizes the orientation and location of DiI in a single monolayer. DiI induced detectable changes in interfacial properties of water ordering, electrostatic potential, and changes in P-N vector orientation of DPPC lipids. The diffusion coefficient of DiI (9.7 +/- 0.02 x 10(-8) cm2 s(-1)) was similar to the diffusion of DPPC molecules (10.7 +/- 0.04 x 10(-8) cm2 s(-1)), supporting the conclusion that DiI dynamics reflect lipid dynamics. These results provide the first atomistic level insight into DiI dynamics, results essential in elucidating lipid dynamics through single molecule fluorescence studies.     

双层类脂膜核酸传感器的研究进展

对近几年发展起来的双层类脂膜（BLM）核酸传感器的研究工作进行了讨论，详细论述了该传感器的特性、工作原理以及在核酸杂交、序列分析研究中的应用，并与其它核酸传感器的研究作了对比；展望了其发展方向，引文31篇。     

Investigation of interaction of Leu-enkephalin with lipid membranes

Enkephalins are peptides with morphine-like activity. To achieve their biological function, they must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. In our study, zeta potential (ZP) method was used to detect the association of Leu-enkephalin and Leu-enkephalinamide with phospholipid liposomes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), cholesterol (Chol), sphingomyelin (SM) as well as soybean phospholipid (SBPL). Transfer of the peptides over lipid membranes was examined by electrophysiology technique (ET) and fluorescence spectroscopy (FS), and further confirmed using 4-fluoro-7-nitrobenzofurazan (NBD-F) labeled Leu-enkephalin (NBD-F-enkephalin) with confocal laser scanning microscopy method (CLSM). Results of zeta potential showed that enkephalinamide associated with lipid membranes and gradually saturated on the membranes either hydrophobically or electrostatically or both. Data from electrophysiology technique indicated that Leu-enkephalin could cause transmembrane currents, suggesting the transfer of peptides across lipid membranes. Transfer examined by fluorescence spectroscopy implied that it could be separated into three steps, adsorption, transportation and desorption, which was afterward reaffirmed by confocal laser scanning microscopy. Transfer efficiencies of enkephalin across SBPL, EPC/DOPE, EPC/DOPE/SM, EPC/SM and EPC/Chol lipid bilayer membranes were evaluated with ET and CLSM experiments. Results showed that the addition of either sphingomyelin or cholesterol, or negatively charged lipid in lipid membrane composition could lower the transfer efficiency.     

Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm^?2) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single‐molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme‐induced compositional heterogeneity within membranes, where NR within liquid‐ordered vs. liquid‐disordered domains shows a ≈4° difference in polar angle and a ≈0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid‐lipid, lipid‐protein, and lipid‐dye interactions with single‐molecule, nanoscale resolution.     

Fluorescent Flipper Probes: Comprehensive Twist Coverage

To image the membrane tension in living cells, planarizable push–pull probes have been introduced. The first operational probe is built around two dithieno[3,2-b:2′,3′-d]thiophenes (DTTs) that are twisted out of co-planarity and polarized with donors and acceptors at either end. In this report, the chemical space available for the twisting of “flipper probes” is assessed comprehensively. The result is, not surprisingly, that every atom matters: Removal of one methyl group in the twist region yields probes that planarize already in solution and are thus less sensitive to membrane tension. Addition of one or more carbons in the same region hinders non-interfering probe alignment along lipid tails and thus partitioning into lipid bilayer membranes as well as mechanosensitivity. However, substitution of one methyl by an isosteric trifluoromethyl group in the twist region, achieved by quite substantial multistep organic synthesis, yields excitation maxima that shift over +100 nm to the red in response to increasing order of the surrounding membrane. This record redshift comes with record changes in fluorescence intensity and lifetime, high push–pull transition dipoles and higher rotational barriers. Supported by distinct dependence on viscosity and twist of the push–pull probes, kinetic competition between dark, fully twisted and bright, fully planarized relaxed excited states emerges as unifying origin of fluorescence quantum yields.