ANSTO :: Browsing by Author "Duff, AP"

Browsing by Author "Duff, AP"

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Annexin V-containing cubosomes for targeted early detection of apoptosis in degenerative retinal tissue
(Royal Society of Chemistry, 2018-10-26) Ding, Y; Chow, SH; Liu, GS; Wang, B; Lin, TW; Hsu, HY; Duff, AP; Le Brun, AP; Shen, HH
New drug delivery materials targeting damaged ocular tissues are of particular interest. In this work, we have formulated annexin/phosphatidylserine/phytantriol and annexin/phosphatidylserine/monoolein cubosomes based on incorporation of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (PS) lipid and annexin V (ANX) protein with phytantriol (Phy) and monoolein (MO) respectively. The incorporation of ANX is important because it can be used as a diagnostic tool for in vivo apoptosis detection due to its high affinity to phosphatidylserine in the presence of Ca2+. We have also prepared PS–Phy and PS–MO cubosomes without ANX as a comparison, and characterized them using dynamic light scattering, cryo-TEM images and small-angle X-ray scattering, showing that PS–Phy cubosomes have greater chemical stability, and that ANX–PS–Phy cubosomes have the potential for in vivo drug delivery. In addition, we have reconstituted an apoptotic biomimetic membrane on a surface to gain insights into cubosome–bilayer interactions using a quartz-crystal microbalance and neutron reflectometry. The neutron reflectivity data reveal that there is exchange of materials between the biomimetic apoptotic bilayer and ANX–PS–Phy cubosomes, with an accumulation of ANX between the membrane and cubosomes possibly being the reason for the reduced cytotoxicity of ANX–PS–Phy cubosomes. A rat model of laser-induced choroidal neovascularization showed that ANX–PS–Phy cubosomes specifically targeted apoptotic cells in vivo. We propose that ANX–PS–Phy cubosomes are a potential candidate for ocular drug delivery for eye diseases. © The Royal Society of Chemistry 2018
The ANSTO contribution to a project to provide experimental standards for SAS profile prediction
(Australian Institure of Nuclear Sicence and Engineering (AINSE), 2020-11-11) Duff, AP; Kirby, N; Ryan, T; Trewhella, J; Whitten, AE; Wood, K
The program CRYSOL1, was the first method developed to rapidly calculate small-angle scattering (SAS) profiles from atomic coordinates of biomolecules. It was a major breakthrough, providing the missing link between high-resolution structures and solution SAS data. The importance of this breakthrough is evident in the fact that decades later, alternate methods continue to be published with various claims of improvement. To date, each of the alternate methods published have been validated using different data sets and models. A consensus set of high quality data would be of considerable value in benchmarking the different approaches. In order to evaluate different approaches to including the hydration layer contribution to the SAS profile, it is desirable to have data obtained using X-rays (SAXS) and neutrons (SANS), the latter in H2O and D2O, as the hydration layer contribution differs significantly for each of these measurements. To develop a consensus set of high quality data, an international project involving the efforts of 37 researchers from 11 different X-ray and 3 different neutron scattering facilities across Asia, Europe and North Ⓒ The Authors.
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.
Binding and dynamics demonstrate the destabilization of ligand binding for the S688Y mutation in the NMDA receptor GluN1 subunit
(MDPI, 2023-05-15) Chen, JZ; Church, WB; Bastard, K; Duff, AP; Balle, T
Encephalopathies are brain dysfunctions that lead to cognitive, sensory, and motor development impairments. Recently, the identification of several mutations within the N-methyl-D-aspartate receptor (NMDAR) have been identified as significant in the etiology of this group of conditions. However, a complete understanding of the underlying molecular mechanism and changes to the receptor due to these mutations has been elusive. We studied the molecular mechanisms by which one of the first mutations within the NMDAR GluN1 ligand binding domain, Ser688Tyr, causes encephalopathies. We performed molecular docking, randomly seeded molecular dynamics simulations, and binding free energy calculations to determine the behavior of the two major co-agonists: glycine and D-serine, in both the wild-type and S688Y receptors. We observed that the Ser688Tyr mutation leads to the instability of both ligands within the ligand binding site due to structural changes associated with the mutation. The binding free energy for both ligands was significantly more unfavorable in the mutated receptor. These results explain previously observed in vitro electrophysiological data and provide detailed aspects of ligand association and its effects on receptor activity. Our study provides valuable insight into the consequences of mutations within the NMDAR GluN1 ligand binding domain. © 2023 by the authors. Licensee MDPI, Basel, Switzerland. Open access CC BY.
Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release
(Cell Press, 2012-08-08) Taylor, JEN; Chow, JYH; Jeffries, CM; Kwan, AH; Duff, AP; Hamilton, WA; Trewhella, J
Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ∼70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca2+ or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca2+-signaling during viral processing. © 2012 Biophysical Society.
CLIC proteins, ezrin, radixin, moesin and the coupling of membranes to the actin cytoskeleton: A smoking gun?
(Elsevier, 2014-02-01) Jiang, L; Phang, JM; Yu, J; Harrop, SJ; Sokolova, AV; Duff, AP; Wilik, KE; Alkhamici, H; Breit, SN; Valenzuela, SM; Brown, LJ; Curmi, PMG
The CLIC proteins are a highly conserved family of metazoan proteins with the unusual ability to adopt both soluble and integral membrane forms. The physiological functions of CLIC proteins may include enzymatic activity in the soluble form and anion channel activity in the integral membrane form. CLIC proteins are associated with the ERM proteins: ezrin, radixin and moesin. ERM proteins act as cross-linkers between membranes and the cortical actin cytoskeleton. Both CLIC and ERM proteins are controlled by Rho family small GTPases. CLIC proteins, ERM and Rho GTPases act in a concerted manner to control active membrane processes including the maintenance of microvillar structures, phagocytosis and vesicle trafficking. All of these processes involve the interaction of membranes with the underlying cortical actin cytoskeleton. The relationships between Rho GTPases, CLIC proteins, ERM proteins and the membrane:actin cytoskeleton interface are reviewed. Speculative models are proposed involving the formation of localised multi-protein complexes on the membrane surface that assemble via multiple weak interactions. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. © 2013, Elsevier B.V.
(Corrected) Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release
(Elsevier, 2023-05-02) Taylor, JEN; Chow, JYH; Jeffries, CM; Kwan, AH; Duff, AP; Hamilton, WA; Trewhella, Jill
(Biophysical Journal 103, August 2012; 541–549) The authors identified an omission in this article. The figure legend for Figure S2 should include the following: “The model displayed here includes full-length calcium-bound CaM and the matrix protein sequence spanning amino acids 1–113; i.e., it does not include the flexible C-terminal tail (amino acids 114–133).” None of the article's conclusions are affected by this omission. In addition, after publication of the article, the SAXS and SANS data and modeling for this article were deposited in the Small Angle Scattering Biological Data Bank (SASBDB; https://www.sasbdb.org/) under accession code SASDKR3. Finally, the first author's name should appear as “James E. N. Taylor” rather than “James E. Taylor.”
Correlation of thermostability and conformational changes of catechol 2, 3-dioxygenases from two disparate micro-organisms
(Elsevier Science BV, 2013-10-01) Sokolova, AV; Huang, SL; Duff, AP; Gilbert, EP; Li, WH
We have investigated the structure of recombinant catechol 2, 3-dioxygenase (C23O) purified from two species in which the enzyme has evolved to function at different temperature. The two species are mesophilic bacterium Pseudomonas putida strain mt-2 and thermophilic archaea Sulfolobus acidocaldarius DSM639. Using the primary sequence analysis, we show that both C23Os have only 30% identity and 48% similarity but contain conserved amino acid residues forming an active site area around the iron ion. The corresponding differences in homology, but structural similarity in active area residues, appear to provide completely different responses to heating the two enzymes. We confirm this by small angle X-ray scattering and demonstrate that the overall structure of C23O from P. putida is slightly different from its crystalline form whereas the solution scattering of C230 from S. acidocaldarius at temperatures between 4 and 85 degrees C ideally fits the calculated scattering from the single crystal structure. The thermostability of C230 from S. acidocaldarius correlates well with conformation in solution during thermal treatment. The similarity of the two enzymes in primary and tertiary structure may be taken as a confirmation that two enzymes have evolved from a common ancestor. © 2013, Elsevier Ltd.
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 dynamics and interactions of Scs proteins from Proteus mirabilis
(International Union of Crystallography, 2021-08-14) Whitten, AE; Furlong, E; Choudhury, F; Kurth, F; Duff, AP; Martin, J
Correct formation of disulfide bonds is critical to the folding of a wide variety of proteins. Bacterial virulence factors are one class of proteins containing disulfide bonds, thus, an approach to disarm virulent bacterial might involve shutting down the machinery involved in the formation of disulfide bonds. The suppressor of copper sensitivity (Scs) proteins form part of the disulfide bond forming machinery in bacteria, and it is hoped that determining the structure of molecules such as this may lead to the development of new classes of antibiotics. There are four Scs proteins (ScsA, B, C and D) present in numerous Gram-negative bacteria, and few have been structurally characterised. In this work we show that the ScsC protein from Proteus mirabilis is trimeric and flexible, where the high level of flexibility is afforded by a glutamine rich motif. We also show that the protein interacts with ScsB and that this interaction rigidifies the ScsC protein © The Authors
Effects of mutations in the NMDA receptor GluN1 subunit on binding and dynamics: a computational approach
(International Union of Crystallography, 2021-08-14) Chen, Z; Church, WB; Bastard, K; Duff, AP; Balle, T
N-methyl-D-aspartate receptors (NMDARs) are central to the pathophysiology of neurodegenerative diseases such as schizophrenia [1], however despite significant structural insights of the receptor [2,3,4,5] the importance of mutations in the NMDAR have been poorly described in the literature. Here we present molecular dynamics simulation data combined with modelling and binding free energy calculations to outline the effects of mutations [6] in the GluN1 subunit of the NMDAR on agonist binding affinity and ligandreceptor interactions. Our data demonstrates the changes caused by the positioning of an introduced tyrosine residue at the binding pocket and its associated changes in the conformation upon ligand binding. Furthermore, molecular dynamics simulations demonstrate the changes in ligand environment in the ligand-receptor complex leading to a loss of key interactions and an associated instability of the bound complex. Lastly, binding free energy calculations show that it is no longer energetically favourable for ionic interactions to form and an associated overall increase in Gibbs free energy for ligand binding. These data are important in explaining the changes in behaviour for mutations in the GluN1 ligand binding region and are consistent with previously reported experiments [7]. We are also pursuing experimental approaches to further understand the action of ligand binding. © 2021 The Authors
An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase
(Springer Nature, 2017-01-18) Golden, EA; Yu, LJ; Meilleur, F; Blakeley, MP; Duff, AP; Karton, A; Vrielink, A
The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.© The Authors - Creative Commons Attribution 4.0 International License
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.
Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide
(National Academy of Sciences, 2012-06-19) Christie, MP; Whitten, AE; King, GJ; Hu, SH; Jarrott, RJ; Chen, KE; Duff, AP; Callow, P; Collins, BM; James, DE; Martin, JL
When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode. © 2012, National Academy of Sciences.
New contrast options - diversity and specifity of deuteration
(Australian Institute of Nuclear Science and Engineering (AINSE), 2018-11-19) Holden, PJ; Cagnes, MP; Davydova, N; Duff, AP; Howard, JK; Krause-Heuer, AM; Yepuri, NR; Darwish, TA
The Australian National Deuteration Facility (NDF) continues to develop new capabilities in molecular deuteration. The tailoring of deuteration approach to solve specific contrast problems has greatly increased the range of systems that can be investigated and recent capability development and new applications will be described. The common problem of how to obtain good contrast during SANS of membrane proteins solubilised in detergent micelles has been solved by specific deuteration of detergent head groups and hydrophobic tails to different levels to contrast match at 100% D20 buffer. Likewise, in investigating the lo cation of a purely hydrophobic transmembrane peptide in bicontinuous cubic phase liquid crystals Page 25 ANBUG-AINSE Neutron Scattering Symposium, AANSS 2018 / Book of Abstracts suitable for drug delivery, a mixture of deuterated and hydrogenated phytanoyl monoethanolamide was used to perfectly contrast match out either gyroid or diamond phase lipids to pinpoint the peptides location. In Soft Matter, investigation of structure of the ligands in the shell layer of self-assembled monolayerprotected nanoparticles has been made possible by use of various combinations of immiscible deuterated or hydrogenated Phenylethanethiol (PET) and Dodecanethiol (DDT) and MONSA modelling of SANS data showed the janus and belt like distribution of the ligands on the surface and the effects of nanoparticle core size and ligand ratio on the patterning. Other case studies will include use of deuterated ionic liquids and new previously unavailable deuterated molecules will be highlighted. This work was supported by the National Collaborative Research Infrastructure Strategy © The Authors.
Probing protein structures in solution by molecular dynamics simulation and small-angle x-ray scattering
(International Union of Crystallography, 2021-08-14) Yang, HC; Lin, SW; Ge, YC; Huang, MY; Yang, CH; Liu, WM; Duff, AP; Wu, CM; Lan, YK; Su, AC; Yeh, YQ; Jeng, US; Chou, PT
Lore of chemical biology guides us that drug discovery of protein binding relies on either optimize the active site complexity of lock and key or induced-fit with conformation selection dynamics; yet, the latter that often-coupled protein interior transport dynamics was much harder to study due to its lack of strong interactions in transient states.[1-2] This study starts to make progress in using in-situ operando X-ray and neutron contrast variation techniques to depict the landscape of protein binding substrate dynamics in solution. We herein demonstrate, for the first time, the 3-D dynamical structures of hydrated CYP450 protein exterior surfaces to interior buried heme site by a distributed connection of channels that direct the reactant in and out. Using CYP450s of prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) as prototypes we have unveiled the unique dynamics of P450 functional channels in/out the haem site, which drive a variety of water molecules motion, water density change and pre-organization toward the heme active site and hence harness the substrate binding selectivity. The result is able to clarify how these two proteins catalyze the same substrate of prostaglandin H2 by entirely different regio-chemical-selective pathways. © The Authors
Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scattering
(Springer, 2022-10-26) Pietras, Z; Duff, AP; Morad, V; Wood, K; Jeffries, CM; Sunnerhagen, M
This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2–Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2–Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2–Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general. © The Author(s) 2022. Open Access CC-BY
Protein deuteration extending structural characterisations by small angle neutron scattering with contrast variation.
(Australian Institute of Nuclear Science and Engineering (AINSE), 2020-11-11) Duff, AP
Protein deuteration enables unique applications of neutron scattering o the life sciences, at both low and high resolutions. In recent years, the National Deuteration Facility at ANSTO have developed and published a robust and efficient method of recombinant protein deuteration.[1] Utilising this method enables us to routinely collaborate with life scientists by removing the difficulty of biomolecule deuteration from their needs for sample preparation to make best use of neutron scattering. In this presentation I will highlight the essential role of protein deuteration in the structural characterization of previously poorly characterized “suppressor of copper sensitivity” proteins, as recently published.[2] Using this, and more recent work in progress, I will illustrate the value of small angle scattering as a complementary method to high resolution techniques so as to including disordered to-ordered transitions that are frequently the basis for functional mechanisms in life and disease. Other applications of protein deuteration, for neutron reflectometry, neutron crystallography, and nuclear magnetic resonance, will be briefly explained.
Protein perdeuteration for neutron crystallography
(The Physical Society of Japan, 2019-03-28) Duff, AP
Protein deuteration by recombinant expression in deuterated minimal media offers advantages to several experimental techniques involving neutron scattering. Perdeuteration improves neutron crystallographic studies of proteins. Although not required, protein perdeuteration offers advantages regarding crystal size, data collection time, and the clarity of the resulting experimental nuclear density maps. The level of advantage of perdeuteration depends on practical matters of the existing method of protein production. If recombinant expression yields are high, and purification is efficient, the staff and consumable costs for protein perdeuration are not so great. ©2019 The Physical Society of Japan under the terms of the Creative Commons Attribution 4.0 License

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