Effects of specific versus nonspecific ionic interactions on the structure and lateral organization of lipopolysaccharides

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na⁺ ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca²⁺ ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na⁺ ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca²⁺ ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na⁺- and Ca²⁺ ions in the subphase, the screening effect of Na⁺ is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca²⁺ ions. The unspecific charge-screening effect of the Na⁺ ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.     

Physical Properties of Lipid Monolayers on Alkylated Planar Glass Surfaces

A method for transferring a lipid monolayer from an air-water interface to an alkylated glass slide is described. Specific antibodies bind tightly to lipid haptens contained in these monolayers on the glass slides. We conclude that the polar head groups of the lipids face the aqueous phase. A monolayer containing a fluorescent lipid was used to show that the monolayer is homogeneous as observed with an epifluorescence microscope. A periodic pattern photobleaching technique was used to measure the lateral diffusion of this fluorescent lipid probe in monolayers composed of dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. Different regions of the pressure-area isotherms of the monolayers at the air-water interface can be correlated with the diffusion of the fluorescent probe molecules on the monolayer-coated glass slide. Monolayers derived from the so-called “solid-condensed” state of a monolayer at the air-water interface showed a very low probe diffusion coefficient in this monolayer when placed on a glass slide, D ≤ 10^-10 cm²/s. Monolayers derived from the “liquid condensed/liquid expanded” (LC/LE) region of the monolayer isotherms at the air-water interface showed rapid diffusion (D > 10^-8 cm²/s) when these same monolayers were observed on an alkylated glass slide. The monolayers attached to the glass slide appear to be homogeneous when derived from monolayers in the LC/LE region of monolayers at the air-water interface. There is no major variation of the diffusion coefficient of a fluorescent lipid probe when this diffusion is measured on a lipid monolayer on a glass slide, for monolayers derived from various regions of the LC/LE monolayers at the air-water interface. This is consistent with the view that the LC/LE region is most likely a single fluid phase. Monolayers supported on a planar glass substrate are of much potential interest for biophysical and biochemical studies of the interactions between model membranes and cellular membranes, and for physical chemical studies relating the properties of lipid monolayers to the properties of lipid bilayers.     

Monomolecular layer formation of ferritin molecules on an amphiphilic cyclodextrin derivative at the air/water interface

A monomolecular layer of ferritin molecules was formed by adsorption from the subphase onto a Langmuir film of an amphiphilic beta-cyclodextrin (beta-CD) derivative at the air/water interface. The course of the adsorption of ferritin molecules was monitored by measuring the surface pressure and the resulting film was observed by transmission electron microscopy (TEM). These results show the potential of the amphiphilic CD derivative to work as a milder template for protein molecules at the air/water interface.     

Effect of Supporting Substrates on the Structure of DNA and DNA–Trivaline Complexes Studied by Atomic Force Microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA–TV complexes onto this surface. On mica, both purified DNA and DNA–TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA–TV displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

Assessment of Elasticity and Topography of Aspergillus nidulans Spores via Atomic Force Microscopy

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA⁺ and rodA⁻ strains. Tapping-mode AFM images of wild-type and rodA⁺ spores in air showed characteristic “rodlet” protein structures covering a granular spore surface. In comparison, rodA⁻ spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA⁺ spores. Rodlets were removed from rodA⁺ spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 ± 10, 120 ± 10, and 300 ± 20 N/m and the elastic moduli to be 6.6 ± 0.4, 7.0 ± 0.7, and 22 ± 2 GPa for wild-type, rodA⁺ and rodA⁻ spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.     

Ferritin protein nanocage ion channels: gating by N-terminal extensions

Ferritin protein nanocages, self-assembled from four-α-helix bundle subunits, use Fe²⁺ and oxygen to synthesize encapsulated, ferric oxide minerals. Ferritin minerals are iron concentrates stored for cell growth. Ferritins are also antioxidants, scavenging Fenton chemistry reactants. Channels for iron entry and exit consist of helical hairpin segments surrounding the 3-fold symmetry axes of the ferritin nanocages. We now report structural differences caused by amino acid substitutions in the Fe²⁺ ion entry and exit channels and at the cytoplasmic pores, from high resolution (1.3–1.8 Å) protein crystal structures of the eukaryotic model ferritin, frog M. Mutations that eliminate conserved ionic or hydrophobic interactions between Arg-72 and Asp-122 and between Leu-110 and Leu-134 increase flexibility in the ion channels, cytoplasmic pores, and/or the N-terminal extensions of the helix bundles. Decreased ion binding in the channels and changes in ordered water are also observed. Protein structural changes coincide with increased Fe²⁺ exit from dissolved, ferric minerals inside ferritin protein cages; Fe²⁺ exit from ferritin cages depends on a complex, surface-limited process to reduce and dissolve the ferric mineral. High concentrations of bovine serum albumin or lysozyme (protein crowders) to mimic the cytoplasm restored Fe²⁺ exit in the variants to wild type. The data suggest that fluctuations in pore structure control gating. The newly identified role of the ferritin subunit N-terminal extensions in gating Fe²⁺ exit from the cytoplasmic pores strengthens the structural and functional analogies between ferritin ion channels in the water-soluble protein assembly and membrane protein ion channels gated by cytoplasmic N-terminal peptides.     

A Ferritin from Dendrorhynchus zhejiangensis with Heavy Metals Detoxification Activity

Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd²⁺, Pb²⁺ and Fe²⁺. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd²⁺ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.     

An on-chip thin film photodetector for the quantification of DNA probes and targets in microarrays

A flat microdevice which incorporates a thin-film amorphous silicon (a-Si:H) photodetector with an upper layer of functionalized SiO₂ is used to quantify the density of both immobilized and hybridized DNA oligonucleotides labeled with a fluorophore. The device is based on the photoconductivity of hydrogenated amorphous silicon in a coplanar electrode configuration. Excitation, with near UV/blue light, of a single-stranded DNA molecule tagged with the fluorophore 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide (PyMPO), results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the optoelectronic detection of the DNA molecules. The detection limit of the present device is of the order of 1 × 10¹² molecules/cm² and is limited by the efficiency of the filtering of the excitation light. A surface density of 33.5 ± 4.0 pmol/cm² was measured for DNA covalently immobilized to the functionalized SiO₂ thin film and a surface density of 3.7 ± 1.5 pmol/cm² was measured for the complementary DNA hybridized to the bound DNA. The detection concept explored can enable on-chip electronic data acquisition, improving both the speed and the reliability of DNA microarrays.     

Correlation between surface morphology and surface forces of protein A adsorbed on mica.

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves.     

Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging

RNA microarrays were created on chemically modified gold surfaces using a novel surface ligation methodology and employed in a series of surface plasmon resonance imaging (SPRI) measurements of DNA–RNA hybridization and RNA aptamer–protein binding. Various unmodified single-stranded RNA (ssRNA) oligonucleotides were ligated onto identical 5′-phosphate-terminated ssDNA microarray elements with a T4 RNA ligase surface reaction. A combination of ex situ polarization modulation FTIR measurements of the RNA monolayer and in situ SPRI measurements of DNA hybridization adsorption onto the surface were used to determine an ssRNA surface density of 4.0 × 10¹² molecules/cm² and a surface ligation efficiency of 85 ± 10%. The surface ligation methodology was then used to create a five-component RNA microarray of potential aptamers for the protein factor IXa (fIXa). The relative surface coverages of the different aptamers were determined through a novel enzymatic method that employed SPRI measurements of a surface RNase H hydrolysis reaction. SPRI measurements were then used to correctly identify the best aptamer to fIXa, which was previously determined from SELEX measurements. A Langmuir adsorption coefficient of 1.6 × 10⁷ M⁻¹ was determined for fIXa adsorption to this aptamer. Single-base variations from this sequence were shown to completely destroy the aptamer–fIXa binding interaction.     

Structural properties of phosphatidylcholine in a monolayer at the air/water interface: Neutron reflection study and reexamination of x-ray reflection measurements

Neutron reflectivities of phosphatidylcholine monolayers in the liquid condensed (LC) phase on ultrapure H₂O and D₂O subphases have been measured on a Langmuir film balance. Using a dedicated liquid surface reflectometer, reflectivities down to R = 10^-6 in the momentum transfer range Q_z = 0-0.4 Å^-1 were accessed.

In a new approach, by refining neutron reflectivity data from chain-perdeuterated DPPC-d₆₂ in combination with x-ray measurements on the same monolayer under similar conditions it is shown that the two techniques mutually complement one another. This analysis leads to a detailed conception of the interface structure. It is found that in the LC phase (which is analogous to the L_β, phase in vesicle dispersions) the head group is interpenetrated with subphase water (4 ± 2.5 molecules per lipid) and the average tilt angle of the hydrophobic chains from the surface normal is 33 ± 3 degrees.

     

Direct visualization of polypeptide shell of ferritin molecule by atomic force microscopy.

The polypeptide shell of the ferritin molecule has been imaged in water by atomic force microscopy (AFM). The central dip and the quaternary structure could be observed on the surface of the ferritin molecule anchored inhomogeneously in two dimensions. These structures observed in the AFM images are quite similar to the electron density map near the top of the apoferritin viewed down from a 4-fold axis structure reported previously (S. H. Banyard, D. K. Stammers, and P. M. Harrison, 1978. Nature (Lond.). 271:282-284). It has been achieved by introducing a "self-screening effect" of the surface charges of the AFM sample (S. Ohnishi, M. Hara, T. Furuno, and H. Sasabe. 1992. Biophys. J. 63:1425-1431) and the specially sharpened stylus of AFM cantilever.     

THE DISTRIBUTION OF EXOGENOUS FERRITIN IN TOAD SPINAL GANGLIA AND THE MECHANISM OF ITS UPTAKE BY NEURONS

Toad spinal ganglion cells are individually enclosed in sheaths consisting of one or more attenuated layers of satellite cell cytoplasm surrounded externally by a basement membrane. Narrow (~150 A) extracellular channels separate these layers from one another and from the underlying neuron. In both in vivo and in vitro experiments it was found that molecules of ferritin, a water-soluble protein, are to some extent able to pass across the basement membrane and through these channels to reach the neuronal plasma membrane. Ferritin particles arriving at the neuronal surface are engulfed by the neuron in 0.1 to 0.2 µ "coated" vesicles. The concentration of ferritin in these vesicles is higher than in the perineuronal space. The ferritin incorporated into the neuron is segregated, apparently intact, in multivesicular bodies. It is inferred that the 150A channels in the satellite cell sheath are patent, aqueous spaces through which molecules with a diameter as large as 95 A are able to pass, and that these neurons are capable of taking up whole protein from their immediate environment by the process of pinocytosis.     

The Refined Crystal Structure of an Eel Pout Type III Antifreeze Protein RD1 at 0.62-Å Resolution Reveals Structural Microheterogeneity of Protein and Solvation

RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62 Å. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the “pretzel fold” includes four short β-strands and a 3₁₀ helix. There is a novel internal cavity of 45 ų surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized.     

Effect of the covalently linked fatty acid 18-MEA on the nanotribology of hair's outermost surface

Highly ordered lipids adsorbed or grafted on surfaces are known to provide protection and lubrication custom engineered surfaces. We have used atomic force microscopy (AFM) to measure adhesion and frictional properties of the outermost surfaces of a variety of human hairs with the aim of both understanding the role of 18-methyleicosanoic acid (18-MEA), an unusual branched-chain fatty acid covalently bound to the cuticle surface, and investigating how treatments or the ethnic origin affect this layer. Results show that an unmodified silicon nitride AFM tip is able to detect changes at the hair surface that can be related to the absence or presence of this layer due to treatment conditions and in particular that this monolayer has a lubricant effect.     

Interactions of acetylcholine receptor and acetylcholinesterase with lipid monolayers

The interaction of acetylcholine receptor and acetylcholinesterase with lipid monolayers was followed by measuring changes in surface pressure.When injected into the subphase of a lipid monolayer, the proteins caused increases in surface pressure from 5 to 10 dynes/cm, indicating a penetration of protein into the monolayer. At pH values below the isoelectric point of the proteins the incorporation was improved. The same was observed when Ca²⁺ (2 mM) was added.The presence of the enzyme in the mixed film could be demonstrated by using diiso[³H]propyl fluorophosphate-labelled acetylcholinesterase as well as by measuring enzyme activity. Acetylcholine receptor was shown to be present in the mixed film by using a complex made of the receptor and α-[³H]neurotoxin.     

Disruption of the Primary Fouling Sequence on Fiber Glass-Reinforced Plastic Submerged in the Marine Environment

Fiber glass-reinforced plastic immersed in an experimental estuarine mesocosm fouled at estimated rates of 0.5, 5.5, and 18.8 ng (wet weight) mm⁻² day⁻¹ over days 0 to 2, 2 to 6, and 6 to 14, respectively. Protists, dominated by diatoms, which developed between days 3 and 6 and covered 90% of the undisturbed surface in 2 weeks, were effectively removed by twice-weekly brushing of the surface to maintain an immature 3-day bacterial film which covered 12% or less of the surface and had a biomass 3 orders of magnitude smaller than surfaces with 2 weeks' unrestricted fouling. Direct brushing of the fiber glass-reinforced plastic tank walls of experimental estuarine mesocosms minimized the “wall effect” by keeping a surface that maintained a low biomass of a slowly accumulating bacterial film rather than a surface which supported the more rapid accumulation of protists which in turn may induce the settlement of invertebrates and macrophytes.     

Photoresist-Free Patterning by Mechanical Abrasion of Water-Soluble Lift-Off Resists and Bare Substrates: Toward Green Fabrication of Transparent Electrodes

This paper describes the fabrication of transparent electrodes based on grids of copper microwires using a non-photolithographic process. The process—“abrasion lithography”—takes two forms. In the first implementation (Method I), a water-soluble commodity polymer film is abraded with a sharp tool, coated with a conductive film, and developed by immersion in water. Water dissolves the polymer film and lifts off the conductive film in the unabraded areas. In the second implementation (Method II), the substrate is abraded directly by scratching with a sharp tool (i.e., no polymer film necessary). The abraded regions of the substrate are recessed and roughened. Following deposition of a conductive film, the lower profile and roughened topography in the abraded regions prevents mechanical exfoliation of the conductive film using adhesive tape, and thus the conductive film remains only where the substrate is scratched. As an application, conductive grids exhibit average sheet resistances of 17 Ω sq^–1 and transparencies of 86% are fabricated and used as the anode in organic photovoltaic cells in concert with the conductive polymer, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Compared to devices in which PEDOT:PSS alone serves as an anode, devices comprising grids of copper/nickel microwires and PEDOT:PSS exhibit lowered series resistance, which manifests in greater fill factor and power conversion efficiency. This simple method of forming micropatterns could find use in applications where cost and environmental impact should be minimized, especially as a potential replacement for the transparent electrode indium tin oxide (ITO) in thin-film electronics over large areas (i.e., solar cells) or as a method of rapid prototyping for laboratory-scale devices.