Fluroescence properties of chlorophyll a and b monomolecular films at the air-water interface.

The fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorphyll molecules. Over the range of the mean distances from 27 to 21 A, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll a were observed. In the case of chlorophyll beta, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 A. When the mean distance was 18 A, the short wavelength component of chlorophyll beta disappeared, and only the long avelength component was observed.     

Anomalous temperature dependence of peptide films at air-water interface

The tetrapeptide derivative Tyr-Gly-Phe-Ala-OBz (1) forms monolayers as confirmed by compressibility studies carried out at various temperatures. Peptide 1 monolayer exhibits an anomalous structural transition at 40 degrees C as evidenced by pi-A isotherms recorded at different temperatures. The structural transition is also observed in aqueous solution of trifluoroacetate of peptide 1 as evidenced by fluorescence and Raman scattering intensity measurements.     

Enrichment of amyloidogenesis at an air-water interface

The aggregation of proteins or peptides into amyloid fibrils is a hallmark of protein misfolding diseases (e.g., Alzheimer's disease) and is under intense investigation. Many of the experiments performed are in vitro in nature and the samples under study are ordinarily exposed to diverse interfaces, e.g., the container wall and air. This naturally raises the question of how important interfacial effects are to amyloidogenesis. Indeed, it has already been recognized that many amyloid-forming peptides are surface-active. Moreover, it has recently been demonstrated that the presence of a hydrophobic interface can promote amyloid fibrillization, although the underlying mechanism is still unclear. Here, we combine theory, surface property measurements, and amyloid fibrillogenesis assays on islet amyloid polypeptide and amyloid-β peptide to demonstrate why, at experimentally relevant concentrations, the surface activity of the amyloid-forming peptides leads to enriched fibrillization at an air-water interface. Our findings indicate that the key that links these two seemingly different phenomena is the surface-active nature of the amyloid-forming species, which renders the surface concentration much higher than the corresponding critical fibrillar concentration. This subsequently leads to a substantial increase in fibrillization.     

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.     

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.     

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.     

Melittin-solid phospholipid mixed films trigger amyloid-like nano-fibril arrangements at air-water interface

We used the Langmuir monolayers technique to study the surface properties of melittin toxin mixed with either liquid-condensed DSPC or liquid-expanded POPC phospholipids. Pure melittin peptide forms stable insoluble monolayers at the air-water interface without interacting with Thioflavin T (Th-T), a sensitive probe to detect protein amyloid formation. When melittin peptide is mixed with DSPC lipid at 50 % of peptide area proportion at the surface, we observed the formation of fibril-like structures detected by Brewster angle microscopy (BAM), but they were not observable with POPC. The nano-structures in the melittin-DSPC mixtures became Th-T positive labeling when the arrangement was observed with fluorescence microscopy. In this condition, Th-T undergoes an unexpected shift in the typical emission wavelength of this amyloid marker when a 2D fluorescence analysis is conducted.Even when reflectivity analysis of BAM imaging evidenced that these structures would correspond to the DSPC lipid component of the mixture, the interpretation of ATR-FTIR and Th-T data suggested that both components were involved in a new lipid-peptide rearrangement. These nano-fibril arrangements were also evidenced by scanning electron and atomic force microscopy when the films were transferred to a mica support. The fibril formation was not detected when melittin was mixed with the liquid-expanded POPC lipid. We postulated that DSPC lipids can dynamically trigger the process of amyloid-like nano-arrangement formation at the interface. This process is favored by the relative peptide content, the quality of the interfacial environment, and the physical state of the lipid at the surface.     

Phase transitions in films of lung surfactant at the air-water interface.

Pulmonary surfactant maintains a putative surface-active film at the air-alveolar fluid interface and prevents lung collapse at low volumes. Porcine lung surfactant extracts (LSE) were studied in spread and adsorbed films at 23 +/- 1 degrees C using epifluorescence microscopy combined with surface balance techniques. By incorporating small amounts of fluorescent probe 1-palmitoyl-2-nitrobenzoxadiazole dodecanoyl phosphatidylcholine (NBD-PC) in LSE films the expanded (fluid) to condensed (gel-like) phase transition was studied under different compression rates and ionic conditions. Films spread from solvent and adsorbed from vesicles both showed condensed (probe-excluding) domains dispersed in a background of expanded (probe-including) phase, and the appearance of the films was similar at similar surface pressure. In quasistatically compressed LSE films the appearance of condensed domains occurred at a surface pressure (pi) of 13 mN/m. Such domains increased in size and amounts as pi was increased to 35 mN/m, and their amounts appeared to decrease to 4% upon further compression to 45 mN/m. Above pi of 45 mN/m the LSE films had the appearance of filamentous materials of finely divided dark and light regions, and such features persisted up to a pi near 68 mN/m. Some of the condensed domains had typical kidney bean shapes, and their distribution was similar to those seen previously in films of dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant. Rapid cyclic compression and expansion of LSE films resulted in features that indicated a possible small (5%) loss of fluid components from such films or an increase in condensation efficiency over 10 cycles. Calcium (5 mM) in the subphase of LSE films altered the domain distribution, decreasing the size and increasing the number and total amount of condensed phase domains. Calcium also caused an increase in the value of pi at which the maximum amount of independent condensed phase domains were observed to 45 mN/m. It also induced formation of large amounts of novel, nearly circular domains containing probe above pi of 50 mN/m, these domains being different in appearance than any seen at lower pressures with calcium or higher pressures in the absence of calcium. Surfactant protein-A (SP-A) adsorbed from the subphase onto solvent-spread LSE films, and aggregated condensed domains in presence of calcium. This study indicates that spread or adsorbed lung surfactant films can undergo expanded to condensed, and possibly other, phase transitions at the air-water interface as lateral packing density increases. These phase transitions are affected by divalent cations and SP-A in the subphase, and possibly by loss of material from the surface upon cyclic compression and expansion.     

Surface behavior,microheterogeneity and adsorption equilibrium of myelin at the air-water interface

Interfacial films of whole myelin membrane adsorb at the air-water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy.     

Linear dichroism of rhodopsin in air-water interface films

Air-water interface films of purified cattle rhodopsin and defined phospholipids are formed by the osmotic lysis of reconstituted membrane vesicles. The interface films thus formed consist of a phospholipid monolayer containing vesicle membrane fragments. Rhodopsin molecules at the interface are restricted within the membrane fragments where they are spectrophotometrically intact and capable of undergoing photoregeneration and chemical regeneration. Multilayers of up to 8 layers can be built from these interface films. The visible absorption band of rhodopsin in these multilayers is linearly dichroic. Quantitative analysis of the linear dichroism reveals that the dipole moment of transition of the retinal chromophore in rhodopsin forms an angle of 15 degrees +/- 4 degrees with the plane of the membrane fragments in the interface film. This orientation of the chromophore relative to the plane of the membrane is essentially the same as that observed in the intact retina. Thus, the orientation of rhodopsin in the interface films is similar to that in the intact disc membranes.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2′,3′-cyclic nucleotide 3′-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G_M1. A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2',3'-cyclic nucleotide 3'-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G(M1). A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Kinetics of adsorption of globular proteins at an air-water interface.

Adsorption of globular proteins at an air-water interface from an infinite stagnant medium was modeled as one-dimensional diffusion in a potential field. The interaction potential experienced by an adsorbing molecule consisted of contributions from electrostatic interactions, work done against the surface pressure to clear area at the interface in order to anchor the adsorbed segments, and the change in the free energy due to exposure of penetrated surface hydrophobic functional groups to air. The assumption of irreversible adsorption is employed in the present analysis. The energy barrier to adsorption, present at sufficiently large surface pressures, was found to be higher for smaller surface hydrophobicities, larger surface pressures, larger size molecules, and oblate orientation of an ellipsoidal molecule. Consequently, more adsorption occurred at larger surface hydrophobicities, smaller size molecules, and for prolate orientation of ellipsoidal molecules. The subphase concentration has been shown to be zero at short times, increasing with time at larger times, and eventually becoming close to the bulk concentration as a result of increasing energy barrier to adsorption. The predicted evolution of surface concentration with time for adsorption of lysozyme at an air-water interface agreed well with the experimental data of Graham and Phillips (1979a).     

Surface enhanced Raman scattering of a lipid Langmuir monolayer at the air-water interface

Surface enhanced Raman spectra were recorded from a phospholipid monolayer directly at the air-water interface. We used an organized monolayer of negatively charged tetramyristoyl cardiolipins as a template for the electrochemical generation of silver deposits. This two-dimensional electrodeposition of silver under potentiostatic control was the substrate for enhancement of Raman spectra. We report the optimized conditions for the Raman enhancement, the microscopic observations of the deposits, and their characterization by atomic force microscopy. Laser excitation at 514.5 nm leads to intense and reproducible surface enhanced Raman scattering spectra recorded in situ from one monolayer of cardiolipin, using 0.5 mol % of 10N nonyl acridine orange or 5 mol % of acridine in the film, and demonstrates the possibility of estimating the pH at the metal/phospholipidic film interface.     

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.     

Structural and functional properties of irradiated nuclear membranes at the air-water phase interface

The monolayer technique, the methods of electron microscopy and IR-spectroscopy were used to study isolated nuclear membranes of calf thymus cells. The data obtained permitted to study the relationship between structural disorders induced by irradiation of membranes, the changes in their functional status, and the role played by a lipid component of membranes in these processes.