ANSTO :: Browsing by Author "Wilde, KL"

Browsing by Author "Wilde, KL"

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ANSTO’s National Deuteration Facility: recent advancements and an overview on molecular deuteration capabilities for neutron applications
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Wilde, KL; Cagnes, MP; Duff, AP; Klenner, MA; Krause-Heuer, AM; Moir, M; Rekas, A; Russell, RA; Yepuri, NR; Darwish, TA
The National Deuteration Facility (NDF) at the Australian Nuclear Science and Technology Organisation (ANSTO) provides deuteration through both biological and chemical techniques for a diversity of molecules and applications and is the only facility of its type in the Southern Hemisphere with the specialised expertise and infrastructure for both biological and chemical molecular deuteration. Molecular deuteration of organic compounds and biomolecules significantly increases the options in complex structure function investigations using neutron scattering and reflectometry, nuclear magnetic resonance (NMR), mass spectrometry (MS) and other techniques. Deuteration (substitution of the naturally occurring hydrogen stable isotope deuterium (2H or D) for 1H (or H)) can provide contrast and improved resolution to assist investigations into the relationship between molecular structure and function of molecules of both biological and synthetic origin. By developing a suite of capabilities in both in vivo deuteration of biomolecules and chemical deuteration of small organic molecules, the NDF provides access to a broad range of deuterated molecules for research and industry. Variably deuterated proteins can be produced via recombinant expression in Escherichia coli and other microbial systems utilised to produce deuterated cellulose and cholesterol. By tailoring deuteration approaches with the ongoing development of chemical deuteration protocols for a broader range of molecular classes than available commercially, the NDF has increased the range of systems that can be investigated using deuterated molecules. Lipids, phospholipids (including head or tail or head/tail deuterated mono-unsaturated lipids such as POPC and DOPC), heterocyclics, aromatics, surfactants, ionic liquids, saturated and unsaturated fatty acids, sugars and match-out detergents have been deuterated. Common neutron applications include partially deuterated proteins for SANS experiments investigating multiprotein systems, neutron crystallography of perdeuterated proteins, neutron reflectometry of lipid bilayers systems and SANS using saturated lipid vesicles, or detergents amongst others. An overview and update on the NDF will be provided which will include details on the NDF User Program (e.g. information on the available modes of access), recent advancements in custom deuterated molecules available and brief highlights of deuterated molecule utilisation for neutron experiments at ANSTO’s Australian Centre for Neutron Scattering (ACNS). © 2020 The Authors.
The Australian National Deuteration Facility for structure function applications using neutrons
(International Conference on Neutron Scattering, 2017-07-12) Darwish, TA; Yepuri, NR; Heuer, AK; Duff, AP; Wilde, KL; Holden, PJ
There have been limited global initiatives in the field of molecular deuteration where the majority of these programs focus on biological deuteration of proteins and lipids, while more complex deuterated small molecules haven’t been widely available to the neutron community. This has limited the experiments that can be performed, and formed a bottle-neck for advancing the applications of neutron scattering. In this paper we will discuss the recent advancements and the impact of deuteration on the research outcomes achieved by using deuterated molecules produced by the National Deuteration Facility of the Australian Nuclear Science and Technology Organisation. Recent high-impact case studies in the fields of molecular electronics, structural biology, and biotechnology will be presented which reveal the exciting and diverse characterisation studies which are now available for the neutron community.
Bacterial nano-particle uptake under extreme white-beam irradiation conditions
(Australian Institute of Physics, 2009-02-05) Wilde, KL; Asquith, NL; Graham, SM; Holden, PJ; Stampfl, APJ; Kempson, I; Yang, BW; Hwu, YK
Not available
Demonstration of the use of Scenedesmus and Carteria biomass to drive bacterial sulfate reduction by Desulfovibrio alcoholovorans isolated from an artificial wetland
(Elsevier, 2003-10) Russell, RA; Holden, PJ; Wilde, KL; Neilan, BA
A major factor limiting application of bacterial sulfate reduction to removal of sulfate and heavy metals in wetland systems is the requirement to supply carbon and energy to drive the process. Primary production by aquatic plants and algae is a cheap option for driving sustainable bacterial sulfate reduction and most operational systems have relied on plants. The use of harvested, non-growing algal biomass to support bacterial sulfate reduction was investigated. Two genera of green algae, strains N9 and A3, were isolated from treatment cells from the Artificial Wetland Filter at the Ranger uranium mine (Northern Territory, Australia) which successfully removes UO22+, Mn2+ and nitrate, but little sulfate, from mine waters. These algae were identified as Carteria sp. and Scenedesmus sp. and were used as the sole carbon and energy source to enrich a sulfate-reducing mixed bacterial culture from the constructed wetland. Bacterial sulfate reduction supported solely by degradation of algal biomass was demonstrated at laboratory scale using both algae. In excess of 300 mg/L, sulfate was reduced in 17 days following an initial period of approximately 8 days during which sulfate levels did not decrease. The amount and rate of reduction was shown to be dependent on the concentration of algal biomass added. Carteria algae at low concentration showed reduction earlier; however, yields at higher concentration were affected by unknown inhibition. Scenedesmus strain N9 produced a maximum specific yield of 94.3 g of sulfate reduced per gram biomass added compared with 43.5 for Carteria strain A3. Sequence analysis of the 16S rRNA gene of members of the bacterial consortium indicated that the sulfate-reducing bacteria (SRB) showed highest homology (98.5%) with Desulfovibrio alcoholovorans. A second bacterium, which showed homologies of 91–92% with organisms of the Clostridial assemblage, was also present in the culture and represents a new species, or possibly a new genus. Crown Copyright 2003 Published by Elsevier B.V.
Deuteration for biological SANS: case studies, success and challenges in chemistry and biology
(Elsevier, 2022-11) Duff, AP; Cagnes, MP; Darwish, TA; Krause-Heuer, AM; Moir, M; Recsei, C; Rekas, A; Russell, RA; Wilde, KL; Yepuri, NR
Small angle neutron scattering is a powerful complementary technique in structural biology. It generally requires, or benefits from, deuteration to achieve its unique potentials. Molecular deuteration has become a mature expertise, with deuteration facilities located worldwide to support access to the technique for a wide breadth of structural biology and life sciences. The sorts of problems well answered by small angle scattering and deuteration involve large (> 10 Å) scale flexible movements, and this approach is best used where high-resolution methods (crystallography, NMR, cryo-EM) leave questions unanswered. This chapter introduces deuteration, reviewing biological deuteration of proteins, lipids and sterols, and then steps through the ever-expanding range of deuterated molecules being produced by chemical synthesis and enabling sophisticated experiments using physiologically relevant lipids. Case studies of recent successful use of deuteration may provide illustrative examples for strategies for future experiments. We discuss issues of nomenclature for synthesised molecules of novel labeling and make recommendations for their naming. We reflect on our experiences, with cost associated with achieving an arbitrary deuteration level, and on the benefits of experimental co-design by user scientist, deuteration scientist, and neutron scattering scientist working together. Although methods for biological and chemical deuteration are published in the public domain, we recommend that the best method to deuterate is to engage with a deuteration facility. © 2022 Elsevier
The effect of the pH on the toxicity and accumulation of Cu and Zn in a tropical freshwater alga (Chlorella sp)
(Royal Australian Chemical Institute & Australasian Society of Ecotoxicology, 2002-07-21) Wilde, KL; Markich, SJ; Stauber, JL; Franklin, NM
As part of a larger study to test the biotic ligand model (BLM) with unicellular algae, the effect of pH (5.5, 6.5 and 7.5) on the toxicity and accumulation of Cu and Zn in the freshwater alga Chorella sp was investigated.
The electronic structure of S-layer proteins from Lactobacillus brevis
(IEEE, 2008-07-28) Graham, SM; Asquith, NL; Wilde, KL; Short, KT; Holden, PJ; Stampfl, APJ; Holmes, AJ; Ruys, AJ; Stojanov, P; Riley, JD; Fang, LJ; Yang, YW; Hwu, YK
The valence electronic structure of the S-layer of Lactobacillus brevis is determined using synchrotron-based photoelectron spectroscopy and soft X-ray absorption spectroscopy. Spectra are compared to experimental work on amino-acids and S-layers of Bacillus sphaericus. While it is indeed possible to identify energy levels with those of natural amino-acids, distinct energy shifts are indeed observed which cannot be reconciled using such simple comparisons. Furthermore a strong nitrogen signal observed in both the occupied and unoccupied energy levels suggests that the Lactobacillus brevis protein is amine-terminated. A discussion of the surface of this protein is given. © 2008 IEEE
Final report on a field study of soil-to-plant transfer of radioactive caesium, strontium and zinc in tropical Northern Australia to the IAEA/FAO/IUR CRP on classification of soils systems on the basis of transfer factors of radionuclides from soil to reference plants
(Australian Nuclear Science and Technology Organisation, 2003-09) Twining, JR; Shotton, P; Tagami, K; Payne, TE; Itakura, T; Russell, RA; Wilde, KL; McOrist, GD; Wong, HKY
Soil-to-plant radionuclide transfer factors for cesium (134Cs), strontium (85Sr) and zinc (65Zn) into sorghum and mung plants grown in tropical Australia have been determined over a four-year study period. The crops were grown on two types of red earth soils. Transfer factors for Cs and Sr are not substantially different from the expected values based on previous studies, reported in the general literature and compiled in the IUR database, mainly performed within temperate climates. In contrast, the values for zinc (Zn) are more than an order of magnitude greater than anticipated. Most of the radioactivity added to the soils has been retained in the top 5 cm of both soils. There has been a general decline in soil-to-plant transfer of Cs and Zn as time has increased.
High yield expression and efficient purification of deuterated human protein galectin-2
(Elsevier, 2012-07-01) Chen, XJ; Wilde, KL; Wang, H; Lake, V; Holden, PJ; Middelberg, APJ; He, LH; Duff, AP
Structural studies of biological macromolecules often require deuterated proteins, necessitating an effective bioprocessing strategy for high yield deuteration and purification. The fermentation and bioseparation studies reported here concern deuterated human protein galectin-2 mutant C57M (hGal-2), a protein showing potential for therapeutic applications. Using the vector pET-28a and a defined D2O based minimal medium with glycerol as the sole carbon source and kanamycin for selection, we have demonstrated that a high density of Escherichia coli expressing deuterated protein at a bench bioreactor scale (7L) can be achieved, with due attention to prevention of oxygen limitation. Yields achieved were 58 g\L biomass (wet weight) containing 0.7 g/L hGal-2. Affinity chromatography and ion-exchange chromatography were combined to achieve high purity as well as removal of hGal-2 aggregates, giving an overall yield of 1200 mg deuterated hGal-2. The deuterated hGal-2 was characterized and compared with the non-deuterated protein by size exclusion chromatography (SEC), HPLC, N-terminal sequencing, mass spectrometry (MS) and a dot blot immunoassay, showing that deuteration and subsequent purification did not impact the lactose binding and antibody recognition abilities of hGal-2. MS for both intact and trypsin-digested hGal-2 demonstrated that the extent of labeling of non-exchangeable hydrogen atoms by deuterium was (66 +/- 1)%, which provides sufficient contrast variation for structural studies using small angle neutron scattering. The fermentation and bioseparation method established in this work can be applied to process other deuterated proteins with high yield and purity, opening the way to advanced structural studies. © Institution of Chemical Engineers 2014.
In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate
(Springer Nature, 2008-05-15) Russell, RA; Holden, PJ; Wilde, KL; Garvey, CJ; Hammerton, KM; Foster, LJR
The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions. © 2008 Crown Copyright
Investigation of the phase morphology of bacterial PHA inclusion bodies by contrast variation SANS
(Elsevier, 2006-11-15) Russell, RA; Holden, PJ; Garvey, CJ; Wilde, KL; Hammerton, KM; Foster, LJR
Under growth-limiting conditions, many bacteria are able to metabolise excess organic acids into polyhydroxyalkanoates (PHA) and store these polymers as intracellular inclusions until the return of favourable conditions. Various models have been proposed for the macromolecular organisation of the boundary layer Surrounding the polymer, and contrast-variation small-angle neutron scattering (SANS) was used to study its organisation. Inclusions formed by Pseudomonas oleovorans under hydrogenating conditions showed lowest scattering intensity at ca. 20% D2O. The inclusions consist of protein and membrane lipids in the boundary layer and polyhydroxyoctanoate (lipid) in the inclusion body. At 20% D2O the contributions of lipids were contrast matched with the solvent, indicating that lipids contributed the bulk of the scattering intensity observed at other D2O/H2O ratios. These results are inconsistent with a model of the boundary layer which proposed outer and inner layers of crystalline protein lattice sandwiching a membrane lipid membrane layer [E.S. Stuart, R.W. Lenz, R.C. Fuller, Can J Microbiol 41(Suppl 1) (1995) 84 93], and is more consistent with a model consisting of a lipid monolayer containing embedded proteins [U. Pieper-furst, M.H. Madkour, F. Mayer, A. Steinbuchel, J. Bacteriol. 176 (1994) 4328-4337.] By altering the H/D content of the precursors, we were able to collect SANS data from preparations of both deuterated and H/D copolymer inclusions, where initial PHA produced was hydrogenated followed by deuteration. Deuterated inclusions showed minimum intensity above 90% D2O/H2O whereas the sequentially produced copolymer (assumed to be in a core/shell arrangement) displayed minimum scattering some 20% lower, which is consistent with the increased hydrogenation of the boundary layer expected from its synthesis during supply of hydrogenated followed by deuterated precursors. © 2006, Elsevier Ltd
Production and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions
(Elsevier, 2007-11-01) Russell, RA; Holden, PJ; Wilde, KL; Hammerton, KM; Foster, LJR
This work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems. © 2007, Elsevier Ltd.
A robust and reliable method for high yield deuterated recombinant protein production using Escherichia coli BL21.
(International Conference on Neutron Scattering, 2017-07-12) Duff, AP; Wilde, KL; Rekas, A; Lake, V; Holden, PJ
We have developed a method that has proven highly reliable for the deuteration of a broad range of proteins by recombinant expression in Escherichia coli BL21. Typical biomass yields are 40-80 g/L wet weight, yielding 50-400mg/L purified protein. This method uses a simple, relatively inexpensive defined medium, and routinely results in a high yield expression without need for optimisation. The key elements are: very tight control of expression, careful starter culture adaption steps, and strict maintenance of aerobic conditions ensuring exponential growth. Temperature is reduced as required to prevent biological oxygen demand exceeding maximum aeration capacity. Glycerol is the sole carbon source. We have not encountered an upper limit for the size of proteins that can be expressed, achieving excellent expression for proteins from 7-112 kDa and the quantity produced at 1L scale ensures that no SANS, NMR or neutron crystallography experiment is limited by the amount of deuterated material. Where difficulties remain, these tend to be cases of protein solubility exacerbated by high protein concentration and slightly increased stickiness of proteins in D O. There are some very few cases in which we have been unable to express a protein by our method despite unlabelled expression being reliable in rich media using induction at low OD. Few proteins tested have not expressed in deuterated medium despite unlabelled expression being reliable.
Seasonal changes of redox potential and microbial activity in two agricultural soils of tropical Australia: some implications for soil-to-plant transfer of radionuclides
(Elsevier, 2004-05-14) Twining, JR; Zaw, M; Russell, RA; Wilde, KL
Very little is known of the factors controlling soil-to-plant transfer of radionuclides in tropical environments. As part of an IAEA/FAO coordinated research project (CRP) designed to elucidate some of those factors, near-surface samples of two agricultural red-earth soils (Blain and Tippera) were collected from a study site in the Northern Territory. The climate is tropical monsoonal with crops being grown over the wet season from December to March/April. It is important to understand soil variables that may be related to this dramatic seasonality. In this investigation, soil redox state and microbial populations were assessed before and after the growing season with a view to generating hypotheses for future evaluation. The X-ray absorption near edge structure (XANES) technique was used to determine overall changes in the solid-state redox speciation of Fe and Mn in soils across the growing period. Fe speciation did not change but approximately 10% of the total Mn was oxidised from Mn(II) to Mn(III) and Mn(IV) in both soils between October 1999 and April 2000. An apparent disconnect between Fe and Mn was not unexpected given the >10 times higher concentration of Fe in the soils compared with Mn. These results have implications for the bioavailability of redox sensitive radionuclides such as Tc and Pu. Similarly, microbial population estimates were derived before and after the growing period. Total bacterial populations did not vary from 106 to 107 colonies per gram. Fungal populations increased over the growing season from 3–6×105 to 1–4×106 colonies per gram of soil. Fungi have the potential to decrease soil pH and hence increase the bioavailability of radionuclides such as Cs. In addition, fungi act to facilitate plant nutrition. This could lead to enhanced accumulation of nutrient analogues (e.g. Sr and Ra for Ca; Tc for Mn), but this effect may be masked by improved biomass production. © 2004 Elsevier Ltd
Solid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: a well-ordered β-sheet core amidst structural heterogeneity
(Wiley-VCH Verlag GMBH, 2012-01-01) Morris, VK; Linser, R; Wilde, KL; Duff, AP; Sunde, M; Kwan, AH
GrEASy fibrils: Hydrophobins are fungal proteins that assemble into an amphipathic fibrillar monolayer with amyloid properties and a hydrophobic face as water-resistant as Teflon. Solid-state NMR studies on EAS hydrophobin fibrils reveal direct evidence of a partial molecular rearrangement on assembly and an ordered β-sheet-rich core in the context of a whole protein in this functional amyloid. © 2012, Wiley-VCH Verlag GmbH & Co. KGaA
Structure of biomimetic casein micelles: critical tests of the hydrophobic colloid and multivalent-binding models using recombinant deuterated and phosphorylated β-casein
(SSRN, 2023-10-24) Raynes, JK; Mata, JP; Wilde, KL; Kelly, SM; Holt, C
Milk contains high concentrations of amyloidogenic casein proteins and is supersaturated with respect to crystalline calcium phosphates such as apatite. Nevertheless, the mammary gland normally remains unmineralized and free of amyloid. Unlike κ-casein, β- and αS-caseins are highly effective mineral chaperones that prevent ectopic and pathological calcification of the mammary gland. Milk invariably contains a mixture of two to five different caseins that act on each other as molecular chaperones. Instead of forming amyloid fibrils, several thousand caseins and hundreds of nanoclusters of amorphous calcium phosphate combine to form fuzzy complexes called casein micelles. To understand the biological functions of the casein micelle its structure needs to be understood better than at present. The location in micelles of the highly amyloidogenic k-casein is disputed. In traditional hydrophobic colloid models, it, alone, forms a stabilizing surface coat that also determines the average size of the micelles. In the recent multivalent-binding model, κ-casein is present throughout the micelle, in intimate contact with the other caseins. To discriminate between these models, a range of biomimetic micelles was prepared using a fixed concentration of the mineral chaperone b-casein and nanoclusters of calcium phosphate, with variable concentrations of κ-casein. A biomimetic micelle was also prepared using a highly deuterated and in vivo phosphorylated recombinant β-casein with calcium phosphate and unlabelled κ-casein. Neutron and X-ray scattering experiments revealed that κ-casein is distributed throughout the micelle, in quantitative agreement with the multivalent-binding model but contrary to the hydrophobic colloid models. © 2023 The Authors

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