Urease and hexadecylamine-urease films at the air-water interface: an x-ray reflection and grazing incidence x-ray diffraction study

We report the results of surface x-ray scattering measurements performed on urease and hexadecylamine-urease films at the air-aqueous solution interface. It is demonstrated that although hexadecylamine does not form a stable monolayer on the pure aqueous surface, it does self-assemble into a stable, well-organized structure when spread on top of a urease film at the air-water interface. It is also likely that protein and hexadecylamine domains coexist at the interface.     

The SC3 hydrophobin self-assembles into a membrane with distinct mass transfer properties

Hydrophobins are a class of small proteins that fulfill a wide spectrum of functions in fungal growth and development. They do so by self-assembling into an amphipathic membrane at hydrophilic-hydrophobic interfaces. The SC3 hydrophobin of Schizophyllum commune is the best-studied hydrophobin. It assembles at the air-water interface into a membrane consisting of functional amyloid fibrils that are called rodlets. Here we examine the dynamics of SC3 assembly at an oil-water and air-water interface and the permeability characteristics of the assembled layer. Hydrophobin assembled at an oil-water interface is a dynamic system capable of emulsifying oil. It accepts soluble-state SC3 oligomers from water in a unidirectional process and sloughs off SC3 vesicles back into the water phase enclosing a portion of the oil phase in their hydrophobic interior. The assembled layer is impermeable to solutes >200 Da from either the water phase or the oil phase; however, due to the emulsification process, oil and the hydrophobic marker molecules in the oil phase can be transferred into the water phase, thus giving the impression that the assembled layer is permeable to the marker molecules. By contrast, the layer assembled at an air-water interface is permeable to water vapor from either the hydrophobic or hydrophilic side.     

Thermodynamic and dynamic characteristics of hydroxypropylmethylcellulose adsorbed films at the air-water interface

Surface pressure isotherms and structural and surface dilatational properties of three hydroxypropylmethycelluloses (HPMCs, called E4M, E50LV, and F4M) adsorbed films at the air-water interface were determined. In this work we present evidence that HPMC molecules are able to diffuse and saturate the air-water interface at very low concentrations in the bulk phase. As bulk concentration increased, structural changes at a molecular level occurred at the interface. These changes corresponded to transition from an expanded structure (structure I) to a condensed one (structure II). When the surface concentration of HPMC was high enough, the collapse of the monolayer was observed. The three HPMCs formed very elastic films at the air-water interface, even at low surface pressures. E4M showed features that make it unique. For instance it showed the highest surface activity, mainly at low bulk concentrations (<10(-4) wt %). The differences observed in surface activity may be attributed to differences in the hydroxypropyl molar substitution and molecular weight of HPMC. All three HPMCs formed films of similar viscoelasticity and elastic dilatational modulus, which can be accounted for by their similar degree of methyl substitution.     

Formation,structure, and spectrophotometry of air-water interface films containing rhodopsin

Summary Air-water interface films of cattle rhodopsin and defined lipids are formed without the use of organic solvents by a method in which vesicle membranes consisting of egg phosphatidyl choline and purified rhodopsin are osmotically shocked at the interface. Lipid and protein molecules organize as insoluble films at the interface. The structure of these films varies with the lipid to protein mole ratio of the source vesicle membranes. Electron microscopic observations reveal that films formed with membranes of 1501 mole ratio consist of nonoverlapping, randomly distributed vesicle membrane fragments separated by a lipid monolayer. These membrane fragments exist as single sheets on the water surface and occupy approximately 35% of this surface. Essentially all the rhodopsin molecules at the interface are spectroscopically intact and are contained within the membrane fragments. The visible absorption spectrum of the interface films is identical to that of suspensions of rod disc membranes. Moreover, flash illumination of rhodopsin in air-dried multilayers formed from the interface films results in the formation of a stable MetarhodopsinI intermediate (_max480 nm) which can be fully bleached by increasing the relative humidity of the multilayers or can be photoconverted into rhodopsin and, presumably, isorhodopsin. Furthermore, rhodopsin is chemically regenerable at the air-water interface. Bleached rhodopsin can generate dark rhodopsin at the interface in the presence of 11-cis retinal in the aqueous subphase. Thus, the spectroscopic structure and the chemical regenerability function of rhodopsin in these interface films are indistinguishable from those exhibited by the protein in intact rod disc membranes.     

Proton transport by bacteriorhodopsin through an interface film

Summary Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (<1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials, are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.     

Langmuir-Blodgett films of a novel cellulose derivative with dihydrophytyl group: the ability to anchor beta-carotene molecules

A novel cellulose derivative, 6-O-dihydrophytylcellulose (DHPC), was first synthesized via a ring-opening polymerization and allowed to self-assemble onto an air-water interface. Langmuir-Blodgett (LB) films were characterized with atomic force microscope (AFM), UV-vis spectroscopy, and Fourier transform infrared spectroscopy. The surface pressure-area (pi-A) isotherms for DHPC and beta-carotene (betaC) mixture indicated strong interaction between these compounds to pack well. Thus, DHPC has the ability to anchor betaC in the monolayer. It was proved that a betaC-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films by UV spectroscopic analysis. The transmission and reflection-absorption IR spectra (RAS) indicated that the dihydrophytyl chains had almost trans-zigzag conformation and were oriented nearly perpendicular to the substrate. AFM section analysis revealed the thickness per layer to be 2.32 nm. Consequently, DHPC was found to be an appropriate matrix to fabricate the mixed LB films containing betaC.     

Interaction of Trastuzumab with biomembrane models at air-water interfaces mimicking cancer cell surfaces

Trastuzumab (Tmab) is a monoclonal antibody administered as targeted therapy for HER2-positive breast cancer whose molecular interactions at the HER2 receptor microenvironment are not completely clarified yet. This paper describes the influence of Tmab in the molecular organization of films of biological-relevant molecules at the air water interface. For that, we spread components of tumorigenic and non-tumorigenic cells directly on the air-water interface. The physicochemical properties of the films were investigated with surface pressure-area isotherms and Brewster angle microscopy, and distinction between the cellular lines with higher or lower amount of HER2 could be detected based on the physicochemical properties of the interfacial films. The systems organized at the air-water interface were transferred to solid supports as Langmuir-Blodgett films and the nano-scale morphology investigated with atomic force microscopy. The overall results related to Tmab interacting with the films lead to the conclusion that Tmab tends to condense rich-HER2 films, causing irregular dimerization of the receptor protein, changing the membrane topography of the films, with formation of phases with different levels of reflectivity and aggregation morphology, and finally revealing that the interaction of the antibody with proteo-lipidic biointerfaces is modulated by the film composition. We believe that novel perspectives concerning the molecular interactions in the plasma membrane microenvironment through Langmuir monolayers can be obtained from this work in order to enhance the Tmab-based cancer therapy.     

气/液界面及固体表面硬脂酸LB膜结构性质研究

对气液/和固/液界面上硬脂酸LB膜的结构性质的研究表明,二价离子能够使气/液界面上LB膜表面压力降低,并出现一个固-固转变的过程.对此可以解释为是由二价离子富集在亚相表面,减弱了膜分子之间的库仑作用,使表面电势降低引起的.同时由于二价离子与硬脂酸分子形成复合物,单层膜的结构发生改变,导致固-固转变点的产生.对固体基质上多层LB膜的椭圆偏振研究表明,有序排列的硬脂酸LB膜具有明显的双折射性质.电镜观察发现两个固相垂直提位获得的多层膜在形貌上存在差异,低压固相膜较之高压固相膜存在明显的不均匀性.分析认为这是在膜从气/液界面向固体表面转移过程中发生重结晶引起的.     

Self-assembled hydrophobin protein films at the air-water interface: structural analysis and molecular engineering

Hydrophobins are amphiphilic proteins produced by filamentous fungi. They function in a variety of roles that involve interfacial interactions, as in growth through the air-water interface, adhesion to surfaces, and formation of coatings on various fungal structures. In this work, we have studied the formation of films of the class II hydrophobin HFBI from Trichoderma reesei at the air-water interface. Analysis of hydrophobin aqueous solution drops showed that a protein film is formed at the air-water interface. This elastic film was clearly visible, and it appeared to cause the drops to take unusual shapes. Because adhesion and formation of coatings are important biological functions for hydrophobins, a closer structural analysis of the film was made. The method involved picking up the surface film onto a solid substrate and imaging the surface by atomic force microscopy. High-resolution images were obtained showing both the hydrophilic and hydrophobic sides of the film at nanometer resolution. It was found that the hydrophobin film had a highly ordered structure. To study the orientation of molecules and to obtain further insight in film formation, we made variants of HFBI that could be site specifically conjugated. We then used the avidin-biotin interaction as a probe. On the basis of this work, we suggest that the unusual interfacial properties of this type of hydrophobins are due to specific molecular interactions which lead to an ordered network of proteins in the surface films that have a thickness of only one molecule. The interactions between the proteins in the network are likely to be responsible for the unusual surface elasticity of the hydrophobin film.     

Raman spectroscopy of model membrane monolayers of dipalmitoylphosphatidic acid at the air-water interface using surface enhancement from buoyant thin silver films

A novel method for the acquisition of surface enhanced Raman (SER) spectra of model membranes of dipalmitoylphosphatidic acid (DPPA) in Langmuir layers at the air-water interface is reported. The approach is based on the electrochemical formation of a buoyant thin layer of coalesced silver colloids in the vicinity of the phosphatidic acid head groups at the interface. This Ag layer is an excellent platform for SER scattering, which shows the spectral features from all parts of the molecule and water between the Ag surface and the DPPA layer. The observation of the spectral response from the phosphatidic acid head groups is of particular significance, allowing insight into their chemical state and orientation at the air-water interface.     

Atomic tritium as an instrument for study of protein behavior at the air-water interface

Atomic tritium was successfully applied as an instrument for study of protein behavior at the air-water interface. Samples of lysozyme solution in 20 mM phosphate buffer (pH 7.0) with concentration of 2 mg/ml incubated at the room temperature for 1 h were exposed to bombardment with tritium atoms generated on hot tungsten wire in special vacuum device. This procedure resulted in substitution of hydrogen atoms by radioactive tritium in the thin surface layer of studied preparations. Analysis of experimental data on intramolecular radioactivity distribution in lysozyme and computer simulation of tritium bombardment allowed us to suggest two equally probable opposite orientations of lysozyme molecule in the adsorption layer at the air-water interface.     

Structure of planar membrane formed from liposomes

Lipid vesicles with incorporated ion channels from polyene antibiotic amphotericin B were used to investigate structures of planar membranes formed by Shindler's techniques. A planar membrane assembled on the aperture in a lavsan film from two layers generated at the air-aqueous liposome suspension interface is not a simple bilayer but a bimolecular membrane containing numerous partly fused liposomes. A complete fusion of liposomal membranes with the planar bilayer is an unlikely event during membrane formation. A planar bimolecular lipid membrane without incorporated liposomes can be made by a method consisting of three stages: formation of a lipid layer on the air-water interface of a suspension containing liposomes, transfer of this layer along the surface of the solution into a chamber containing a solution without liposomes where a lipid monomolecular layer forms gradually (within about 20 min) at the air-water interface, assembling of the planar bilayer membrane from this monolayer. The knowledge of the planar membrane structure may be useful in experiments on incorporation of membrane proteins into a planar lipid bilayer.     

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.     

Molecular interaction between organophosphorus acid anhydrolase and diisopropylfluorophosphate

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.     

Scanning Force Microscopy at the Air-Water Interface of an Air Bubble Coated with Pulmonary Surfactant

To study the structure-function relationship of pulmonary surfactant under conditions close to nature, molecular films of a model system consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and surfactant-associated protein C were prepared at the air-water interface of air bubbles about the size of human alveoli (diameter of 100 μm). The high mechanical stability as well as the absence of substantial film flow, inherent to small air bubbles, allowed for scanning force microscopy (SFM) directly at the air-water interface. The SFM topographical structure was correlated to the local distribution of fluorescent-labeled dipalmitoylphosphatidylcholine, as revealed from fluorescence light microscopy of the same bubbles. Although SFM has proven before to be exceptionally well suited to probe the structure of molecular films of pulmonary surfactant, the films so far had to be transferred onto a solid support from the air-water interface of a film balance, where they had been formed. This made them prone to artifacts imposed by the transfer. Moreover, the supported monolayers disallowed the direct observation of the structural dynamics associated with expansion and compression of the films as upon breathing. The current findings are compared in this respect to our earlier findings from films, transferred onto a solid support.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids.

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.     

The displacement of calcium ions from phospholipid monolayers by pharmacologically active and other organic bases

1. The binding of (45)Ca(2+) to a monolayer of phosphatidylinositol at the air-water interface was maximal when the separation of the phospholipid head groups approximated to the diameter of a hydrated Ca(2+) ion. 2. The displacement of Ca(2+) adsorbed on monomolecular films of phosphatidylinositol by a series of drugs (both narcotic and excitatory) and other organic bases was related to the ability of the bases to penetrate into the film. 3. With films of phosphatidylinositol at constant area, and at an initial surface pressure of 10dynes/cm., the displacement of Ca(2+) by increasing concentrations of the local anaesthetic, tetracaine, was linearly related to the change in surface pressure (Deltapi) caused by the penetration of the drug. 4. Deltapi and the displacement of Ca(2+) showed a related fall when the initial surface pressure of the phosphatidylinositol film was increased from 4 to 40dynes/cm. both at a constant bulk tetracaine concentration and when this latter concentration was adjusted to keep it at a constant ratio to the surface density of phosphatidylinositol molecules. 5. The displacement of Ca(2+) from phosphatidylinositol films by cetyltri-methylammonium ions was directly compared with the surface concentration of the base in the film, measured by using labelled base and a surface-radioactivity technique. 6. The ability of a series of straight-chain aliphatic amines to displace Ca(2+) from phosphatidylinositol films increased with the number of carbon atoms up to C(12). However, there was a marked jump in the displacing activity after hexylamine, and this could probably be correlated with the carbon chain's being of sufficient length to just reach the hydrophobic fatty acid chains of the orientated phospholipid molecules with the charges on both substances in juxtaposition.     

Gelation of gelatin observation in the bulk and at the air-water interface

Gelation of gelatin under various conditions has been followed by atomic force microscopy (AFM) with the objective of understanding more fully the structure formed during the gelation process. AFM images were obtained of the structures formed from both the bulk sol and in surface films during the onset of gelation. While gelation occurred in the bulk sol, the extent of helix formation was monitored by measurements of optical rotation, and the molecular aggregation was imaged by AFM. Interfacial gelatin films formed at the air-water interface were also studied. Measurements of surface tension and surface rheology were made periodically and Langmuir-Blodgett films were drawn from the interface to allow AFM imaging of the structure of the interfacial layer as a function of time. Structural studies reveal that at low levels of helical content the gelatin molecules assemble into aggregates containing short segments of dimensions comparable to those expected for gelatin triple helices. With time larger fibrous structures appear whose dimensions suggest that they are bundles of triple helices. As gelation proceeds, the number density of fibers increases at the expense of the smaller aggregates, eventually assembling into a fibrous network. The gel structure appears to be sensitive to the thermal history, and this is particularly important in determining the structure and properties of the interfacial films. © 1998 John Wiley & Sons, Inc. Biopoly 46: 245–252, 1998     

Mixed monolayers of amphiphile-modified nucleic bases and diynoic acids. I. Phase states at air-water interface and in Langmuir-Blodgett films

Monolayers of amphiphile-modified nucleic bases with diynoic acid were obtained and characterized. The synthesized nucleic bases contained in the monolayer complementarily bind the nucleotide molecules contained in the aqueous subphase, and the structure of the resulting monolayers can be fixed by the photopolymerization of diynoic acid. The resulting monolayer exemplifies a novel type of model systems for investigating molecular recognition at the surface of biological membranes. Procedures for the transfer of the monolayers onto solid substrates and photopolymerization of the diynoic acid in mixtures with the derivatives of nucleic bases were developed. The films obtained were structurally characterized using atomic force microscopy. Compression isotherms of the mixed monolayers as well as individual components of monolayers at the air-water interface allowed one to determine the concentration range at which the diynoic acid form true mixtures or domain structures with the derivatives of nucleic base. A study of the films transferred to the solid substrate by atomic force microscopy indicated that this concentration dependence of miscibility behavior was conserved in the transferred films.