Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Conformational changes in monolayers of a cationic amphiphilic porphyrin on saline subphases

Langmuir monolayer films of the tetracationic porphyrin tetrakis(octadecyl-4-pyridin ium)porphyrinatozinc(II) bromide on various salt containing subphases were analyzed using surface pressure-area isotherms and X-ray reflectivity. The use of these complementary techniques showed that the porphyrin molecules undergo changes in conformation upon compression. Two main phases were identified, one in which the porphyrin moiety is parallel to the subphase and one in which the porphyrin moiety is tilted out of the plane. The addition of different salts into the subphase brought about changes in film behaviour, which are explained in terms of a lyotropic series. Copyright (C) 2002 Society of Porphyrins,& Phthalocyanines.

Characterization of molecular damage induced by UV photons and carbon ions on biomimetic heterostructures

The study of the effect of radiation on living tissues is a rather complex task to address mainly because they are made of a set of complex functional biological structures and interfaces. Particularly if one is looking for where damage is taking place in a first stage and what are the underlying reaction mechanisms. In this work a new approach is addressed to study the effect of radiation by making use of well identified molecular hetero-structures samples which mimic the biological environment. These were obtained by assembling onto a solid support deoxyribonucleic acid (DNA) and phospholipids together with a soft water-containing polyelectrolyte precursor in layered structures and by producing lipid layers at liquid/air interface with DNA as subphase. The effects of both ultraviolet (UV) radiation and carbon ions beams were systematically investigated in these heterostructures, namely damage on DNA by means vacuum ultraviolet (VUV), infrared (IR), X-Ray Photoelectron (XPS) and impedance spectroscopy. Experimental results revealed that UV affects furanose, PO2-, thymines, cytosines and adenines groups. The XPS spectrometry carried out on the samples allowed validate the VUV and IR results and to conclude that ionized phosphate groups, surrounded by the sodium counterions, congregate hydration water molecules which play a role of UV protection. The ac electrical conductivity measurements revealed that the DNA electrical conduction is arising from DNA chain electron hopping between base-pairs and phosphate groups, with the hopping distance equal to the distance between DNA base-pairs and is strongly dependent on UV radiation exposure, due loss of phosphate groups. Characterization of DNA samples exposed to a 4 keV C3+ ions beam revealed also carbon-oxygen bonds break, phosphate groups damage and formation of new species. Results from radiation induced damage carried out on biomimetic heterostructures having different compositions revealed that damage is dependent on sample composition, with respect to functional targeted groups and extent of damage. Conversely, LbL films of 1,2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG) liposomes, alternated with poly(allylamine hydrochloride) (PAH) revealed to be unaffected, even by prolonged UV irradiation exposure, in the absence of water molecules. However, DPPG molecules were damaged by the UV radiation in presence of water with cleavage of C-O, C=O and –PO2- bonds. Finally, the study of DNA interaction with the ionic lipids at liquid/air interfaces revealed that electrical charge of the lipid influences the interaction of phospholipid with DNA. In the presence of DNA in the subphase, the effects from UV irrladiation were seen to be smaller, which means that ionic products from biomolecules degradation stabilize the intact DPPG molecules. This mechanism may explain why UV irradiation does not cause immediate cell collapse, thus providing time for the cellular machinery to repair elements damaged by UV.

High-affinity small molecule-phospholipid complex formation: Binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction Of X-PA = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.

Interaction of small amounts of bovine serum albumin with phospholipid monolayers investigated by surface pressure and atomic force microscopy

The influence of small amounts of bovine serum albumin (BSA) (nM concentration) on the lateral organization of phospholipid monolayers at the air-water interface and transferred onto solid substrates as one-layer Langmuir-Blodgett (LB) films was investigated. The kinetics of adsorption of BSA onto the phospholipid monolayers was monitored with surface pressure isotherms in a Langmuir trough, for the zwitterionic dipalmitoylphosphatidyl ethanolamine (N,N-dimethyl-PE) and the anionic dimyristoylphosphatidic acid (DMPA). A monolayer of N,N-dimethyl-PE or DMPA incorporating BSA was transferred onto a solid substrate using the Langmuir-Blodgett technique. Atomic force microscopy (AFM) images of one-layer LB films displayed protein-phospholipid domains, whose morphology was characterized using dynamic scaling theories to calculate roughness exponents. For DMPA-BSA films the surface is characteristic of self-affine fractals, which may be described with the Kardar-Parisi-Zhang (KPZ) equation. on the other hand, for N,N-dimethyl-PE-BSA films, the results indicate a relatively flat surface within the globule. The height profile and the number and size of globules varied with the type of phospholipid. The overall results, from kinetics of adsorption on Langmuir monolayers and surface morphology in LB films, could be interpreted in terms of the higher affinity of BSA to the anionic DMPA than to the zwitterionic N,N-dimethyl-PE. Furthermore, the effects from such small amounts of BSA in the monolayer point to a cooperative response of DMPA and N,N-dimethyl-PE monolayers to the protein. (c) 2005 Elsevier B.V. All rights reserved.

Poliuretana contendo corante azóico e espaçador tipo bis-carbonato como material para fabricação de filmes nanoestruturados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fabricação e caracterização de filmes finos de perileno: arquitetura molecular e aplicações sensoriais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da interação da natamicina com membranas fosfolipídicas incorporadas de esteróis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The negligible effects of the antifungal natamycin on cholesterol-dipalmitoyl phosphatidylcholine monolayers may explain its low oral and topical toxicity for mammals

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sistemas bioinspirados aplicados no estudo de interação e sensoriamento de derivados xantênicos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo de interação entre um hormônio estrógeno e modelos simples de membrana biológica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Coupling Between GABA(A)-R Ligand-Binding Activity and Membrane Organization in beta-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

Correlations between GABA(A) receptor (GABA(A)-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with beta-cyclodextrin (beta-CD). The mere pre-incubation (PI) at 37A degrees C accompanying the beta-CD treatment was an underlying source of perturbations increasing [H-3]-FNZ maximal binding (70%) and K (d) (38%), plus a stiffening of SMs' hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A (DPH)) in SMs submitted to a heating-cooling cycle (4-37-4A degrees C) with A (DPH,heating) < A (DPH,cooling). Compared with PI samples, the beta-CD treatment reduced B (max) by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A (TMA-DPH). PI, but not beta-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with beta-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, beta-CD is not completely eliminated from the system through centrifugation washings. It was concluded that beta-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual beta-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABA(A)-R density, relative to untreated samples.

Structure formation of amphiphilic molecules at the air,water interface and after film transfer

One of the basic concepts of molecular self-assembly is that the morphology of the aggregate is directly related to the structure and interaction of the aggregating molecules. This is not only true for the aggregation in bulk solution, but also for the formation of Langmuir films at the air/water interface. Thus, molecules at the interface do not necessarily form flat monomolecular films but can also aggregate into multilayers or surface micelles. In this context, various novel synthetic molecules were investigated in terms of their morphology at the air/water interface and in transferred films. rnFirst, the self-assembly of semifluorinated alkanes and their molecular orientation at the air/water interface and in transferred films was studied employing scanning force microscopy (SFM) and Kelvin potential force microscopy. Here it was found, that the investigated semifluorinated alkanes aggregate to form circular surface micelles with a diameter of 30 nm, which are constituted of smaller muffin-shaped subunits with a diameter of 10 nm. A further result is that the introduction of an aromatic core into the molecular structure leads to the formation of elongated surface micelles and thus implements a directionality to the self-assembly. rnSecond, the self-assembly of two different amphiphilic hybrid materials containing a short single stranded desoxyribonucleic acid (DNA) sequence was investigated at the air/water interface. The first molecule was a single stranded DNA (11mer) molecule with two hydrophobically modified 5-(dodec-1-ynyl)uracil nucleobases at the terminal 5'-end of the oligonucleotide sequence. Isotherm measurements revealed the formation of semi-stable films at the air/water interface. SFM imaging of films transferred via Langmuir-Blodgett technique supported this finding and indicated mono-, bi- and multilayer formation, according to the surface pressure applied upon transfer. Within these films, the hydrophilic DNA sequence was oriented towards air covering 95% of the substrate.rnSimilar results were obtained with a second type of amphiphile, a DNA block copolymer. Furthermore, the potential to perform molecular recognition experiments at the air/water interface with these DNA hybrid materials was evaluated.rnThird, polyglycerol ester molecules (PGE), which are known to form very stable foams, were studies. Aim was to elucidate the molecular structure of PGE molecules at the air/water interface in order to comprehend the foam stabilization mechanism. Several model systems mimicking the air/water interface of a PGE foam and methods for a noninvasive transfer were tested and characterized by SFM. It could be shown, that PGE stabilizes the air/water interface of a foam bubble by formation of multiple surfactant layers. Additionally, a new transfer technique, the bubble film transfer was established and characterized by high speed camera imaging.The results demonstrate the diversity of structures, which can be formed by amphiphilic molecules at the air/water interface and after film transfer, as well as the impact of the chemical structure on the aggregate morphology.

Controlling the luminescence properties of poly(p-phenylene vinylene) entrapped in Langmuir and Langmuir-Blodgett films of stearic acid

Properties of hybrid films can be enhanced if their molecular architecture is controlled. In this paper, poly (p-phenylene vinylene) was mixed with stearic acid in order to form stable hybrid Langmuir monolayers. Surface properties of these films were investigated with measurements of surface pressure, and also with polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The films were transferred from the air-water interface to solid supports through the Langmuir-Blodgett technique, and the viability of the film as optical device was investigated with fluorescence spectroscopy. Comparing the fluorescent spectra for the polymer in solution, as a casting film, and as an LB film, the emission bands for LB films were narrower and appeared at lower wavelengths. The interactions between the film components and the design for the LB film may take advantage of the method to immobilize luminescent polymers in mixed ultrathin films adsorbed in solid matrices. (C) 2011 Elsevier B.V. All rights reserved.

The structures of Langmuir-Blodgett films of fatty acids and their salts

Recent advances in several experimental techniques have enabled detailed structural information to be obtained for floating (Langmuir) monolayers and Langmuir-Blodgett films. These techniques are described briefly and their application to the study of films of fatty acids and their salts is discussed. Floating monolayers on aqueous subphases have been shown to possess a complex polymorphism with phases whose structures may be compared to those of smectic mesophases. However, only those phases that exist at high surface pressures are normally used in Langmuir-Blodgett (LB) deposition. In single LB monolayers of fatty acids and fatty acid salts the acyl chains are in the all-cans conformation with their long axes normal to the substrate. The in-plane molecular packing is hexagonal with long-range bond orientational order and short-range positional order: known as the hexatic-B structure. This structure is found irrespective of the phase of the parent floating monolayer. The structures of multilayer LB films are similar to the structures of their bulk crystals, consisting of stacked bilayer lamellae. Each lamella is formed from two monolayers of fatty acid molecules or ions arranged head to head and held together by hydrogen bonding between pairs of acids or ionic bonding through the divalent cations. With acids the acyl chains are tilted with respect to the substrate normal and have a monoclinic structure, whereas the salts with divalent cations may have the chains normal to the substrate or tilted. The in-plane structures are usually centred rectangular with the chains in the trans conformation and packed in a herringbone pattern, Multilayer films of the acids show only a single-step order-disorder transition at the malting point, This temperature tends to rise as the number of layers increases. Complex changes occur when multilayer films of the salts are heated. Disorder of the chains begins at low temperatures but the arrangement of the head groups does not alter until the melting temperature is reached, Slow heating to a temperature just below the melting temperature gives, with some salts, a radical change in phase. The lamellar structure disappears and a new phase consisting of cylindrical rods lying parallel to the substrate surface and stacked in a hexagonal pattern is formed, In each rod the cations are aligned along the central axis surrounded by the disordered acyl chains. (C) 2001 Elsevier Science B,V. All rights reserved.