Richard G. Haverkamp

The Hydrophobin EAS Is Largely Unstructured in Solution and Functions by Forming Amyloid-Like Structures

BACKGROUND Fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. These monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. The molecular details underlying the formation of these monolayers, however, have been elusive. We have studied EAS, the hydrophobin from the ascomycete Neurospora crassa, in an effort to understand the structural aspects of hydrophobin polymerization. RESULTS We have purified both wild-type and uniformly 15N-labeled EAS from N. crassa conidia, and used a range of physical methods including multidimensional NMR spectroscopy to provide the first high resolution structural information on a member of the hydrophobin family. We have found that EAS is monomeric but mostly unstructured in solution, except for a small region of antiparallel beta sheet that is probably stabilized by four intramolecular disulfide bonds. Polymerised EAS appears to contain substantially higher amounts of beta sheet structure, and shares many properties with amyloid fibers, including a characteristic gold-green birefringence under polarized light in the presence of the dye Congo Red. CONCLUSIONS EAS joins an increasing number of proteins that undergo a disorder-->order transition in carrying out their normal function. This report is one of the few examples where an amyloid-like state represents the wild-type functional form. Thus the mechanism of amyloid formation, now thought to be a general property of polypeptide chains, has actually been applied in nature to form these remarkable structures.

Silver Nanoparticles Disrupt Wheat (Triticum aestivum L.) Growth in a Sand Matrix

Hydroponic plant growth studies indicate that silver nanoparticles (Ag NPs) are phytotoxic. In this work, the phytotoxicity of commercial Ag NPs (10 nm) was evaluated in a sand growth matrix. Both NPs and soluble Ag were recovered from water extracts of the sand after growth of plants challenged with the commercial product; the surface charge of the Ag NPs in this extract was slightly reduced compared to the stock NPs. The Ag NPs reduced the length of shoots and roots of wheat in a dose-dependent manner. Furthermore, 2.5 mg/kg of the NPs increased branching in the roots of wheat (Triticum aestivum L.), thereby affecting plant biomass. Micron-sized (bulk) Ag particles (2.5 mg/kg) as well as Ag ions (63 μg Ag/kg) equivalent to the amount of soluble Ag in planted sand with Ag NPs (2.5 mg/kg) did not affect plant growth compared to control. In contrast, higher levels of Ag ions (2.5 mg/kg) reduced plant growth to a similar extent as the Ag NPs. Accumulation of Ag was detected in the shoots, indicating an uptake and transport of the metal from the Ag NPs in the sand. Transmision electron microscopy indicated that Ag NPs were present in shoots of plants with roots exposed to the Ag NPs or high levels of Ag ions. Both of these treatments caused oxidative stress in roots, as indicated by accumulation of oxidized glutathione, and induced expression of a gene encoding a metallothionein involved in detoxification by metal ion sequestration. Our findings demonstrate the potential effects of environmental contamination by Ag NPs on the metabolism and growth of food crops in a solid matrix.

Accumulation of Gold Nanoparticles in Brassic Juncea

Enzymatic digestion is proposed as a method for concentrating gold nanoparticles produced in plants. The mild conditions of digestion are used in order to avoid an increase in the gold particle size, which would occur with a high-temperature process, so that material suitable for catalysis may be produced. Gold nanoparticles of a 5–50-nm diameter, as revealed by transmission electron microscopy (TEM), at concentrations 760 and 1120 ppm Au, were produced within Brassica juncea grown on soil with 22–48 mg Au kg−1. X-ray absorption near edge spectroscopy (XANES) reveals that the plant contained approximately equal quantities of Au in the metallic (Au0) and oxidized (Au+1) states. Enzymatic digestion dissolved 55–60 wt% of the plant matter. Due to the loss of the soluble gold fraction, no significant increase in the total concentration of gold in the samples was observed. However, it is likely that the concentration of the gold nanoparticles increased by a factor of two. To obtain a gold concentration suitable for catalytic reactions, around 95 wt% of the starting dry biomass would need to be solubilized or removed, which has not yet been achieved.

Multifunctional inorganic-binding beads self-assembled inside engineered bacteria.

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.

Direct observation of the asphaltene structure in paving‐grade bitumen using confocal laser‐scanning microscopy

The structure of the asphaltene phase in the bitumen is believed to have a significant effect on its rheological properties. It has traditionally been difficult to observe the asphaltene phase in unaltered samples of bitumen. The maltenes are thought to form a continuous phase in which the asphaltenes are ‘dispersed’. In this study, confocal laser‐scanning microscopy (CLSM) operating in fluorescence mode was used to examine the structure of paving‐grade Safaniya and San Joaquin bitumen. The asphaltene fraction fluoresces in the 515–545 nm wavelength range when irradiated with light with a wavelength of 488 nm. The major advantages of CLSM are that the bitumen sample requires little pretreatment or preparation that may affect the original dispersion of asphaltenes and the bitumen is observed at ambient temperature and pressure. This reduces the possibility of producing images that are not representative of the original material. CLSM was able to show the distribution of maltene and asphaltene components in bitumen. The asphaltene aggregates in the bitumen were observed to be 2–7 µm in size and formed a dispersed ‘sol’ structure in the continuous maltene matrix rather than a network ‘gel’ structure. Surprisingly, the structure and fluorescence of the asphaltene phase does not appear to alter radically upon oxidative ageing. The structure of the asphaltene phase of an AR4000 San Joaquin bitumen was found to be more homogeneous than that of Safaniya bitumen, illustrating the range of structures that can be observed in bitumens by this method.

Modelling the dissolution of alumina powder in cryolite

Alumina dissolution in cryolite is a complex process at elevated temperatures. However, it is desirable to have a simple model for predictive purposes. In this study, several models for the dissolution of alumina powder in cryolite were formulated and compared with experimental results obtained using modified fast linear sweep voltammetry. As a first approximation the alumina was assumed to be smooth spheres which decrease in size as dissolution proceeds. The models were based on rate control by either first order chemical reaction at the alumina surface, heat transfer, or diffusion. The shape of the curves generated gave a reasonable fit to experimental data but the heat transfer and diffusion models appear to be the best. It is certain that these models can be used to correlate the experimental data on dissolution. It is envisioned that the actual mechanism may be a combination of both heat transfer and diffusion control. The models developed may also be applicable to other systems involving dissolving particles.

Studies of the microstructure of polymer-modified bitumen emulsions using confocal laser scanning microscopy

Polymer‐modified bitumen emulsions present a safer and more environmentally friendly binder for enhancing the properties of roads. Cationic bitumen emulsion binders containing polymer latex were investigated using confocal laser scanning microscopy. The latex was incorporated into the bitumen emulsion by using four different addition methods and all emulsions were processed with a conventional colloid mill. The emulsion binder films were studied after evaporation of the emulsion aqueous phase. We show how the microstructure and distribution of the polymer varies within the bitumen binder depending on latex addition method, and that the microstructure of the binder remains intact when exposed to elevated temperature. It was found that a distinctly fine dispersion of polymer results when the polymer is blended into the bitumen before the emulsifying process (a monophase emulsion). In contrast, bi‐phase emulsion binders produced by either post‐adding the latex to the bitumen emulsion, or by adding the latex into the emulsifier solution phase before processing, or by comilling the latex with the bitumen, water and emulsifier all resulted in a network formation of bitumen particles surrounded by a continuous polymer film. The use of emulsified binders appears to result in a more evenly distributed polymer network compared to the use of hot polymer‐modified binders, and they therefore have greater potential for consistent binder cohesion strength, stone retention and therefore improved pavement performance.

Leather structure determination by small-angle X-ray scattering (SAXS): cross sections of ovine and bovine leather.

SAXS has been applied to structural determination in leather. The SAXS beamline at the Australian Synchrotron provides 6 orders of magnitude dynamic range, enabling a rich source of structural information from scattering patterns of leather sections. SAXS patterns were recorded for q from 0.004 to 0.223 A(-1). Collagen d spacing varied across ovine leather sections from 63.8 nm in parts of the corium up to 64.6 nm in parts of the grain. The intensity of the collagen peak at q = 0.06 A(-1) varied by 1 order of magnitude across ovine leather sections with the high-intensity region in the corium and the low intensity in the grain. The degree of fiber orientation and the dispersion of the orientation has been quantified in leather. It is shown how the technique provides a wealth of useful information that may be used to characterize and compare leathers, skin, and connective tissue.

Biophysical characterization of ovine forestomach extracellular matrix biomaterials

Ovine forestomach matrix (OFM) is a native and functional decellularized extracellular matrix biomaterial that supports cell adhesion and proliferation and is remodeled during the course of tissue regeneration. Small angle X-ray scattering demonstrated that OFM retains a native collagen architecture (d spacing = 63.5 ± 0.2 nm, orientation index = 20°). The biophysical properties of OFM were further defined using ball-burst, uniaxial and suture retention testing, as well as a quantification of aqueous permeability. OFM biomaterial was relatively strong (yield stress = 10.15 ± 1.81 MPa) and elastic (modulus = 0.044 ± 0.009 GPa). Lamination was used to generate new OFM-based biomaterials with a range of biophysical properties. The resultant multi-ply OFM biomaterials had suitable biophysical characteristics for clinical applications where the grafted biomaterial is under load.

Collagen Fibril Orientation in Ovine and Bovine Leather Affects Strength: A Small Angle X-ray Scattering (SAXS) Study

There is a large difference in strength between ovine and bovine leather. The structure and arrangement of fibrous collagen in leather and the relationship between collagen structure and leather strength has until now been poorly understood. Synchrotron based SAXS is used to characterize the fibrous collagen structure in a series of ovine and bovine leathers and to relate it to tear strength. SAXS gives quantitative information on the amount of fibrous collagen, the orientation (direction and spread) of the collagen microfibrils, and the d-spacing of the collagen. The amount of collagen varies through the thickness of the leather from the grain to the corium, with a greater concentration of crystalline collagen measured toward the corium side. The orientation index (OI) is correlated strongly with strength in ovine leather and between ovine and bovine leathers. Stronger leather has the fibrils arranged mostly parallel to the plane of the leather surface (high OI), while weaker leather has more out-of-plane fibrils (low OI). With the measurement taken parallel to the animals backbone, weak (19.9 N/mm) ovine leather has an OI of 0.422 (0.033), stronger (39.5 N/mm) ovine leather has an OI of 0.452 (0.033), and bovine leather with a strength of (61.5 N/mm) has an OI of 0.493 (0.016). The d-spacing profile through leather thickness also varies according to leather strength, with little variation being detected in weak ovine leather (average=64.3 (0.5) nm), but with strong ovine leather and bovine leather (which is even stronger) exhibiting a dip in d-spacing (from 64.5 nm at the edges dropping to 62 nm in the center). This work provides a clear understanding of a nanostructural characteristic of ovine and bovine leather that leads to differences in strength.