Self-Aggregation and Orientation of the Ion Channel-Forming Zervamicin IIA in the Membranes of ePC Vesicles Studied by cw EPR and ESEEM Spectroscopy

The ion channel-forming peptide antibiotic zervamicin A was studied in egg phosphocholin lipid membranes of large multilamellar vesicles (LMV) at 77 K. Continuous wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) methods combined with site-specific electron spin labeling were used to study the aggregation and immersion depth of two analog molecules, i.e., each monolabeled either at the N- or C-terminal end of the helical molecule. Analysis of the shape of the EPR spectra indicates that zervamicin molecules form aggregates in which the dipolar interaction between the spin labels at the N-terminus is substantially larger than that between the labels at the C-terminus. The ESEEM method was used to study the interaction between the nitroxide radical spin labels of the zervamicin molecules and deuterium nuclei in LMV, which were prepared using a D₂O buffer. It is established that the largest amplitude of deuterium modulation of the unpaired electron is observed for zervamicin molecules labeled at the N-terminus. Based on the analysis of the Fourier parameters of the deuterium modulated spectrum, a model of the immersion depth of the terminal ends of the zervamicin molecule in a lipid bilayer is formulated. All of the spin labels at the N-terminus are grouped at the lipid–water interface, whereas 60% of labels at the C-terminus are located at the lipid–water interface and 40% are more deeply inserted into the lipid bilayer.     

Vectorial photoinduced electron transfer in phospholipid vesicles and LB bilayers

Summary Photoinduced electron transfer (PET) was studied in phospholipid vesicles and in Langmuir-Blodgett bilayers in the attempt to produce a model for electron transfer processes in biological media. Spatial organization of the reaction centers in lipid membranes needs to be controlled in order to provide high efficiency of light-to-chemical energy conversion. We designed a phospholipid system where the donor is localized in the inner bilayer whereas the acceptor is at the polar groups-water interface. We used dipalmitoylphosphatidic acid vesicles containing low molar fractions of dipalmitoylphosphatidylcholine with pyrene (donor) bound to one of the alkyl chains. Methylviologen (acceptor) was added to the external aqueous phase; upon photoexcitation of the donor we observed the electron transfer to take place in a unidirectional manner from the inside of the bilayer to the interface. Information about the location of the donor was obtained studying the photophysical properties of the pyrene chromophore in vesicles and in LB layers. The photoinduced electron transfer reaction was evidenced by quenching of pyrene fluorescence in the presence of increasing concentrations of acceptor, the process was studied both with steady-state and time-resolved fluorescence emission. Fluorescence intensity was found to decrease with increasing concentration of methylviologen, similar results were obtained for vesicles and LB layers of analog composition immersed in a methylviologen solution. Lifetimes of the excited species were found to be of the same order of magnitude in vesicle and LB-layer systems. Paper presented at the I International Conference on Scaling Concepts and Complex Fluids, Copanello, Italy, July 4–8, 1994.     

Tryptophan radicals as reaction intermediates in versatile peroxidases: Multifrequency EPR, ENDOR and density functional theory studies

Versatile peroxidases are a new class of ligninolytic enzymes secreted by fungi from the group of white-rot basidiomycetes. Versatile peroxidase (VP) is a structural hybrid between lignin and manganese peroxidase. This hybrid combines the catalytic properties of the two above peroxidases being able to catalyze Mn(II)-dependent and Mn(II)-independent reactions. A long-range electron transfer mechanism has been inferred for the oxidation of big substrate molecules starting from an exposed tryptophan to the heme cofactor. A neutral tryptophan radical has been identified in VP fromBjerkandera adusta andPleurotus eryngii, after H₂O₂ activation and assigned to a specific tryptophan residue using multifrequency electron paramagnetic resonance and electron-nuclear double resonance spectroscopy. Comparative density functional theory calculations were performed for tryptophan neutral and cation radical species, considering also the effect of the polar environment surrounding the radical. The functional role of the radicals is discussed in the context of mechanistic models for VP.     

Electron Spin Resonance Study on the Photoinduced Electron Transfer in Chlorophyll a in Reconstituted Lipid Bilayer Vesicles

Abstract  

The photoinduced electron transfer from chlorophyll a through the interface of positively charged dioctadecyltrimethylammonium chloride (DODAC), neutral dipalmitoylphosphatidylcholine (DPPC) and negatively charged dihexadecylphosphate (DHP) headgroup of the lipid bilayers was studied. The photoinduced radicals were identified by electron spin resonance (ESR) and radical yields of chlorophyll a were determined by double integration of the ESR spectra. The formation of vesicles was identified indirectly by measuring change of the λ _max value of optical absorption spectrophotometer from diethyl ether solution to vesicle solutions, and observed directly with scanning and transmission electron microscopic images. The interaction distance between chlorophyll a and interface water (D₂O) determined by deuterium modulation depth with electron spin echo modulation (ESEM) showed a decreasing order DODAC > DPPC > DHP. The interface charge of each vesicle was determined with zeta potential measurement. The interface charge of the lipid bilayers affected the radical yields of chlorophyll a more critically than the interaction distance between chlorophyll a and interface water.     

Shapes of nearly cylindrical, axisymmetric bilayer membranes

Shapes of nearly cylindrical sections of axisymmetric phospholipid membranes are studied theoretically. Describing the shape of such sections by their deviation from a reference cylinder, the well-established shape equation for axisymmetric bilayer membranes is expanded in terms of this deviation, and it is then solved analytically. The phase diagram shows the resulting stationary shapes as functions of system parameters and external conditions, i.e., the pressure difference across the membrane, the membrane tension, the difference between the tensions of the two monolayers, and the axial force acting on the vesicle. The accuracy of the approximate analytical solution is demonstrated by comparison with numerical results. The obtained analytical solution allows to extend the analysis to include shapes where numerical methods have failed. Received 27 September 2000 and Received in final form 26 March 2001     

合成双分子膜三元体系的激发态能量转移

在４ （４’ 癸氧基 ４ 氧基联苯）丁基三甲基溴化铵双分子膜体系内,通过静电相互作用使达旦黄、荧光黄和四苯基卟啉磺酸阴离子键合在囊泡表面;研究了达旦黄给体通过荧光黄传递到四苯基卟啉磺酸阴离子受体的三元激发态能量转移。能量转移的结果改善了光的输出,扩展了光波的输出范围。     

Exciplex parameters: key factors in the dielectric behaviour of magnetodynamics

Photoinduced electron transfer reactions in solution produce two primary geminate radical ion pairs: contact ion pair or exciplex and solvent-separated ion pair. The magnetodynamics of radical ion pairs involves suppression of the spin-evolution between singlet and triplet states of a fraction of solvent-separated ion pairs, the partners of which undergo prior diffusion to attain the distance where exchange interaction is negligible, in the presence of an external low magnetic field of the order of the hyperfine interactions present in the system. This results in an increase in geminate recombination of the singlet solvent-separated ion pairs and enhancement in exciplex luminescence since the precursor radical ion pair is singlet. Although seemingly magnetodynamics is a diffusion-controlled phenomenon that should depend mainly on the dielectric constant of the medium (keeping viscosity almost constant), it is not true for all the exciplex systems since the nature of the magnetic field effect versus medium dielectric curves differ from each other in peak positions, peak heights and onset points. To investigate this differential nature, magnetic field effects among exciplex systems consisting of different derivatives of carbazole as electron donors are compared with a universal acceptor, 1,4-dicyano-benzene, with the pyrene—N,N-dimethylaniline exciplex system as reference. It was found that, apart from the solvent dielectric, the exciplex energy and the bulk effect of the steric constraints present on either donor or acceptor site, regulating the optimum inter-radical distance in the initially formed radical ion pair, are the key factors in controlling the magnetodynamic behaviour.     

Regulation of the intermittent release of giant unilamellar vesicles under osmotic pressure

Osmotic pressure can break the fluid balance between intracellular and extracellular solutions. In hypo-osmotic solution, water molecules, which transfer into the cell and burst, are driven by the concentration difference of solute across the semi-permeable membrane. The complicated dynamic processes of intermittent bursts have been previously observed. However, the underlying physical mechanism has yet to be thoroughly explored and analyzed. Here, the intermittent release of inclusion in giant unilamellar vesicles was investigated quantitatively, applying the combination of experimental and theoretical methods in the hypo-osmotic medium. Experimentally, we adopted a highly sensitive electron multiplying charge-coupled device to acquire intermittent dynamic images. Notably, the component of the vesicle phospholipids affected the stretch velocity, and the prepared solution of vesicles adjusted the release time. Theoretically, we chose equations and numerical simulations to quantify the dynamic process in phases and explored the influences of physical parameters such as bilayer permeability and solution viscosity on the process. It was concluded that the time taken to achieve the balance of giant unilamellar vesicles was highly dependent on the molecular structure of the lipid. The pore lifetime was strongly related to the internal solution environment of giant unilamellar vesicles. The vesicles prepared in viscous solution were able to visualize long-lived pores. Furthermore, the line tension was measured quantitatively by the release velocity of inclusion, which was of the same order of magnitude as the theoretical simulation. In all, the experimental values well matched the theoretical values. Our investigation clarified the physical regulatory mechanism of intermittent pore formation and inclusion release, which provides an important reference for the development of novel technologies such as gene therapy based on transmembrane transport as well as controlled drug delivery based on liposomes.     

Influence of Nature of Functional Groups on Interaction of Tetrasubstituted at Lower Rim <Emphasis Type="Italic">p</Emphasis>-<Emphasis Type="Italic">tert</Emphasis>-Butyl Thiacalix[4]arenes in 1,3-Alternate Configuration with Model Lipid Membranes

The influence of p-tert-butyl thiacalix[4]arenes (1,3-alternate) functionalized by N-propyl morpholine, N,N-dipropyliden-amine and aminodiacetate fragments on dynamic structure of liposomal membranes prepared from dipalmitoyl phosphatidylcholine was investigated by means of electron spin resonance spin-probe technique and Fourier transform infrared spectroscopy. Nuclear magnetic resonance and transmission electron microscopy techniques were applied to characterize the interacting systems. The obtained results have shown that all studied calixarenes interact with polar domains of bilayer. Depending on functional groups and hydrophobic/hydrophilic properties of calixarenes they can dip into bilayer, locate close to the surface of bilayer or form bridges between positively charged groups of adjacent lipid molecules, thus assisting to disordering or putting in order the lipid molecules.     

Electronic Spectra of Radical Ions

Abstract

Organic molecules may accept an extra electron to form radical anions, R⁺, or give up an electron by ionization to form radical cations, R⁺. Some of the radical ions are relatively stable in their ground states in solution phase and can be readily characterized by spectroscopic methods. Electron spin resonance spectroscopy provides an ideal tool for the study of the unshared electron in these radical ions. Most radical ions are intensely colored and give characteristic absorption bands in the electronic spectra. Further, electronic spectroscopy is one of the few tools available for the study of the dianions, which are generally diamagnetic. Electronic spectroscopy is used not merely for the characterization of the ionic species in solution but also for the study of the chemical equilibria involving various kinds of ionic species.     

Nanobionics of Pharmacologically Active Derivatives of Fullerene C60

The physical–chemical mechanisms of pharmacologic functioning of amino acid derivatives of fullerene C₆₀ (ADF) have been studied. ADF were shown to penetrate through the lipid bilayer of liposomes without destruction of membrane integrity. ADF are able to carry bivalent metal ions through phospholipid bilayer owing to the formation of complexes. It was shown that stereoisomers of ADF selectively penetrate into phospholipid membranes. In contrast to D-isomers, L-isomers penetrate through the phosphatidylcholine membrane into liposome interior. Stereo-specific effect of ADF enantiomers was also observed in reaction of peroxidation of lipids. Besides that, ADF bring about a substantial increase in the catalytic activity of monoaminoxidases A and B. The directed intraprotein electron transfer was studied by creating a donor–acceptor pair in a water solution in the presence of ADF. To realize the intraprotein electron transfer, the model system was produced on the base of apomyoglobin by incorporating ADF (electron acceptor) into the heme pocket of protein instead of removed heme. It was established that the fullerene C₆₀ and its derivatives did not produce specific anti-C₆₀ antibodies, both IgG and IgE classes, while ADF themselves are efficient adjuvants, i.e. they increased the antibody response to poor antigens. Some ADF were found to inhibit the human immunodeficiency virus and human cytomegalovirus infection.     

Diffusion of gases across lipid membranes with OmpA channel: a molecular dynamics study

Molecular transport across biological membranes occurs in a range of important chemical and biological processes. The biological membrane can usually be modelled as a phospholipid bilayer, but to correctly represent biological transport, the embedded transmembrane proteins must also be included. In previous molecular simulation studies on transport of small gas molecules in dipalmitoylphosphatidylcholine (DPPC) bilayer membrane, a coarse-grained model was used to provide direct insight into collective phenomena in biological membranes. Coarse graining allowed investigation of longer time and length scales by reducing the degrees of freedom and employing suitable potentials. In this work, membranes that include transmembrane proteins are modelled. This allows one to compare the molecular transport across a lipid membrane with and without the assistance of transmembrane channels. Outer membrane protein A (OmpA) – a porin from Escherichia coli with a small pore size – was chosen in this study because its detailed structure is known, it has high stability and is known to form a nonspecific diffusion channel that permits the penetration of various solutes. In this work the pore characteristics and interaction between lipid and protein were investigated and transport of water and other small gas molecules within the channel were studied. The MD simulation results obtained are compared with previous simulation results and available experimental data. The results obtained from this study will lead to better understanding of protein functionality and advance the development of biochips and drug delivery systems.     

Packing state in bilayer membranes of diacylphosphatidylcholines with varying acyl chain lengths under high pressures

The bilayer packing states of a series of diacylphosphatidylcholines (CnPC) containing linear saturated acyl chains were examined by a high-pressure fluorescence method. We revealed from the second derivatives of Prodan fluorescence spectra for all bilayer membranes that the Prodan molecules can be distributed into multiple sites in these bilayer membranes and move around the head-group region, depending on the phase state. The hydrophobicity of the PC molecules markedly affected the distribution quantities of the Prodan molecules between the gel and liquid crystalline phases. The distribution of the Prodan molecules into the gel phase decreased with the increasing acyl chain length while that into the liquid crystalline phase conversely increased. The present study suggests that Prodan can sensitively recognize the packing states and strengths in the bilayer membranes and becomes a good packing indicator.     

ADDENDUM

Abstract

Experiments have been performed to provide basic heat transfer data for thermal convection in a horizontal concentric annulus partially filled with water. A real-time holographic interferometry was employed to map the temperature distribution and a laser shadowgraph system was used to measure local heat transfer coefficients along the heated inner cylinder. Experimental results demonstrate that a minimum average Nusselt number for the water-wetted portion of the inner cylinder occurs at the air-water interface situated flush with the top of the inner cylinder. Corresponding numerical simulations were conducted for the cases of either fully or half-filled with water and the predictions agree well with the experimental results.     

Time resolved CIDNP study of electron transfer reactions in proteins and model compounds

Intramolecular electron transfer (IET) from tyrosine to tryptophan cation radicals is investigated using time resolved chemically induced dynamic nuclear polarization (CIDNP) spectroscopy in combination with laser flash photolysis. In both the tryptophan-tyrosine dipeptide and the denatured state of hen lysozyme in aqueous solution, the transformation TrpH⁺ → TyrO by IET leads to an increase in the tyrosine radical concentration, growth in the tyrosine CIDNP signal, fast decay of the tryptophan CIDNP, and inversion of the phase of the CIDNP of the photosensitizing dye, 2,2′-dipyridyl. IET effects are not observed for mixtures of the amino acid or for the native state of lysozyme. The steady state CIDNP effects seen for denatured lysozyme thus depend not only on the accessibility of the amino acid residues on the surface of the protein but also on the reactivity of the radical intermediates.     

Dissociation of vortex stacks into fractional-flux vortices

We discuss the zero field superconducting phase transition in a finite system of magnetically coupled superconducting layers. Transverse screening is modified by the presence of other layers resulting in topological excitations with fractional flux. Vortex stacks trapping a full flux and present at any finite temperature undergo a dissociation transition which corresponds to the depairing of fractional-flux vortices in individual layers. We propose an experiment with a bilayer system allowing us to identify the dissociation of bound vortex molecules.     

EPR-detected photoinduced electron transfer in three structurally related molecular triads

A comparative electron paramagnetic resonance (EPR) study has been performed on a series of structurally related molecular triads which undergo photoinduced electron transfer and differ one from the other in terms of the acceptor or donor moieties. The molecular triads, C-P-C₆₀, TTF-P-C₆₀ and C-P-P_F, share the same free-base, tetraarylporphyrin (P) as the primary electron donor, which after light excitation initiates the electron transfer process, but differ either in terms of the electron acceptor (fullerene derivative, C₆₀, versus fluorinated free-base porphyrin, P_F), or in terms of the final electron donor (carotenoid polyene, C, versus tetrathiafulvalene, TTF). All these molecular triads can be considered artificial photosynthetic reaction centers in their ability to mimic several key properties of the reaction center primary photochemistry. Photoinduced charge separation and recombination have been followed by time-resolved EPR in a glass of 2-methyltetrahydrofuran and in the nematic phase of the uniaxial liquid crystal E-7. All the triads undergo photoinduced electron transfer, with the generation of charge-separated states in both the low-dielectric environment of the 2-methyl-tetrahydrofuran glass and in anisotropic E-7 medium. Different photochemical pathways have been recognized depending on the specific donor and acceptor moieties constituting the molecular triads. In the presence of the tetrathiafulvalene electron donor singlet- and triplet-initiated electron transfer routes are concurrently active. Recombination to the low-lying carotenoid triplet state occurs in the carotene-based triads, while singlet recombination is the only active route for the TTF-P-C₆₀ triad, where a low-lying triplet state is lacking. Long-lived charge separation has been observed in the case of TTF-P-C₆₀: about 8 μs for the singlet-born radical pair in the glassy isotropic matrix and about 7 μs for the triplet-born radical pair in the nematic phase of E-7. For all the molecular triads, a weak exchange interaction (J?1 G) between the electrons in the final spin-correlated radical pair has been evaluated by simulation of the EPR spectra, providing evidence for superexchange electronic interactions mediated by the tetraarylporphyrin bridge.     

Tyrosyl Radical in the W164Y Mutant of P. eryngii Versatile Peroxidase: an EPR and DFT/PCM Study

Versatile peroxidases (VP) constitute a new class of high redox potential fungal enzymes that are able to degrade lignin and large substrate molecules. These enzymes catalyze the oxidation of substrates at an exposed tryptophan radical formed by a long-range electron transfer mechanism to heme following the activation by H₂O₂. In a previous paper, it was demonstrated using electron paramagnetic resonance (EPR) and electron-nuclear double resonance experiments on wild-type VP that Trp164 was the radical site and that it was in a hydrogen-bonded neutral form. In this paper, the W164Y variant is analyzed and it is shown that also the variant is able to form the so-called Compound I (VPI) in the form of protein radical, although in different yields with respect to the wild type. The X-band EPR experiments in combination with density functional theory/polarizable continuum model calculations show that the W164Y mutant is able to form a neutral radical on Tyr164 residue, after activation by H₂O₂, but in contrast to Trp164, tyrosine is not expected to be hydrogen bonded.     

2-甲基蒽醌光敏氧化芳香类氨基酸的激光光解

以355 nm激光为激励光源,研究 2-甲基蒽醌(MAQ)光敏氧化芳香氨基酸(色氨酸、酪氨酸、苯丙氨酸)的光化学行为。在乙腈-水体积比1∶1的混合体系中,2-甲基蒽醌三重激发态(3MAQ*)的吸收峰位于390,450和590 nm,利用3MAQ*与电子给体的电子转移反应确定其阴离子自由基的特征吸收峰为390和490 nm。2-甲基蒽醌与色氨酸、酪氨酸、苯丙氨酸的瞬态反应都显示随着590 nm激发三重态的衰减,出现490 nm 2-甲基蒽醌阴离子自由基的生成,并进一步确定了2-甲基蒽醌与色氨酸、酪氨酸和苯丙氨酸的电子转移反应的速率分别为3.0×109,1.1×109和1.8×108 L·mol-1·s-1。同时吉布斯自由能(ΔG)的计算结果也表明2-甲基蒽醌的激发三重态与色氨酸、酪氨酸、苯丙氨酸电子转移反应在热力学上是可行的。     

药物小分子穿越磷脂双层膜输运过程中的时滞效应

本文基于扩散动力学,建立了一种新的药物小分子穿越磷脂双层膜输运的理论模型,研究药物小分子穿越磷脂双层膜输运的动态过程,考察药物小分子跨膜输运过程中的时间延迟(时滞)效应。研究发现,药物小分子在数分钟内穿越磷脂双层膜各区域进入细胞,由于时滞效应,穿膜过程呈现了周期性演化特性。当药物小分子数量增加到一定程度,磷脂分子层会出现微小孔,让积累的药物小分子快速通过。通过分析模型中各参数的敏感性,我们还发现,药物小分子在磷脂双层膜内不同区域的扩散特性,以及输运过程的时滞性,都会对药物小分子穿越磷脂双层膜的动力学有较大程度的影响。理论结果符合模拟、实验观测,进一步深刻揭示了药物小分子穿越磷脂双层膜的穿膜特性,可为设计确切的疗法药物提供必要的参考和新方案。