Publications by authors named "Andrew E Whitten"

53 Publications

Membrane Protein Structures in Lipid Bilayers; Small-Angle Neutron Scattering With Contrast-Matched Bicontinuous Cubic Phases.

Front Chem 2020 9;8:619470. Epub 2021 Feb 9.

Department of Chemical Engineering, Monash University, Clayton, VIC, Australia.

This perspective describes advances in determining membrane protein structures in lipid bilayers using small-angle neutron scattering (SANS). Differentially labeled detergents with a homogeneous scattering length density facilitate contrast matching of detergent micelles; this has previously been used successfully to obtain the structures of membrane proteins. However, detergent micelles do not mimic the lipid bilayer environment of the cell membrane . Deuterated vesicles can be used to obtain the radius of gyration of membrane proteins, but protein-protein interference effects within the vesicles severely limits this method such that the protein structure cannot be modeled. We show herein that different membrane protein conformations can be distinguished within the lipid bilayer of the bicontinuous cubic phase using contrast-matching. Time-resolved studies performed using SANS illustrate the complex phase behavior in lyotropic liquid crystalline systems and emphasize the importance of this development. We believe that studying membrane protein structures and phase behavior in contrast-matched lipid bilayers will advance both biological and pharmaceutical applications of membrane-associated proteins, biosensors and food science.
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http://dx.doi.org/10.3389/fchem.2020.619470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903247PMC
February 2021

BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Antioxid Redox Signal 2021 Apr 12. Epub 2021 Apr 12.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia.

Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from termed bovine colonization factor protein H (BcfH) and defined its role in virulence. In the conserved bovine colonization factor () fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a proline instead of the conserved -proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to fimbrial biogenesis. Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity.
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http://dx.doi.org/10.1089/ars.2020.8218DOI Listing
April 2021

Small angle X-ray scattering analysis of ligand-bound forms of tetrameric apolipoprotein-D.

Biosci Rep 2021 Jan;41(1)

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia.

Human apolipoprotein-D (apoD) is a glycosylated lipocalin that plays a protective role in Alzheimer's disease due to its antioxidant function. Native apoD from human body fluids forms oligomers, predominantly a stable tetramer. As a lipocalin, apoD binds and transports small hydrophobic molecules such as progesterone, palmitic acid and sphingomyelin. Oligomerisation is a common trait in the lipocalin family and is affected by ligand binding in other lipocalins. The crystal structure of monomeric apoD shows no major changes upon progesterone binding. Here, we used small-angle X-ray scattering (SAXS) to investigate the influence of ligand binding and oxidation on apoD oligomerisation and conformation. As a solution-based technique, SAXS is well suited to detect changes in oligomeric state and conformation in response to ligand binding. Our results show no change in oligomeric state of apoD and no major conformational changes or subunit rearrangements in response to binding of ligands or protein oxidation. This highlights the highly stable structure of the native apoD tetramer under various physiologically relevant experimental conditions.
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http://dx.doi.org/10.1042/BSR20201423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786332PMC
January 2021

Effect of red blood cell shape changes on haemoglobin interactions and dynamics: a neutron scattering study.

R Soc Open Sci 2020 Oct 14;7(10):201507. Epub 2020 Oct 14.

Jülich Centre for Neutron Science (JCNS-1) and Institute of Biological Information Processing (IBI-8: Neutron Scattering and Biological Matter), Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.

By using a combination of experimental neutron scattering techniques, it is possible to obtain a statistical perspective on red blood cell (RBC) shape in suspensions, and the inter-relationship with protein interactions and dynamics inside the confinement of the cell membrane. In this study, we examined the ultrastructure of RBC and protein-protein interactions of haemoglobin (Hb) in them using ultra-small-angle neutron scattering and small-angle neutron scattering (SANS). In addition, we used the neutron backscattering method to access Hb motion on the ns time scale and Å length scale. Quasi-elastic neutron scattering (QENS) experiments were performed to measure diffusive motion of Hb in RBCs and in an RBC lysate. By using QENS, we probed both internal Hb dynamics and global protein diffusion, on the accessible time scale and length scale by QENS. Shape changes of RBCs and variation of intracellular Hb concentration were induced by addition of the Na-selective ionophore monensin and the K-selective one, valinomycin. The experimental SANS and QENS results are discussed within the framework of crowded protein solutions, where free motion of Hb is obstructed by mutual interactions.
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http://dx.doi.org/10.1098/rsos.201507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657910PMC
October 2020

Vortex fluidic mediated encapsulation of functional fish oil featuring in situ probed small angle neutron scattering.

NPJ Sci Food 2020 9;4:12. Epub 2020 Sep 9.

Flinders Institute for Nanoscale Science and Technology, College of Science and Engineering, Flinders University, Bedford Park, SA 5042 Australia.

Major challenges for optimizing the benefits of fish oil on human health are improved bioavailability while overcoming the strong odor and avoiding significant oxidation of the omega-3 polyunsaturated fatty acids (PUFAs). The scalable continuous flow thin film vortex fluidic device (VFD) improves the Tween 20 encapsulation of fish oil relative to conventional homogenization processing, with the fish oil particles significantly smaller and the content of the valuable omega-3 fatty acids higher. In addition, after 14 days storage the remaining omega-3 fatty acids content was higher, from ca 31.0% for raw fish oil to ca 62.0% of freeze-dried encapsulated fish oil. The VFD mediated encapsulated fish oil was used to enrich the omega-3 fatty acid content of apple juice, as a model water-based food product, without changing its sensory values. The versatility of the VFD was further demonstrated in forming homogenous suspensions of fish oil containing water-insoluble bioactive molecules, curcumin and quercetin. We have also captured, for the first time, real-time structural changes in nanoencapsulation by installing a VFD with in in situ small angle neutron scattering. Real-time measurements afford valuable insights about self-assembly in solution.
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http://dx.doi.org/10.1038/s41538-020-00072-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481235PMC
September 2020

Structure of soft and hard protein corona around polystyrene nanoplastics-Particle size and protein types.

Biointerphases 2020 09 11;15(5):051002. Epub 2020 Sep 11.

School of Chemical Sciences, The University of Auckland, Auckland 1010, New Zealand.

A major challenge in understanding nanoplastic toxicity (or nanoparticles in general) lies in establishing the causal relationships between its physical properties and biological impact. This difficulty can be attributed to surface alterations that follow the formation of a biological complex around the nanoplastic, as exemplified by protein coronae. The protein corona is known to be responsible for the biological response elicited, although its own structure and attributes remain unknown. We approach this knowledge gap by independently studying the structure of soft and hard coronae using neutron scattering techniques. We investigated the formation and the structure of corona proteins (human serum albumin and lysozyme) and the resulting protein corona complexes with polystyrene nanoplastics of different sizes (20 and 200 nm) and charges. Soft corona complexes (regardless of protein type) adopted a structure where the nanoplastics were surrounded by a loose protein layer (∼2-3 protein molecules thick). Hard corona complexes formed fractal-like aggregates, and the morphology of which is known to be harmful to cellular membranes. In most cases, hard-corona coated nanoplastics also formed fractal-like aggregates in solution. Nanoplastic size affected the structures of both the protein corona and the intrinsic protein: more significant conformational change was observed in the hard corona proteins around smaller nanoparticles compared to larger ones, as the self-association forces holding the nanoplastic/protein complex together were stronger. This also implies that protein-dependent biochemical processes are more likely to be disrupted by smaller polystyrene nanoplastics, rather than larger ones.
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http://dx.doi.org/10.1116/6.0000404DOI Listing
September 2020

Nanostructured mesoporous gold biosensor for microRNA detection at attomolar level.

Biosens Bioelectron 2020 Nov 1;168:112429. Epub 2020 Aug 1.

Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD, 4072, Australia; Department of Plant and Environmental New Resources, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu Yongin-si Gyeonggi-do, 17104, South Korea; School of Chemical Engineering, Faculty of Engineering, Architecture and Information Technology (EAIT), The University of Queensland, Brisbane, QLD, 4072, Australia. Electronic address:

Advances in nanoarchitectonics enable a wide variety of nanostructured electrodes with tunable shapes and surface for constructing sensitive biosensors. Herein we demonstrate the fabrication of a mesoporous gold (Au) biosensor for the specific and sensitive detection of miRNA in a relatively simple and portable manner. The electrocatalytic activity of the mesoporous Au electrode (MPGE) towards the redox reaction of Fe(CN)] expansively examined. Leveraging the electrocatalytic activity and signal enhancement capacity of the MPGE, an ultrasensitive and specific electrochemical sensor was developed for the detection of microRNA (miRNA). The target miRNA from spiked samples is selectively isolated and purified using magnetic bead-capture probe followed by the direct adsorption on the MPGE through direct affinity interaction between miRNA and mesoporous Au surface. The MPGE-bound miRNA is then quantified by differential pulse voltammetry (DPV) using [Fe(CN)] redox system (Faradaic current decrease with reference to the bare MPGE). This method evades the cumbersome PCR (polymerase chain reaction) and enzymatic amplification steps. This is a single-step assay building which can detect a wide dynamic linear range (100 aM to 1 nM) of miRNA with an ultra-low limit detection of 100 aM and present high translational potentiality for the development of high-performance detection tools for clinics.
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http://dx.doi.org/10.1016/j.bios.2020.112429DOI Listing
November 2020

Structural basis of the zinc-induced cytoplasmic aggregation of the RNA-binding protein SFPQ.

Nucleic Acids Res 2020 04;48(6):3356-3365

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.

SFPQ is a ubiquitous nuclear RNA-binding protein implicated in many aspects of RNA biogenesis. Importantly, nuclear depletion and cytoplasmic accumulation of SFPQ has been linked to neuropathological conditions such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Here, we describe a molecular mechanism by which SFPQ is mislocalized to the cytoplasm. We report an unexpected discovery of the infinite polymerization of SFPQ that is induced by zinc binding to the protein. The crystal structure of human SFPQ in complex with zinc at 1.94 Å resolution reveals intermolecular interactions between SFPQ molecules that are mediated by zinc. As anticipated from the crystal structure, the application of zinc to primary cortical neurons induced the cytoplasmic accumulation and aggregation of SFPQ. Mutagenesis of the three zinc-coordinating histidine residues resulted in a significant reduction in the zinc-binding affinity of SFPQ in solution and the zinc-induced cytoplasmic aggregation of SFPQ in cultured neurons. Taken together, we propose that dysregulation of zinc availability and/or localization in neuronal cells may represent a mechanism for the imbalance in the nucleocytoplasmic distribution of SFPQ, which is an emerging hallmark of neurodegenerative diseases including AD and ALS.
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http://dx.doi.org/10.1093/nar/gkaa076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102971PMC
April 2020

Interfacial Structures of Droplet-Stabilized Emulsions Formed with Whey Protein Microgel Particles as Revealed by Small- and Ultra-Small-Angle Neutron Scattering.

Langmuir 2019 09 26;35(37):12017-12027. Epub 2019 Aug 26.

Riddet Institute , Massey University , Private Bag 11 222 , Palmerston North 4442 , New Zealand.

Droplet-stabilized emulsions (DSEs) were made from oil droplets coated with whey protein microgel (WPM) particles. The WPM particles with -average hydrodynamic diameters of 270.9 ± 4.7 and 293.8 ± 6.7 nm were obtained by heating whey proteins with 10 mM phosphate buffer, pH 5.9 (-PB) and no buffer (-NPB), respectively. The primary emulsions coated by WPM-NPB and WPM-PB particles had mass fractal dimensions of ∼2.75, as determined by small- and ultra-small-angle neutron scattering (SANS and USANS). The size of the subsequently formed DSEs ( ≈ 7-23 μm), which were stabilized by the primary emulsion droplets, made with either WPM-NPB (termed DSE-NPB) or WPM-PB (termed DSE-PB) was dependent on the concentration of the primary emulsion (10-60 wt %) in the aqueous phase. At the DSE-NPB interface, the adsorbed primary emulsion droplets formed a fractal network with a surface fractal dimension of about 3, indicating a rough interfacial layer. Combined SANS and USANS allowed a comprehensive understanding of the multilength scale structures from WPM particles to DSEs.
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http://dx.doi.org/10.1021/acs.langmuir.9b01966DOI Listing
September 2019

Unique structural features of a bacterial autotransporter adhesin suggest mechanisms for interaction with host macromolecules.

Nat Commun 2019 04 29;10(1):1967. Epub 2019 Apr 29.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, VIC, Australia.

Autotransporters are the largest family of outer membrane and secreted proteins in Gram-negative bacteria. Most autotransporters are localised to the bacterial surface where they promote colonisation of host epithelial surfaces. Here we present the crystal structure of UpaB, an autotransporter that is known to contribute to uropathogenic E. coli (UPEC) colonisation of the urinary tract. We provide evidence that UpaB can interact with glycosaminoglycans and host fibronectin. Unique modifications to its core β-helical structure create a groove on one side of the protein for interaction with glycosaminoglycans, while the opposite face can bind fibronectin. Our findings reveal far greater diversity in the autotransporter β-helix than previously thought, and suggest that this domain can interact with host macromolecules. The relevance of these interactions during infection remains unclear.
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http://dx.doi.org/10.1038/s41467-019-09814-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488583PMC
April 2019

Pore-tuning to boost the electrocatalytic activity of polymeric micelle-templated mesoporous Pd nanoparticles.

Chem Sci 2019 Apr 12;10(14):4054-4061. Epub 2019 Mar 12.

Key Laboratory of Eco-chemical Engineering , College of Chemistry and Molecular Engineering , Qingdao University of Science and Technology (QUST) , Qingdao 266042 , China.

Understanding how mesoporous noble metal architectures affect electrocatalytic performance is very important for the rational design and preparation of high-performance electrocatalysts. Herein, by using polymeric micelle-assembled structures as templates, mesoporous Pd nanoparticles with tunable porous constructions are synthesized by simply tuning the solvent compositions. The effect of porous Pd nanoparticles on the electrocatalytic performance is thoroughly studied. Their superior electrocatalytic activity can be attributed to the mass transport efficiency and open porous structures.
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http://dx.doi.org/10.1039/c8sc03911aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457336PMC
April 2019

Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase ScsC from Proteus mirabilis.

Acta Crystallogr D Struct Biol 2019 Mar 26;75(Pt 3):296-307. Epub 2019 Feb 26.

Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia.

Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance, which is a key virulence trait of this uropathogen. Each protomer of the enzyme has an N-terminal trimerization stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, two PmScsC variants, PmScsCΔN and PmScsCΔLinker, are characterized. PmScsCΔN is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsCΔN reported here reveals, as expected, a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant, PmScsCΔLinker, was designed to remove the 11-amino-acid linker, and it is shown that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsCΔLinker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small-angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsCΔLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function.
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http://dx.doi.org/10.1107/S2059798319000081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450059PMC
March 2019

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase.

Acta Crystallogr D Struct Biol 2019 Mar 26;75(Pt 3):283-295. Epub 2019 Feb 26.

Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia.

Disulfide-bond-forming (DSB) oxidative folding enzymes are master regulators of virulence that are localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K-12 consists of a dithiol-oxidizing redox-relay pair (DsbA/B), a disulfide-isomerizing redox-relay pair (DsbC/D) and the specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast, the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these, α-DsbA1 and α-DsbB, form a redox-relay pair analogous to DsbA/B from E. coli. The third enzyme, α-DsbA2, incorporates a DsbA-like sequence but does not interact with α-DsbB. In comparison to other DsbA enzymes, α-DsbA2 has ∼50 extra N-terminal residues (excluding the signal peptide). The crystal structure of α-DsbA2ΔN, an N-terminally truncated form in which these ∼50 residues are removed, confirms the DsbA-like nature of this domain. However, α-DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. Firstly, α-DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase: i.e. its role is to shuffle rather than to introduce disulfide bonds. Moreover, small-angle X-ray scattering (SAXS) of α-DsbA2 reveals a homotrimeric arrangement that differs from those of the other characterized bacterial disulfide isomerases DsbC from Escherichia coli (homodimeric) and ScsC from Proteus mirabilis (PmScsC; homotrimeric with a shape-shifter peptide). α-DsbA2 lacks the shape-shifter motif and SAXS data suggest that it is less flexible than PmScsC. These results allow conclusions to be drawn about the factors that are required for functionally equivalent disulfide isomerase enzymatic activity across structurally diverse protein architectures.
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http://dx.doi.org/10.1107/S2059798318018442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450060PMC
March 2019

Author Correction: A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2019 03 1;10(1):976. Epub 2019 Mar 1.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD, 4072, Australia.

This Article contains errors in Fig. 1, Table 1 and the Methods section. In panel c, the labels for PmScsC and EcDsbC in the upper two curves are interchanged. In Table 1 and the Methods section entitled 'Extended structure', the space group of the extended PmScsC structure is incorrectly referred to as H3 and should read H32. Correct versions of Fig. 1 and Table 1 are presented below; the errors have not been corrected in the Article.
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http://dx.doi.org/10.1038/s41467-019-08920-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397206PMC
March 2019

Soft and Hard Interactions between Polystyrene Nanoplastics and Human Serum Albumin Protein Corona.

Bioconjug Chem 2019 04 19;30(4):1067-1076. Epub 2019 Mar 19.

School of Chemical Sciences , The University of Auckland , Auckland 1010 , New Zealand.

Upon contact with biological fluids, the surface of nanoparticles is surrounded by many types of proteins, forming a so-called "protein corona". The physicochemical properties of the nanoparticle/corona complex depend predominantly on the nature of the protein corona. An understanding of the structure of the corona and the resulting complex provides insight into the structure-activity relationship. Here, we structurally evaluate the soft and hard components of the protein corona, formed from polystyrene (PS) nanoplastics and human serum albumin (HSA). Using circular dichroism spectroscopy to elucidate the structure of HSA within the complex, we establish the effect of nanoparticle size and pH on the nature of the protein corona formed- whether hard or soft. Despite the weak interaction between PS and the HSA corona, small angle neutron scattering revealed the formation of a complex structure that enhanced the intermolecular interactions between HSA proteins, PS particles, and the HS/PSA complexes. Fractal formation occurred under conditions where the interaction between PS and HSA was strong, and increasing HSA concentrations suppressed the degree of aggregation. The size of the nanoparticles directly influenced the nature of the protein corona, with larger particles favoring the formation of a soft corona, due to the decreased PS-HSA attraction.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00015DOI Listing
April 2019

Studying Munc18:Syntaxin Interactions Using Small-Angle Scattering.

Methods Mol Biol 2019 ;1860:115-144

Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia.

The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.
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http://dx.doi.org/10.1007/978-1-4939-8760-3_7DOI Listing
June 2019

Mesoporous Metallic Iridium Nanosheets.

J Am Chem Soc 2018 Oct 31;140(39):12434-12441. Epub 2018 Aug 31.

College of Chemistry and Molecular Engineering , Qingdao University of Science and Technology , Qingdao 266042 , China.

Two-dimensional (2D) metals are an emerging class of nanostructures that have attracted enormous research interest due to their unusual electronic and thermal transport properties. Adding mesopores in the plane of ultrathin 2D metals is the next big step in manipulating these structures because increasing their surface area improves the utilization of the material and the availability of active sites. Here, we report a novel synthetic strategy to prepare an unprecedented type of 2D mesoporous metallic iridium (Ir) nanosheet. Mesoporous Ir nanosheets can be synthesized with close-packed assemblies of diblock copolymer (poly-(ethylene oxide)- b-polystyrene, PEO- b-PS) micelles aligned in the 2D plane of the nanosheets. This novel synthetic route opens a new dimension of control in the synthesis of 2D metals, enabling new kinds of mesoporous architectures with abundant catalytically active sites. Because of their unique structural features, the mesoporous metallic Ir nanosheets exhibit a high electrocatalytic activity toward the oxygen evolution reaction (OER) in acidic solution as compared to commercially available catalysts.
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http://dx.doi.org/10.1021/jacs.8b05206DOI Listing
October 2018

Identification of a novel tetrameric structure for human apolipoprotein-D.

J Struct Biol 2018 09 6;203(3):205-218. Epub 2018 Jun 6.

Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia; School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia; Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia. Electronic address:

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded β-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-β burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.
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http://dx.doi.org/10.1016/j.jsb.2018.05.012DOI Listing
September 2018

Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-ray Scattering.

ACS Nano 2018 06 4;12(6):5791-5799. Epub 2018 Jun 4.

EMBL Australia Node for Single Molecule Science, School of Medical Sciences , The University of New South Wales , Sydney 2052 , Australia.

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.
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http://dx.doi.org/10.1021/acsnano.8b01669DOI Listing
June 2018

Disulfide isomerase activity of the dynamic, trimeric ScsC protein is primed by the tandem immunoglobulin-fold domain of ScsB.

J Biol Chem 2018 04 28;293(16):5793-5805. Epub 2018 Feb 28.

From the Institute for Molecular Bioscience, University of Queensland, St, Lucia, Queensland 4072, Australia,

Correct disulfide bond formation is essential for proper folding of many proteins, including bacterial virulence factors. The suppressor of copper sensitivity (Scs) proteins have roles in dithiol/disulfide interchange and the bacterial response to copper stress. Encoded in a four-gene cassette (ScsABCD) present in many Gram-negative bacteria, the Scs proteins are enigmatic and poorly characterized. Here, we show that the periplasmic α-domain of the membrane protein ScsB in the Gram-negative bacterium forms a redox relay with the soluble periplasmic protein PmScsC. We also found that the periplasmic α-domain is sufficient to activate the disulfide isomerase activity of PmScsC. The crystal structure of PmScsBα at a resolution of 1.54 Å revealed that it comprises two structurally similar immunoglobulin-like folds, one of which includes a putative redox-active site with the sequence CC. We confirmed the importance of these cysteine residues for PmScsBα function, and in addition, we engineered cysteine variants that produced a stable complex between PmScsC and PmScsBα. Using small-angle X-ray and neutron scattering analyses with contrast variation, we determined a low-resolution structure of the PmScsC-PmScsBα complex. The structural model of this complex suggested that PmScsBα uses both of its immunoglobulin-like folds to interact with PmScsC and revealed that the highly dynamic PmScsC becomes ordered upon PmScsBα binding. These findings add to our understanding of the poorly characterized Scs proteins.
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http://dx.doi.org/10.1074/jbc.RA118.001860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912455PMC
April 2018

Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis.

Acta Crystallogr F Struct Biol Commun 2018 01 1;74(Pt 1):31-38. Epub 2018 Jan 1.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia.

The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallographic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbD, n-NmDsbD, c-NmDsbD and c-NmDsbD diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P23, P321, P4 and P121, respectively.
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http://dx.doi.org/10.1107/S2053230X17017800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947690PMC
January 2018

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

PLoS One 2017 31;12(10):e0187302. Epub 2017 Oct 31.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland, Australia.

The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187302PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663490PMC
November 2017

2017 publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution: an update.

Acta Crystallogr D Struct Biol 2017 Sep 18;73(Pt 9):710-728. Epub 2017 Aug 18.

ANSTO, New Illawarra Road, Lucas Heights, NSW 2234, Australia.

In 2012, preliminary guidelines were published addressing sample quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular small-angle scattering (SAS) experiments. Biomolecular SAS has since continued to grow and authors have increasingly adopted the preliminary guidelines. In parallel, integrative/hybrid determination of biomolecular structures is a rapidly growing field that is expanding the scope of structural biology. For SAS to contribute maximally to this field, it is essential to ensure open access to the information required for evaluation of the quality of SAS samples and data, as well as the validity of SAS-based structural models. To this end, the preliminary guidelines for data presentation in a publication are reviewed and updated, and the deposition of data and associated models in a public archive is recommended. These guidelines and recommendations have been prepared in consultation with the members of the International Union of Crystallography (IUCr) Small-Angle Scattering and Journals Commissions, the Worldwide Protein Data Bank (wwPDB) Small-Angle Scattering Validation Task Force and additional experts in the field.
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http://dx.doi.org/10.1107/S2059798317011597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586245PMC
September 2017

The nature of the Syntaxin4 C-terminus affects Munc18c-supported SNARE assembly.

PLoS One 2017 25;12(8):e0183366. Epub 2017 Aug 25.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, QLD, Australia.

Vesicular transport of cellular cargo requires targeted membrane fusion and formation of a SNARE protein complex that draws the two apposing fusing membranes together. Insulin-regulated delivery and fusion of glucose transporter-4 storage vesicles at the cell surface is dependent on two key proteins: the SNARE integral membrane protein Syntaxin4 (Sx4) and the soluble regulatory protein Munc18c. Many reported in vitro studies of Munc18c:Sx4 interactions and of SNARE complex formation have used soluble Sx4 constructs lacking the native transmembrane domain. As a consequence, the importance of the Sx4 C-terminal anchor remains poorly understood. Here we show that soluble C-terminally truncated Sx4 dissociates more rapidly from Munc18c than Sx4 where the C-terminal transmembrane domain is replaced with a T4-lysozyme fusion. We also show that Munc18c appears to inhibit SNARE complex formation when soluble C-terminally truncated Sx4 is used but does not inhibit SNARE complex formation when Sx4 is C-terminally anchored (by a C-terminal His-tag bound to resin, by a C-terminal T4L fusion or by the native C-terminal transmembrane domain in detergent micelles). We conclude that the C-terminus of Sx4 is critical for its interaction with Munc18c, and that the reported inhibitory role of Munc18c may be an artifact of experimental design. These results support the notion that a primary role of Munc18c is to support SNARE complex formation and membrane fusion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183366PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571939PMC
October 2017

A shape-shifting redox foldase contributes to Proteus mirabilis copper resistance.

Nat Commun 2017 07 19;8:16065. Epub 2017 Jul 19.

Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland 4072, Australia.

Copper resistance is a key virulence trait of the uropathogen Proteus mirabilis. Here we show that P. mirabilis ScsC (PmScsC) contributes to this defence mechanism by enabling swarming in the presence of copper. We also demonstrate that PmScsC is a thioredoxin-like disulfide isomerase but, unlike other characterized proteins in this family, it is trimeric. PmScsC trimerization and its active site cysteine are required for wild-type swarming activity in the presence of copper. Moreover, PmScsC exhibits unprecedented motion as a consequence of a shape-shifting motif linking the catalytic and trimerization domains. The linker accesses strand, loop and helical conformations enabling the sampling of an enormous folding landscape by the catalytic domains. Mutation of the shape-shifting motif abolishes disulfide isomerase activity, as does removal of the trimerization domain, showing that both features are essential to foldase function. More broadly, the shape-shifter peptide has the potential for 'plug and play' application in protein engineering.
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http://dx.doi.org/10.1038/ncomms16065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524982PMC
July 2017

Whaddaya Know: A Guide to Uncertainty and Subjectivity in Structural Biology.

Trends Biochem Sci 2017 02 12;42(2):155-167. Epub 2017 Jan 12.

School of Chemistry and Biochemistry, The University of Western Australia, Crawley, WA 6014, Australia.

The methods of structural biology, while powerful, are technically complex. Although the Protein Data Bank (PDB) provides a repository that allows anyone to download any structure, many users would not appreciate the caveats that should be considered when examining a structure. Here, we describe several key uncertainties associated with the application of X-ray crystallography, NMR spectroscopy, single-particle electron microscopy (SPEM), and small-angle scattering (SAS) to biological macromolecules. The take-home message is that structures are not absolute truths - they are models that fit the experimental data and therefore have uncertainty and subjectivity associated with them. These uncertainties must be appreciated - careful reading of the associated paper, and any validation report provided by the structure database, is highly recommended.
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http://dx.doi.org/10.1016/j.tibs.2016.11.002DOI Listing
February 2017

Effects of Alkali Cations and Halide Anions on the Self-Assembly of Phosphatidylcholine in Oils.

Langmuir 2016 11 8;32(46):12166-12174. Epub 2016 Nov 8.

Institute of Polymer Science and Engineering, National Taiwan University , Taipei 10617, Taiwan.

The interactions between ions and phospholipids are closely associated with the structures and functions of cell membrane. Instead of conventional aqueous systems, we systematically investigated the effects of inorganic ions on the self-assembly of lecithin, a zwitterionic phosphatidylcholine, in cyclohexane. Previous studies have shown that addition of inorganic salts with specific divalent and trivalent cations can transform lecithin organosols into organogels. In this study, we focused on the effect of monovalent alkali halides. Fourier transform infrared spectroscopy was used to demonstrate that the binding strength of the alkali cations with the phosphate of lecithin is in the order Li > Na > K. More importantly, the cation-phosphate interaction is affected by the paired halide anions, and the effect follows the series I > Br > Cl. The salts of stronger interactions with lecithin, including LiCl, LiBr, LiI, and NaI, were found to induce cylindrical micelles sufficiently long to form organogels, while others remain organosols. A mechanism based on the charge density of ions and the enthalpy change of the ion exchange between alkali halides and lecithin headgroup is provided to explain the contrasting interactions and the effectiveness of the salts to induce organogelation.
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http://dx.doi.org/10.1021/acs.langmuir.6b03449DOI Listing
November 2016

Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments.

Nat Protoc 2016 Nov 6;11(11):2122-2153. Epub 2016 Oct 6.

European Molecular Biology Laboratory (EMBL) Hamburg Outstation, DESY, Hamburg, Germany.

Small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume, including the solvent and buffer components, as well as the macromolecules of interest. To obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis, so it is essential that the samples be pure and monodisperse for the duration of the experiment. This protocol outlines the basic physics of SAXS and SANS, and it reveals how the underlying conceptual principles of the techniques ultimately 'translate' into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size-exclusion chromatography (SEC) and light scattering. Also included are procedures that are specific to X-rays (in-line SEC-SAXS) and neutrons, specifically preparing samples for contrast matching or variation experiments and deuterium labeling of proteins.
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http://dx.doi.org/10.1038/nprot.2016.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402874PMC
November 2016

Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

Nat Struct Mol Biol 2016 Mar 8;23(3):197-203. Epub 2016 Feb 8.

Structural and Computational Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.
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http://dx.doi.org/10.1038/nsmb.3172DOI Listing
March 2016

SNARE-ing the structures of Sec1/Munc18 proteins.

Curr Opin Struct Biol 2014 Dec 2;29:44-51. Epub 2014 Oct 2.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia. Electronic address:

Membrane fusion is essential for cellular transport in eukaryotes. Abnormalities contribute to a wide range of diseases including diabetes and neurological disorders. A key regulator of SNARE-mediated membrane fusion is the Sec1/Munc18 (SM) protein family. Universal structural features of SM proteins have been identified that affect the way these interact with their partner Syntaxin SNARE proteins. Whilst the molecular basis for SM-regulated SNARE complex formation has been extensively studied, it remains poorly understood. Recent crystal structures of SM proteins alone or in complex have provided new insight. Here we examine the available structural information on SM proteins for clues to how these enigmatic proteins might regulate SNARE complex assembly and membrane fusion.
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http://dx.doi.org/10.1016/j.sbi.2014.09.003DOI Listing
December 2014