Nanoscale properties of mixed fengycin/ceramide monolayers explored using atomic force microscopy

To gain insight into the interactions between fengycin and skin membrane lipids, mixed fengycin/ceramide monolayers were investigated using atomic force microscopy (AFM) (monolayers supported on mica) and surface pressure-area isotherms (monolayers at the air-water interface). AFM topographic images revealed phase separation in mixed monolayers prepared at 20 degrees C/pH 2 and composed of 0.25 and 0.5 fengycin molar ratios, in the form of two-dimensional (2-D) hexagonal crystalline domains of ceramide surrounded by a fengycin-enriched fluid phase. Surface pressure-area isotherms as well as friction and adhesion AFM images confirmed that the two phases had different molecular orientations: while ceramide formed a highly ordered phase with crystalline chain packing, fengycin exhibited a disordered fluid phase with the peptide ring lying horizontally on the substrate. Increasing the temperature and pH to values corresponding to the skin parameters, i.e., 37 degrees C/pH 5, was found to dramatically affect the film organization. At low fengycin molar ratio (0.25), the hexagonal ceramide domains transformed into round domains, while at higher ratio (0.5) these were shown to melt into a continuous fengycin/ceramide fluid phase. These observations were directly supported by the thermodynamic analysis (deviation from the additivity rule, excess of free energy) of the monolayer properties at the air-water interface. Accordingly, this study demonstrates that both the environmental conditions (temperature, pH) and fengycin concentration influence the molecular organization of mixed fengycin/ceramide monolayers. We believe that the ability to modulate the formation of 2-D domains in the skin membrane may be an important biological function of fengycin, which should be increasingly investigated in future pharmacological research.     

Two-dimensional protein crystals on a solid substrate: effect of surface ligand concentration

Proteins imbedded in solid-supported lipid bilayers can serve as model systems for investigations of cellular membranes and protein behavior on surfaces. We have investigated the self-assembly of streptavidin on mica-supported bilayer membranes. Using fluorescence microscopy and atomic force microscopy, our studies reveal that the concentration of surface ligand influences the molecular packing of the resulting protein arrays, which in turn affects overall crystal morphology. Two-dimensional streptavidin crystals are obtained when the biotinylated lipid density on the substrate reaches 1.5% mole fraction, yielding high-aspect morphologies that comprise primarily of crystals with P1 symmetry. At 3% and above, crystals with C222 symmetry are formed and result in H-shaped and confluent structures. In intermediate densities between 2 and 3%, a coexistence of P1 and C222 crystal forms is observed. The relationship between macroscopic morphology and molecular configuration is similar to previously reported data obtained at the air/water interface. This suggests that, under our experimental conditions, protein interactions with the supporting substrate are less significant for defining self-assembly behavior than interactions between protein molecules. Ligand-inhibition and fluorescence recovery after photobleaching were used to elucidate the concentration-dependent mechanism for the divergent crystal forms. We have measured the diffusion coefficient of molecules in P1-forming conditions to be approximately twice that of molecules in C222-forming concentrations, which is consistent with proteins bound to the surface through one and two ligands, respectively. The differential flexibility associated with the binding state is therefore likely to alter the subtle protein interactions involved in crystallization.     

Crystalline protein domains and lipid bilayer vesicle shape transformations

Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.     

Two-dimensional arrays of human C-reactive protein formed on a dense aqueoys subphase underneath a lipid layer

A recently developed protein spreading technique, using a subphase of higher density than that of the injected protein solution, was combined with the lipid monolayer approach for two-dimensional crystallization experiments of human C-reactive protein (hCRP) at the air-water interface. Densely packed two-dimensional arrays of hCRP adsorbed on a stearylamine/egg yolk phosphatidylcholine lipid layer have been obtained in the pH range from 5.4 to 5.8. Correct choices of the lipid mixture and of the pH of the subphase were important for the formation of large arrays. Image analysis of transmission electron micrographs showed that the densely packed arrays do not possess crystalline order. The orientational homogeneity of the protein molecules adsorbed on the lipid layer could be used, however, to facilitate single particle averaging techniques. At a resolution of about 1.5 nm a clear handedness of the five-fold symmetric molecule becomes visible, and an asymmetric mass distribution of each subunit is revealed.     

Structural properties and Raman spectroscopy of lipid Langmuir monolayers at the air-water interface

Spectra of octadecylamine (ODA) Langmuir monolayers and egg phosphatidylcholine (PC)/ODA-mixed monolayers at the air-water interface have been acquired. The organization of the monolayers has been characterized by surface pressure-area isotherms. Application of polarized optical microscopy provides further insight in the domain structures and interactions of the film components. Surface-enhanced Raman scattering (SERS) data indicate that enhancement in Raman spectra can be obtained by strong interaction between headgroups of the surfactants and silver particles in subphase. By mixing ODA with phospholipid molecules and spreading the mixture at the air-water interface, we acquired vibrational information of phospholipid molecules with surfactant-aided SERS effect.     

Partitioning of clay colloids at air-water interfaces

Colloid sorption onto air-water interfaces in a variety of natural environments has been previously recognized, but better quantification and understanding is still needed. Affinities of clay colloids for the air-water interface were measured using a bubble-column method and reported as partition coefficients (K). Four types of dilute clay suspensions were measured in NaCl solutions under varying pH and ionic strength conditions: kaolinite KGa-1, illite IMt-2, montmorillonite SWy-2, and bentonite. The K values of three types of polystyrene latex particles with different surface-charge properties were also measured for comparison. Kaolinite exhibited extremely high affinity to the air-water interface at pH values below 7. Illite has lower affinity to air-water interfaces than kaolinite, but has similar pH dependence. Na-montmorillonite and bentonite clay were found excluded from the air-water interface at any given pH and ionic strength. Positively and negatively charged latex particles exhibited sorption and exclusion, respectively, at the air-water interface. These results show the importance of electrostatic interactions between the air-water interface and colloids, especially the influence of pH-dependent edge charges, and influence of particle shape.     

Photoswitchable Dissipative Two‐Dimensional Colloidal Crystals

Control over particle interactions and organization at fluid interfaces is of great importance both for fundamental studies and practical applications. Rendering these systems stimulus‐responsive is thus a desired challenge both for investigating dynamic phenomena and realizing reconfigurable materials. Here, we describe the first reversible photocontrol of two‐dimensional colloidal crystallization at the air/water interface, where millimeter‐sized assemblies of microparticles can be actuated through the dynamic adsorption/desorption behavior of a photosensitive surfactant added to the suspension. This allows us to dynamically switch the particle organization between a highly crystalline (under light) and a disordered (in the dark) phase with a fast response time (crystallization in ≈10 s, disassembly in ≈1 min). These results evidence a new kind of dissipative system where the crystalline state can be maintained only upon energy supply.     

Hierarchical nanoparticle/block copolymer surface features via synergistic self-assembly at the air-water interface

This work demonstrates the first example of the controlled organization of semiconducting nanoparticles (NPs) using amphiphilic block copolymer self-assembly at the air-water interface. Preferential interactions between polystyrene-functionalized NPs and the polystyrene block of an amphiphilic polystyrene-b-poly(ethylene oxide) block copolymer result in synergistic self-assembly at the air-water interface, forming a range of highly stable one-dimensional NP/polymer surface features, including branched nanowires, nanocables up to 100 microm in length, and nanowires with nanoring connectors. This strategy offers new routes to hierarchical hybrid assemblies with potential photonics applications because the nanoscale organization of NPs is coupled to features with dimensions that are commensurate with optical wavelengths.     

Interfacial recognition reactions as seen by fluorescence-, surface plasmon-and atomic force microscopies

We report on various microscopic investigations of the specific recognition and binding reaction between a biotinylated lipid layer and streptavidin. First, we present fluorescence microscopic evidence for the preferential adsorption of the protein to only the fluid matrix of a monolayer at the water-air interface if the latter is compressed to the phase transition region where crystalline domains coexist with expanded phase. Surface plasmon microscopy shows that this selectivity is preserved also if the monofilm is first transferred to a solid support (but still in contract with the aqueous phase) and then exposed to a streptavidin-containing solution. Finally, atomic force microscopic pictures taken at the monolayer-electrolyte interface are presented that confirm this preferential binding.     

Comparative studies of nontoxic and toxic amyloids interacting with membrane models at the air-water interface

Many in vitro studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. In a previous study, we generated a yeast toxic mutant (M8) of the harmless model amyloid protein HET-s((218-289)). In this study, we compared the self-assembling process of the nontoxic wild-type (WT) and toxic (M8) protein at the air-water interface and in interaction with various phospholipid monolayers (DOPE, DOPC, DOPI, DOPS and DOPG). We first demonstrate using ellipsometry measurements and polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS) that the air-water interface promotes and modifies the assembly of WT since an amyloid-like film was instantaneously formed at the interface with an antiparallel β-sheet structuration instead of the parallel β-sheet commonly observed for amyloid fibers generated in solution. The toxic mutant (M8) behaves in a similar manner at the air-water interface or in bulk, with a fast self-assembling and an antiparallel β-sheet organization. The transmission electron microscopy (TEM) images established the fibrillous morphology of the protein films formed at the air-water interface. Second, we demonstrate for the first time that the main driving force between this particular fungus amyloid and membrane interaction is based on electrostatic interactions with negatively charged phospholipids (DOPG, DOPI, DOPS). Interestingly, the toxic mutant (M8) clearly induces perturbations of the negatively charged phospholipid monolayers, leading to a massive surface aggregation, whereas the nontoxic (WT) exhibits a slight effect on the membrane models. This study allows concluding that the toxicity of the M8 mutant could be due to its high propensity to interact with membranes.     

Tilting operator for phospholipidic molecular domains at the liquid–gas interface

We derive a coordinate independent operator expression for the tilting operator of molecular domains at the liquid–gas interface. The domains are made up of phospholipidic molecules modeled as spherocylinders. The molecules of the domain are oriented parallel to each other. The centers of symmetry of the molecules form a lattice. The tilting operator keeps track of the deformations suffered by this lattice as the domain molecules are tilted relative to the normal to the interface. The results obtained are important for dynamic calculations of inclination dependent collective film characteristics, as in the simulation of surface density versus surface pressure curves in a Langmuir film. The tilting operation can be decomposed into three separate simple operations: a global rotation, a local oblique realignment, and a global vertical translation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure and phase behavior of archaeal lipid monolayers

We report X-ray reflectivity (XRR) and grazing incidence X-ray diffraction (GIXD) measurements of archaeal bipolar tetraether lipid monolayers at the air-water interface. Specifically, Langmuir films made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at three different temperatures, i.e., 68, 76, and 81 °C, were examined. The dependence of the structure and packing properties of PLFE monolayers on surface pressure were analyzed in a temperature range between 10 and 50 °C at different pH values. Additionally, the interaction of PLFE monolayers (using lipids derived from cells grown at 76 °C) with the ion channel peptide gramicidin was investigated as a function of surface pressure. A total monolayer thickness of approximately 30 ? was found for all monolayers, hinting at a U-shaped conformation of the molecules with both head groups in contact with the interface. The monolayer thickness increased with rising film pressure and decreased with increasing temperature. At 10 and 20 °C, large, highly crystalline domains were observed by GIXD, whereas at higher temperatures no distinct crystallinity could be observed. For lipids derived from cells grown at higher temperatures, a slightly more rigid structure in the lipid dibiphytanyl chains was observed. A change in the pH of the subphase had an influence only on the structure of the lipid head groups. The addition of gramicidin to an PLFE monolayer led to a more disordered state as observed by XRR. In GIXD measurements, no major changes in lateral organization could be observed, except for a decrease of the size of crystalline domains, indicating that gramicidin resides mainly in the disordered areas of the monolayer and causes local membrane perturbation, only.     

Grazing Incidence X-ray diffraction on Langmuir films: toward atomic resolution

We have analyzed grazing incidence diffration (GIXD) data from condensed phases of Langmuir films of long-chain fatty acids at the air-water interface by using a new method consisting of a careful extraction of the structure factors followed by fitting of molecular parameters. We show that, contrary to the general belief, the information contained in GIXD spectra is enough to obtain near-atomic structural information. In particular, we directly determine for the first time the orientation of the chain backbone planes and of the carboxylic headgroups and we evaluate chain conformation defects. This new method allowed us to evidence a new phase of symmetry p2gm at high pressure, corresponding to a minimum in lattice energy, but never observed.     

Incorporation of Blood-Clotting Proteins into Phospholipid Langmuir Monolayers: A Fluorescence Microscopy Study

Phospholipid monolayers adsorbed at an air-water interface are model cell membranes and have been used in this work to study interactions with blood-clotting proteins. Factor I (non-membrane binding) was used as a control protein, and its association with L-alpha-dipalmitoylphosphatidylcholine Langmuir monolayers was compared to factor VII, a membrane-binding protein. Fluorescence micrographs indicated that factor I penetration of the lipid monolayers in the phase transition region occurred extensively, causing condensation of the lipid film. The association of factor I with phospholipid monolayers was deemed nonspecific. Factor VII was shown to associate with the periphery of lipid domains in the absence of calcium ions, causing flattening of domain edges. In the presence of calcium, factor VII induced expansion of the lipid monolayer. This effect is a specific interaction attributed to exposure of hydrophobic residues upon calcium binding, followed by protein association with lipid hydrocarbon chains. Copyright 2001 Academic Press.     

Specific interaction of desthiobiotin lipids and water-soluble biotin compounds with streptavidin

As shown for biotin lipids (Ref. 1), the formation of perfect 2-D crystalline streptavidin domains can also be observed in the plane of desthiobiotin lipid monolayers. The binding constant of streptavidin with desthiobiotin (K_a = 5·10¹³ mol⁻¹) is lower than that with biotin (K_a = 10¹⁵ mol⁻¹) (Ref. 2). By adding free biotin into the subphase a competitive replacement and a detaching of the streptavidin domains from the desthiobiotin lipid monolayer takes place. Streptavidin domains built at receptor lipid monolayers are still functional. As could be shown, there are two biotin binding sites at each protein molecule that are fully accessible to biotin (Ref. 1). This can be proven by the interaction with biotinylated ferritin and fluoresceinated biotin. Further coupling of an anti-FITC-antibody can proceed and a second protein layer can be formed. Using a bifunctional biotin linker a second crystalline streptavidin layer underneath the first one can be obtained.     

Interfacial characterization of beta-lactoglobulin networks: displacement by bile salts

The competitive displacement of a model protein (beta-lactoglobulin) by bile salts from air-water and oil-water interfaces is investigated in vitro under model duodenal digestion conditions. The aim is to understand this process so that interfaces can be designed to control lipid digestion thus improving the nutritional impact of foods. Duodenal digestion has been simulated using a simplified biological system and the protein displacement process monitored by interfacial measurements and atomic force microscopy (AFM). First, the properties of beta-lactoglobulin adsorbed layers at the air-water and the olive oil-water interfaces were analyzed by interfacial tension techniques under physiological conditions (pH 7, 0.15 M NaCl, 10 mM CaCl2, 37 degrees C). The protein film had a lower dilatational modulus (hence formed a weaker network) at the olive oil-water interface compared to the air-water interface. Addition of bile salt (BS) severely decreased the dilatational modulus of the adsorbed beta-lactoglobulin film at both the air-water and olive oil-water interfaces. The data suggest that the bile salts penetrate into, weaken, and break up the interfacial beta-lactoglobulin networks. AFM images of the displacement of spread beta-lactoglobulin at the air-water and the olive oil-water interfaces suggest that displacement occurs via an orogenic mechanism and that the bile salts can almost completely displace the intact protein network under duodenal conditions. Although the bile salts are ionic, the ionic strength is sufficiently high to screen the charge allowing surfactant domain nucleation and growth to occur resulting in displacement. The morphology of the protein networks during displacement is different from those found when conventional surfactants were used, suggesting that the molecular structure of the surfactant is important for the displacement process. The studies also suggest that the nature of the oil phase is important in controlling protein unfolding and interaction at the interface. This in turn affects the strength of the protein network and the ability to resist displacement by surfactants.     

Molecular-scale neural nets: an approach to the self-assembly of molecular networks

It is suggested that molecular self-assembly techniques may be utilised in molecular scale electronic systems. Taking a neural network as an example, it is shown how the affinity of proteins for their complementary ligand could be used to form the molecular scale network essential to such a system. The approach is based on the polymerisation of the tetrameric proteins avidin and streptavidin using biotinylated ligands. By synthesising a range of aromatic bisbiotin ligands, some simple ground rules are established for ensuring complexation of protein and ligand. Based on these findings, a tetrabiotinylated tetrapyridylporphyrin salt (TBPP) was synthesised with a view to forming a two-dimensional protein polymer. Gel chromatography experiments and a UV-visible investigation of solutions containing the protein/TBPP mixture provide strong evidence for complex formation. The specific immobilisation of streptavidin to a monolayer of TBPP formed at the air-water interface is also demonstrated using fluorescence microscopy. Based on the promising results of these initial studies, a number of suggestions are made for the further exploitation of the techniques described herein.     

High-affinity tags fused to s-layer proteins probed by atomic force microscopy

Two-dimensional, crystalline bacterial cell surface layers, termed S-layers, are one of the most commonly observed cell surface structures of prokaryotic organisms. In the present study, genetically modified S-layer protein SbpA of Bacillus sphaericus CCM 2177 carrying the short affinity peptide Strep-tag I or Strep-tag II at the C terminus was used to generate a 2D crystalline monomolecular protein lattice on a silicon surface. Because of the genetic modification, the 2D crystals were addressable via Strep-tag through streptavidin molecules. Atomic force microscopy (AFM) was used to investigate the topography of the single-molecules array and the functionality of the fused Strep-tags. In high-resolution imaging under near-physiological conditions, structural details such as protein alignment and spacing were resolved. By applying molecular recognition force microscopy, the Strep-tag moieties were proven to be fully functional and accessible. For this purpose, streptavidin molecules were tethered to AFM tips via approximately 8-nm-long flexible polyethylene glycol (PEG) linkers. These functionalized tips showed specific interactions with 2D protein crystals containing either the Strep-tag I or Strep-tag II, with similar energetic and kinetic behavior in both cases.     

Drop-shape analysis of receptor-ligand binding at the oil/water interface

Drop-shape analysis was used to study the binding of streptavidin to biotin at the interface between water and a pendant chloroform droplet. Polyethylene oxide molecules were synthesized with a hydrophobic tail at one end of the molecule and a hydroxyl or biotin group at the other end. The interfacial tension of the water/chloroform interface was measured before and after addition of these amphiphiles to the chloroform phase and before and after addition of streptavidin to the aqueous phase. The hydroxyl-terminated amphiphiles eliminate nonspecific adsorption of the streptavidin to the interface, while streptavidin binds irreversibly to the biotin-terminated molecules. Molecular interactions within this bound layer were studied by measuring changes in the interfacial pressure as the layer is contracted and expanded by changing the volume of the chloroform droplet. A picture of the interfacial structure was obtained from quantitative comparisons between the experimental results and a molecular theory of protein binding to tethered ligands. These comparisons suggest that protein binding is controlled by the extension of the PEO tethers away from the interface.     

Unexpected differences in the behavior of ovotransferrin at the air-water interface at pH 6.5 and 8.0

Adsorption of purified apo-ovotransferrin at the air-water interface was studied by ellipsometry, surface tension, polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS), and shear elastic constant measurements. No significant difference was observed between pH 6.5 and 8.0 as regards the final value of surface concentration and surface pressure. However at low concentration, a weak barrier to adsorption is evidenced at pH 6.5 and confirmed by PM-IRRAS measurements. At a pH where the protein net charge is negative (pH 8.0), the behavior of ovotransferrin at the air-water interface is more influenced by charge effects rather than bulk concentration effects. At this pH, the interface exhibits a low shear elastic constant and a spectral signature not usual for globular proteins.