Revisiting the interpretation of casein micelle SAXS data.

An in-depth, critical review of model-dependent fitting of small-angle X-ray scattering (SAXS) data of bovine skim milk has led us to develop a new mathematical model for interpreting these data. Calcium-edge resonant soft X-ray scattering data provides unequivocal evidence as to the shape and location of the scattering due to colloidal calcium phosphate, which is manifested as a correlation peak centred at q = 0.035 Å(-1). In SAXS data this feature is seldom seen, although most literature studies attribute another feature centred at q = 0.08-0.1 Å(-1) to CCP. This work shows that the major SAXS features are due to protein arrangements: the casein micelle itself; internal regions approximately 20 nm in size, separated by water channels; and protein structures which are inhomogeneous on a 1-3 nm length scale. The assignment of these features is consistent with their behaviour under various conditions, including hydration time after reconstitution, addition of EDTA (a Ca-chelating agent), addition of urea, and reduction of pH.

Solving the mystery of the internal structure of casein micelles.

The interpretation of milk X-ray and neutron scattering data in relation to the internal structure of the casein micelle is an ongoing debate. We performed resonant X-ray scattering measurements on liquid milk and conclusively identified key scattering features, namely those corresponding to the size of and the distance between colloidal calcium phosphate particles. An X-ray scattering feature commonly assigned to the particle size is instead due to protein inhomogeneities.

Chemical techniques for pretreating and regenerating active slag filters for improved phosphorus removal.

Active slag filters are an emerging technology for removing phosphorus (P) from wastewaters. Recent research revealed that adsorption onto Fe oxides/oxyhydroxides at near-neutral pH and oxidizing Eh is the key mechanism of P removal by melter slag filters. Currently, filter lifespan is limited by available adsorption sites. This study examined whether the performance and longevity of active filters could be improved via chemical treatment to create additional reactive sites as well as regenerate exhausted ones. Fresh original melter slag as well as slag from an exhausted full-scale filter was tested. Chemical reagents that could manipulate the pH/Eh of the slag granule surfaces and potentially activate them for further P removal were used, namely hydrochloric acid (HCI), sodium hydroxide (NaOH) and sodium dithionite (Na2S2O4). Waste stabilization pond effluent was then applied to the treated slag to assess the effectiveness of the treatments at improving P removal. Fresh slag treated with Na2S204 and HCl, respectively, retained 1.9 and 1.4 times more P from the effluent than the untreated fresh slag. These reagents were even more effective at regenerating the exhausted slag, increasing total retained P by a factor of 13 and six, respectively, compared with untreated slag. Sodium hydroxide was ineffective at increasing P removal. The higher P retention by the 'treated exhausted slag' compared with the 'treated fresh media' indicates that adsorption sites on melter slag filters become increasingly reactive with time. This research is the first study to provide evidence that P retention by active slag filters can be increased by both (1) chemical pre treatment and (2) chemical post-treatment once their P removal is exhausted, thereby potentially transforming them from a single use system to a more viable, reusable treatment technology.

Assessment of physical techniques to regenerate active slag filters removing phosphorus from wastewater.

Active slag filters are an emerging technology for removing phosphorus (P) from wastewater. Currently, the lifespan of these filters is limited by their available reactive sites. An increasing number of researchers suggest that drying active filters can renew their P removal capacity, although there has only been one research paper that has achieved regeneration. Hence, this study investigated techniques to regenerate the P removal efficiency of exhausted melter slag filter media which had successfully treated effluent for 5 years in the field before becoming inactive. Several techniques were performed to expose fresh surface sites on the slag, including: (1) drying; (2) agitation; and (3) crushing of the slag granules. Crushing resulted in the best regeneration of P removal efficiency by the slag, however, after just 2 months the renewed P removal efficiency of the crushed slag fell back to a similar level to that exhibited by exhausted slag. Furthermore, none of the other physical methods caused long-term rejuvenation of the exhausted slag to remove P from effluent. Scanning electron microscopy revealed that none of the regeneration techniques exposed substantial new phosphorus adsorption sites on the slag granules. These findings therefore challenge the validity of the existing dogma that active slag filters can be effectively regenerated by simple physical treatments, indicating further research is required to optimise active filter performance.

Effect of collagen packing and moisture content on leather stiffness.

Applications for skin derived collagen materials, such as leather and acellular dermal matrices, usually require both strength and flexibility. In general, both the tensile modulus (which has an impact on flexibility) and strength are known to increase with fiber alignment, in the tensile direction, for practically all collagen-based tissues. The structural basis for flexibility in leather was investigated and the moisture content was varied. Small angle X-ray scattering was used to determine collagen fibril orientation, elongation and lateral intermolecular spacing in leather conditioned by different controlled humidity environments. Flexibility was measured by a three point bending test. Leather was prepared by tanning under biaxial loading to create leather with increased fibril alignment and thus strength, but this treatment also increased the stiffness. As collagen aligns, it not only strengthens the material but it also stiffens because tensile loading is then applied along the covalent chain of the collagen molecules, rather than at an angle to it. Here it has been shown that with higher moisture content greater flexibility of the material develops as water absorption inside collagen fibrils produces a larger lateral spacing between collagen molecules. It is suggested that water provides a lubricating effect in collagen fibrils, enabling greater freedom of movement and therefore greater flexibility. When collagen molecules align in the strain direction during tanning, leather stiffens not only by the fiber alignment itself but also because collagen molecules pack closer together, reducing the ability of the molecules to move relative to each other.

Data on collagen structures in leather with varying moisture contents from small angle X-ray scattering and three point bend testing.

The data presented in this article are related to the research article entitled "Effect of collagen packing and moisture content on leather stiffness" (Kelly et al., 2018). This article describes how moisture content affects collagen packing and leather stiffness. Structural changes were experimentally introduced into ovine leather through biaxial strain during tanning (׳stretch tanning׳). Leather samples produced normally without strain (׳non-stretch tanned׳) and those produced by stretch tanning, were conditioned in a range of relative humidity environments and then analysed by small angle X-ray scattering and three point bend testing. The collagen D-spacing, lateral intermolecular spacing and flexural properties were measured under these varying moisture contents.

Investigation of the effects of fine structure on the nanomechanical properties of pectin.

Pectin is an important structural polysaccharide found in the cell walls of all land plants. While in detail its composition and its organization in muro are complex, it is predominantly a copolymer of galacturonic acid and its methylesterified counterpart. Previous single-molecule stretching studies carried out on a sparsely methylesterified pectin sample indicated the importance of force-induced conformational transitions of the pyranose ring during extension, and the possible biological role of such transitions was discussed. More heavily methylesterified samples are better biomimetic models of the polymeric components as found in the plant cell wall, in particular being less restricted by the shackles of the significant intermolecular interactions expected to constrain the behavior of bare galacturonic acid sequences. Density functional theory calculations revealed that upon extending galacturonic acid monomers, whether methylesterified or not, the initial ((4)C1) chair structure is transformed to a ((3)S5) skew boat and that subsequently upon further elongation, via an intermediate inverted skew boat ((5)S3), the inverted chair ((1)C4) is reached. Experimentally, the force-extension curve of highly methylesterified pectin was found to be solvent dependent in the same manner as the un-esterified sample, indicating that minimal changes in the strength of interring hydrogen bonding result from such a substitution, and finally, as only subtle changes in the force-extension behavior of pectin resulted from changes in the degree of methylesterification, previous speculations about the role of force-induced transformations in vivo are supported.

Model for stretching elastic biopolymers which exhibit conformational transformations.

We derive an expression that represents the physical behavior of a polysaccharide molecule as it is stretched from the entropic region, through one or more ring conformational transformations, into the Hookean regime. The model adapts existing models in order to accommodate one or more force-induced conformational transformations of the glycan rings and is based on the concept of equilibrium between the clicked (longer conformers) and unclicked states. This equilibrium is determined by the Gibbs energy difference between these two states which is perturbed in favor of the clicked states by the force applied to the molecule. The derived expression is used to generate force-extension curves for model polymers and can illustrate the effect of the Gibbs energy for each transformation on the shape of these curves. It is also used to fit the force-extension curves of polysaccharides to obtain the Gibbs energy differences between the conformers. Good agreement was found between this model and experimental data on carboxymethylamylose, dextran, alginate, and pectin.

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.

Collagen fibril orientation and tear strength across ovine skins.

Variability of physical properties across hides and skins requires careful consideration when manufacturing goods from leather. Therefore, an understanding of the extent of this variation and its nanostructural basis is useful. Tear strength tests were performed on ovine leather from a grid of 81 positions on skins. Synchrotron small-angle X-ray scattering measurements were made from three positions on the skin, from 26 skins. The X-ray structural measurements are compared with tear strengths of the samples. It is found that the thickness normalized tear strength does not vary greatly between different positions on the skin, in contrast to bovine hides. There is more variation between different skins than within the same skin. The collagen fibril orientation and orientation index, which has previously been shown to be correlated with tear strength, do not vary significantly between the different sampling positions in ovine skins. The collagen fibril orientation varies through the thickness of the skin in a consistent way. The consistency of collagen orientation in ovine leather between different positions on the skin is in marked contrast to bovine hides and informs the use of ovine leather for manufacturing applications.

Effects of redox potential and pH changes on phosphorus retention by melter slag filters treating wastewater.

The release of phosphorus (P) and iron (Fe) from a melter slag filter in solutions of varying Eh and pH was examined. The filter had been removing P from waste stabilization pond effluent for several years. The study revealed that the highest P (95% of total P) and Fe (25% of total Fe) release from the slag occurred in the solution with the lowest Eh (-400 mV, relative to the standard hydrogen electrode, SHE) and lowest pH (4.9). Solutions with high pH (9.1) also proved favorable for P release (20 to 40% of total P) from the slag, at both reducing (-400 mV) and oxidizing (+300 mV) Eh. By contrast, solutions with pH 4.9 and 6.7 and oxidizing Eh (+300 mV) liberated the lowest P and Fe contents into the aqueous phase (<1% for both elements). The findings showed that Eh and pH are important parameters affecting P release from slag filters. At low Eh and low pH, P is released due to the dissolution of Fe oxides/oxyhydroxides, as supported by scanning electron microscopy (SEM)/energy dispersive spectrometry (EDS). At high pH, P is desorbed from negatively charged Fe oxide/oxyhydroxide surfaces. The results of this investigation are pertinentto the design and operation of melter slag filters that treat all forms of P-rich waters, such as wastewater, stormwater, and farm runoff. The study demonstrated that P retention by melter slag filters is optimal in water bodies characterized by near-neutral pH and oxidizing Eh because these conditions favor P adsorption onto Fe oxides/oxyhydroxides.

Structural basis for rodlet assembly in fungal hydrophobins.

Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a beta-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar beta-structures that continue to be discovered in the protein world.

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 micro m 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.

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.