Bio21 - Theses
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ItemExploring novel blue turn-on fluorescent probes for the direct detection of nitric oxide and free radicals in living cells( 2017)Bacterial biofilms are causing considerable damage to different areas in industry such as food industry, oil industry and dentistry. Traditional methods to control biofilm formation and to treat the surfaces affected by these microorganisms has mostly focused on biocidal and antibacterial strategies. The drawbacks of these approaches is related to the development of tolerances that decrease effectiveness of chemicals apply to eradicate these microorganisms. The growth of biofilms therefore is linked to a significant adaptation by bacteria cells to control changes in their environment. In this regard, the development of efficient methods to control biofilms formation as well as their irreversible eradication from affected surfaces is an important area of scientific research. Bacterial biofilms at times undergo regulated and coordinated dispersal events, where sessile biofilm cells convert to free-swimming, planktonic bacteria. Nitric oxide (NO) is an important biochemical signalling molecule that has been linked to the inhibition of biofilm formation and activation of dispersal through the generation of nitrosative and oxidative stress. Therefore, the availability of methods that enable sensing and visualizing NO is critical to reveal details of the biological functioning of this molecule. Knowledge of these will provide important guidelines for the development of strategies to combat biofilm formation. In this thesis two different approaches for detecting NO and oxidative stress were explored, that are based on fluorescence measurements using coumarin as fluorophore. The first strategy explores “turn-on” fluorescence for direct detection of endogenously produced NO. A family of five blue fluorescent probes CB1-5 were designed and synthesized and the photophysical properties studied in detail. These probes feature a substituted 7-hydroxy coumarin chromophore coupled to 2-methyl-8-aminoquinoline, which act as tridentate ligand for Cu(II) and active site for monitoring NO using the replacement strategy. The UV-vis absorption and fluorescence emission characteristics of the probes are significantly influenced by the substitution pattern on the coumarin ring, as well as by solvent polarity and pH. Time-dependent Density Functional Theory (TD-DFT) calculations for CB4 and CB5 showed that the absorptions are due to π ® π* transitions localised on the coumarin system, with a small charge transfer contribution from the quinoline system at higher pH where the 7-hydroxycoumarin moiety is deprotonated. Complexation of the probes with Cu(II) leads to fluorescence quenching, which switches back on upon reaction with NO. In vitro studies revealed that the probes detect NO with high selectivity in nM concentrations and do not respond to other oxidizing species. In vivo studies for CB4 and CB5 showed that these probes enable detection of NO in living bacterial cells in multi-dye imaging experiments. Furthermore, CB5 also enables to detect NO in macrophages, where it is an important effector molecule in host defence against bacterial pathogens. Using confocal microscopy, it was shown that the probe can be trapped by the cells and reacts directly and specifically with NO, rendering it a promising tool for imaging NO in response to pharmacological agents that modulate its level, for example during bacterial infections. The second strategy explored in this work was the development of a profluorescent nitroxide probe, which can be utilized for detecting the formation of reactive nitrogen and oxygen intermediates and associated changes in redox states within microcolonies. Attachment of a nitroxide to a fluorophore leads to fluorescence quenching, which upon free radical scavenging, metabolism or redox processes, returns the molecule to its native fluorescent state. A large variety of synthetic approaches and procedures were explored to construct such structure, but unfortunately none of them were successful.
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ItemNew insights into the redox chemistry of protein thiols( 2017)Redox reactions play a crucial role in many biological processes. These include the role of Cu(II)/Cu(I) couples in enzyme reactions and that of the tripeptide glutathione (GSH) as a key redox buffer in cells via the GSSG/2GSH redox couple. Copper is an essential trace metal but, if redox balance is not properly maintained, excess or ‘free’ copper is toxic. Consequently, specific metabolic routes exist to safely maintain the copper balance within cells. A particular challenge in understanding molecular copper metabolism is purification of cupro-proteins in high purity and yield. A variety of affinity tags, such as the popular poly-histidine tag, have been developed to facilitate purification but they generally rely on expensive affinity resins and their presence may interfere with protein characterization. This work demonstrates that a poly-lysine tag at the C-terminus enables, for certain cupro-proteins, ready purification on large scales via cost-effective cation-exchange chromatography. Cleavage of the tag is normally not necessary since the poly-lysine tag is shown to have no detectable affinity for either Cu(I) or Cu(II) and imposes no interference to the copper binding properties of the target proteins. In contrast, the poly-histidine tag possesses a sub-picomolar affinity for Cu(I) and sub-nanomolar affinity for Cu(II) and may need to be removed for reliable characterization of the target proteins. The GSSG/2GSH couple partners protein thiols in reversible thiol-disulfide exchange reactions that act as redox switches and play important roles in cell signalling and redox homeostasis. Disruption of these processes induces oxidative stress that is linked directly to aging processes and to a range of conditions including cancer and neurodegenerative diseases. Glutaredoxins (Grxs) are a class of oxidoreductase enzymes that specifically catalyse GSH-dependent thiol-disulfide exchange reactions and play essential roles in the regulation and maintenance of redox balance in cells. They protect protein thiols from irreversible oxidation and regulate their cellular activities under a variety of conditions. However, the molecular basis and underlying mechanisms of Grx action remain elusive are controversial and uncertain in many situations. Grxs feature dithiol active sites and can shuttle rapidly between three oxidation states, namely, fully reduced dithiol Grx(SH)2, semi-oxidized mixed disulfide Grx(SH)(SSG) and fully oxidized disulfide Grx(SS). Each is characterized by a distinct standard reduction potential (E^o' ). E_(P(SS))^o' values for the redox couple Grx(SS)/Grx(SH)2 are available but a recent estimate differs by over 100 mV from literature values. No estimates are available for E_(P(SSG))^o' for the mixed disulfide couple Grx(SH)(SSG)/(Grx(SH)2+GSH). The work reported in this thesis determined both E_(P(SS))^o' and E_(P(SSG))^o' for two representative Grx1 enzymes, human hGrx1 and E. coli EcGrx1. The empirical approaches were verified rigorously to overcome the sensitivity of these redox-labile enzymes to experimental conditions. Classic ‘acid quenching’ is demonstrated to shift the thiol-disulfide redox equilibrium. The reduction potentials are in the range that favours dual catalytic functions for Grxs as either an oxidase at low [GSH] or a reductase at high [GSH]. These enzymes were demonstrated to catalyze glutathionylation and deglutathionylation of a target protein monothiol under conditions of low and high GSH concentration, respectively. The catalysis was demonstrated to proceed via a monothiol ping-pong mechanism relying on a single Cys residue only in the dithiol active site. Grxs also catalyze oxidation of a protein dithiol and reduction of a disulfide via conserved parallel monothiol and dithiol mechanisms. Consequently, Grxs are shown to be a class of versatile enzymes with diverse catalytic functions that are driven by specific interactions with GSH.
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ItemInhibitors of an essential M. tuberculosis cytochrome P450 enzyme CYP121( 2017)Tuberculosis (TB) is an infectious disease caused by a rod-shaped mycobacterium called Mycobacterium tuberculosis (Mtb). Since the discovery of streptomycin in the 1940s, several effective anti-TB drugs have been discovered and employed clinically. However, the battle against TB is ongoing with approximately one-third of world’s population estimated to be currently infected with Mtb, with an increased prevalence of multidrug resistant (MDR-TB) and extensively drug resistant (XDR-TB) tuberculosis observed over the last two decades. Thus, there is an on-going need to develop new antimicrobial drugs with novel modes of action. Genome sequencing of Mtb, completed in 1998, revealed the presence of 20 genes encoding cytochrome P450 enzymes (CYPs). One of these CYPs – CYP121A1 – has been shown to be essential for Mtb viability and catalyses a unique reaction involving carbon–carbon bond formation between two tyrosyl side-chains of the cyclodityrosine substrate. This suggests that it may be possible to develop selective inhibitors against CYP121A1 as potential novel anti-Mtb drug leads. During this project, a range of analogues of cyclodityrosine, the natural substrate of CYP121A1, have been synthesized and biochemically analyzed with the aim of defining the binding requirements for the enzyme active site and providing insights into the design of selective inhibitors against CYP121A1. Our analysis shows that modifications at positions 3 and 4 of the tyrosyl aromatic ring are well accommodated within the active site whereas substituents at position-2 are not tolerated. Further, substrate analogue with an iodine at position-3 on the tyrosyl aromatic ring exhibits a 100-fold greater binding affinity compared to the natural substrate. Antimicrobial assays of the substrate analogues including the 3-iodo analogue, show that they are not effective antimicrobial agents (MIC values > 100 μM).
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ItemMechanisms of inclusion assembly in Huntington’s disease( 2017)Formation of microscopically visible intracellular aggregates (inclusions) containing exon 1 fragments of mutant Huntingtin (mHttex1) is a hallmark of Huntington’s Disease (HD) pathology. The role of inclusions in HD pathogenesis is not fully understood and is described by two alternative models. One model considers Huntingtin inclusions being adaptive by sequestering toxic soluble mHttex1 and prolonging cellular survival. In the other, inclusions are toxic by coaggregating with growth factors and proteins crucial for normal cellular activity. Through a fortuitous discovery of early-formed and mature types of mHttex1 inclusions by applying a genetically-encoded biosensor of mHttex1 conformation, we describe a mechanism that ties these two models together. Cells with soluble mHttex1 lived for a shorter period than the ones with inclusions and exclusively died via apoptosis. Early-formed inclusions contained ribonucleoproteins, mRNA splicing and processing machinery. As aggregation progressed, chaperones and aggregation-prone proteins were recruited into mature inclusions that converted into amyloid state. Cells with mature inclusions became metabolically inactive and died exclusively via necrosis. Our data suggests inclusions arise via mHttex1 halting protein translation. Soluble mHttex1 appears to be the trigger for apoptosis in the system. Inclusion formation sequesters soluble mHttex1 and correlates with a reduced risk of apoptosis. The prolonged survival of cells with inclusions correlates with progressive metabolic dysfunction, a failed attempt to clear inclusions and ultimate switch from apoptosis to necrosis.
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ItemMetabolomics reveals the relationship between the host and the gut in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome( 2017)Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disorder that likely has a complex etiological mechanism entailing predisposing, triggering, and maintaining factors. ME/CFS patients present with incapacitating long-term post-exertional malaise and a broad array of neurological, immunological and gastrointestinal symptoms. Few studies have looked to detect the basis of the energy depletion and the gastrointestinal symptoms; both are observable by metabolomics. Metabolomics is the study of metabolites, any small organic molecule, including amino acids, sugars, lipids and more. Measuring metabolites in biofluids has been performed for decades, but the development of fast, automated and standardised methods has led to the “-omics” suffix. Previous metabolite studies on ME/CFS have revealed alterations that presented some clues of amino acid and oxidative stress disturbance in ME/CFS. These studies were not using standardised metabolomics techniques and added no gastrointestinal context. In this thesis, the first application of metabolomics to study ME/CFS is described from an initial preliminary study on blood to a larger study on host metabolite changes and their association with gut metabolites and bacteria. All metabolomics research in this thesis was conducted using NMR spectroscopy. The preliminary study was on blood samples from 11 ME/CFS and 10 non-ME/CFS subjects. Glutamine and ornithine were found to be reduced in ME/CFS, and correlations of these metabolites with other metabolites revealed relationships existing between glucogenic amino acids and metabolites that participate in the urea cycle. The second study was on a much larger all-female cohort, 34 ME/CFS and 25 non-ME/CFS participants. Participants donated faeces, urine and blood samples in a timeframe for associations between biofluids to be made. In ME/CFS patients, the metabolite changes indicated a decreased glycolysis (or increased gluconeogenesis) was occurring. We surmised that this was reducing the adequate supply of acetyl-CoA to the citric acid cycle and proposed that amino acids were being increasingly utilised instead, as evidenced by a reduction in blood and urine amino acids. The prominent decrease in blood glutamate and increase in blood aspartate highlighted that aspartate transaminase (AST) might be upregulated to allow amino acids to fuel the TCA cycle. Further evidence for reduced glycolysis was an increase in creatinine, an alternate resource for anaerobic ATP production within muscle. A reperfusion pathway that produces superoxide and results in urinary allantoin accumulation was also observed in ME/CFS patients adding more evidence for increased oxidative stress. In the faeces, Clostridium spp. was increased and Bacteroides spp. were decreased, possibly indicating an alteration in microbes in ME/CFS was towards a scavenging phenotype. The faecal metabolites changes highlighted an increase in fermentation was occurring in the colon of ME/CFS producing SCFA accumulation. Furthermore, the increase in isovalerate suggests that amino acids were increasingly being used for fermentation in ME/CFS patients. We propose that the increasing use of amino acids for ATP production in the host has reduced the production of digestive enzymes, gut bacteria are provided with increased dietary substrate and increased provisions of amino acids in the colon result in increased SCFA and isovalerate via fermentation.
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ItemUnderstanding the role of Maurer’s clefts in virulence protein trafficking( 2017)The malaria parasite Plasmodium falciparum modifies the host red blood cell to establish virulence protein-trafficking pathways. The major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) is exported from the parasite to the red blood cell surface, where it mediates attachment of the infected cell to ligands on the host vascular endothelium. This process of sequestration enables infected red blood cells to avoid immune detection in the spleen and contributes to the development of severe malaria. The Maurer’s clefts are organelles formed by the parasite and are present in the red blood cell cytoplasm. The primary function of Maurer’s clefts is thought to be the transport of PfEMP1 to the red blood cell membrane. We investigate a Maurer’s clefts protein, ring-exported protein-1 (REX1) and its role in PfEMP1 trafficking. We show that Maurer’s clefts morphology is disrupted by knocking down REX1 and that PfEMP1 surface display is decreased. Using transfectant parasites expressing truncated forms of the protein, we identify a repeat region of REX1 that mediates Maurer’s clefts morphology and is required for efficient PfEMP1 trafficking. We have developed a method to enrich the Maurer’s clefts from infected red blood cells and define their protein composition by tandem mass spectrometry. We epitope-tag a number of putative and established Maurer’s clefts proteins and confirm the location of several novel Maurer’s clefts proteins. Using super-resolution microscopy, we localise Maurer’s clefts proteins to subcompartments within these organelles. Finally, we use co- precipitation to describe a protein interaction network at the Maurer’s clefts.
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ItemRole of Rab11 and Rab30 in regulating the endosome-trans-Golgi network( 2017)The trans-Golgi network (TGN) and the recycling endosomes constitute two major intracellular sorting hubs which are spatially close to each other and are extensively involved in both anterograde and retrograde trafficking pathways. There is considerable membrane flux between the two compartments to account for the flow of cargo. It is therefore pertinent for the TGN and the recycling endosomes to maintain their organelle identities and spatial positioning, processes mediated through recruitment of cytosolic proteins such as Rabs. Here I have analysed the role of two Rabs on TGN and endosomal biology in detail, Rab11 and Rab30, which I have shown are localised to both the recycling endosomes and the TGN. Two members of the Rab11 family, Rab11a and Rab11b are primarily localised to the recycling endosomes with a small proportion localised to the TGN whereas Rab30 is found extensively in both compartments. Rab11a and Rab11b share a high level of sequence homology and although extensively studied, functional disparities between the two Rab11 isoforms have not been completely defined. In Chapter 3, I generated HeLa stable cell lines devoid of either Rab11a or Rab11b using the CRISPR/Cas9 system and functionally dissected their roles in trafficking within the endosome-TGN system. Both Rab11 and Rab11b were required to maintain the morphology of the recycling endosomes, however, only the deletion of Rab11a had a significant effect on the recycling of transferrin receptor. Deletion of either Rab11a or Rab11b resulted in the formation of enlarged early endosomes, leading to a perturbation in the endosomal-lysosomal maturation pathway in Rab11a knockout cells with an associated functional defect in intracellular recycling of the cation-independent mannose 6-phosphate receptor between the late endosomes and the TGN. In addition, deletion of Rab11a, but not Rab11b, resulted in a perturbation in the biogenesis of primary cilia, generating longer primary cilia. Overall, these results indicate that Rab11a and Rab11b have some overlap in function however there are some clear functional differences associated with the biology of endosomal-lysosomal maturation and ciliogenesis. In Chapter 4, I demonstrated that Rab30 is localised not only to the TGN but also the recycling endosomes in a range of cell types. By analysing cargo trafficking in Rab30-silenced HeLa cells, I have shown that Rab30 plays a role in retrograde trafficking but has no apparent role in cargo recycling to the plasma membrane. Silencing of Rab30 resulted in the dispersal of both the endosomal and Golgi compartments in HeLa cells whereas embryonic cortical neurons from CRISPR/Cas9 Rab30-/- mice had aberrant dendrite morphogenesis. These data suggest a role for Rab30 in maintaining organelle positioning possibly through associations with cytoskeletal proteins. I also demonstrated that Rab30 regulates autophagosome biogenesis from the recycling endosome and TGN membranes possibly through the AP1-mediated membrane transport pathway. In summary, although Rab11 and Rab30 are mainly localised at the TGN-recycling endosome nexus, the importance of these Rab proteins extend beyond their primary location and I have shown that they influence not only cargo trafficking but also organelle morphology and distribution, endosomal-lysosomal maturation, ciliogenesis and autophagy. Rab proteins are thus master regulators of intracellular processes made possible via associations with other components of the trafficking machinery.
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ItemMechanistic studies on radical reactions initiated by oxidative electron transfer( 2017)This thesis is concerned with intermolecular radical addition reactions to alkynes. The objective of this work was to explore the generation of azidyl radicals by oxidation of inorganic azide and the reaction of azidyl radicals with C≡C triple bonds. Preliminary experiments revealed cerium (IV) ammonium nitrate is a good oxidation source to generate azidyl radicals from sodium azide. Reactions of azidyl radicals with terminal aromatic alkynes led to three unique chemical conversions, which were identified through a combination of characterisation techniques, such as X-ray crystallography. Electronic effects appear to play a role in the outcome of the functionality at the C≡C triple bond. Reaction of an azidyl radical with electron rich alkyne 65 results in the formation of acyl cyanide 69. In contrast, reaction of an azidyl radical with electron poor alkyne 66 provides nitrile 93. Following investigation of the reaction using experimental and computational studies, the azidyl radical appears to undergo intermolecular radical addition to terminal aromatic alkyne 94 to form a cyclic radical intermediate 98, which then undergoes oxidation to form cyclic cation intermediate 99. Addition of a nitrate ligand at the or position to the aryl ring, followed by ring opening can account for the structurally different products. Acyl cyanide 69 may undergo post-reaction oxygen exchange with water, to account for the incorporation of oxygen-18 labeled water in 69 through isotopic labeling experiments. Aryl alkyne 111, with a para-ethyl substituent, afforded isoxazole 112 and acyl cyanide 113 as major products. Formation of isoxazole 112 may occur via a cyclisation reaction between acyl cyanide and azido-nitrate intermediates, followed by expulsion of NO2– and loss of N3+. Finally, reactions of azidyl radicals with internal aromatic alkynes resulted in the formation of diketones. The persulfate radical anion, generated photochemically or thermally from potassium peroxydisulfate, was investigated as an oxidant. Persulfate radical anions were also found to be an efficient oxidation source, generating azidyl radicals from azide anions. Reaction of azidyl radicals with terminal aromatic alkyne 65 in the presence of potassium peroxydisulfate afforded NH-1,2,3-triazole 162 as the major product, along with nitrile 163 and isoxazole 164. Formation of NH-1,2,3-triazole 162 supported the computational studies, indicating azidyl radical addition to alkynes occurs to form a cyclic radical intermediate. Further calculations indicated that the cyclic radical intermediate could undergo reduction by an azide anion for subsequent triazole formation. Reactions performed with potassium peroxydisulfate as the oxidant were more efficient when initiated thermally than photochemically to generate persulfate radical anions. Reaction of internal aromatic alkyne 27 with azidyl radicals resulted in 2H-1,2,3-triazole 179 and 2-substituted-1,2,3-triazole 180, in the presence of potassium peroxydisulfate. Numerous other oxidants were investigated at length to generate azidyl radicals from inorganic azide. Experiments with these oxidants resulted in complex mixtures, low yielding products and/or no reaction. No reactions were observed between azidyl radicals and aliphatic alkynes.
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ItemClimate adaptation in Eucalyptus microcarpa (Grey Box) and implications for conservation( 2017)Restoration is an important component of conservation management, especially in highly modified landscapes. In the face of rapid environmental change, the mere presence of vegetation doesn’t necessarily equate to the long term sustainability of populations. Rather, there is a need to consider evolutionary potential in conservation planning, including restoration. This thesis investigates two key components of evolutionary potential pertinent to restoration – namely genetic diversity and climate adaptation – in an important restoration tree species in south-eastern Australia, Eucalyptus microcarpa. This thesis aims to understand how genetic diversity and local adaptation are distributed across the range of E. microcarpa and how this knowledge may help inform seed sourcing and enhance resilience of restoration plantings under climate change. To begin, I use a landscape genomic approach to explore genomic diversity in E. microcarpa and how diversity in small habitat remnants and revegetation (restored) sites compare to large remnants. This work found that small, habitat remnants and revegetation sites largely, but not completely captured patterns of genomic diversity across the landscape. Whilst overall genomic diversity was similar between site types, patterns of diversity across the genome varied between site types. These results suggest important genomic differences between site types that may influence future adaptive potential of revegetation sites and small habitat remnants. I then investigated adaptation to climate in E. microcarpa using multiple approaches. Firstly, using a landscape genomic approach, I found evidence of genomic climate adaptation in E. microcarpa. These results suggest climate adaptation to be a genome-wide phenomenon, involving many genes and genomic regions. Exploration of genomic changes that may be required to match projected climate change suggest adaptation to be via shifts in allele frequency from standing variation. In addition to suggesting a number of climate variables associated with adaptation in E. microcarpa, these results highlight the importance of genetic diversity and standing variation for maintaining adaptive potential. Utilising existing genetic resources for this species, I found evidence of heritable, genetic variation in growth and leaf traits of E. microcarpa growing in a provenance trial. Furthermore, significant trait variation between provenances and associations with climate variables suggest climate as a driver of adaptive differences. Finally, I combined the independent genomic and phenotypic analyses to provide stronger support for climate adaptation in E. microcarpa, including links between genomic variants and adaptive traits. Associations between traits and single nucleotide polymorphisms (SNPs) using putatively adaptive SNPs genotyped in provenance trial trees validated genomic results, suggesting some trait variation could be explained by these SNPs. Furthermore, links between all three sources of variation relevant to local adaptation – genotype, phenotype and climate – corroborated findings of the two independent analyses. This approach therefore provides greater support for adaptation to climate in E. microcarpa. Together these analyses address the current genetic state of restoration in E. microcarpa as well as the structure of genetic diversity and climate adaptation across its distribution. These results suggest adaptive differences within E. microcarpa that could be utilised to enhance evolutionary potential within restoration plantings.
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ItemMolecular characterisation of the amyloid precursor protein: a key biomolecule in Alzheimer's disease( 2017)The amyloid precursor protein (APP) is strongly implicated in Alzheimer’s disease pathogenesis due to the observed aggregation of its Aβ sequence in the brain of AD patients. Despite being a molecule of intense interest, a sound molecular understanding of the normal functions of APP remains elusive. This work provides molecular insights into two ostensibly significant interactions involving the extracellular domain of APP, namely: (i) copper binding; and (ii) heparin-induced homo-dimerization. Biological copper is available in two oxidation states: Cu(I) and Cu(II). Well-characterised spectroscopic probes for Cu(I) binding studies are available, but reliable probes for Cu(II) binding studies are lacking. This work has developed a new method for in vitro characterisation of Cu(II) binding affinities. This involved the design and characterisation of four fluorescent peptide-based probes which respond to the binding of paramagnetic Cu(II) ions. Each displays a different affinity for the metal and together they are capable of detecting Cu(II) binding from micromolar to femtomolar range. This allows quantification of the Cu(II) binding affinities of individual protein targets on a unified scale. These probes facilitated studies of copper binding in the APP ectodomain. Biological studies performed over the last two decades have suggested that APP has a role in maintaining cellular copper homeostasis and it is commonly cited as a copper-binding protein. Several putative copper binding sites in the extracellular domain have previously been identified using structural techniques (NMR, X-ray crystallography). However, the chemistry of these has never been systematically studied under uniform conditions and the preferred metal coordination site is unknown. This work has characterised the thermodynamics of Cu(I) and Cu(II) binding in the APP ectodomain. The preferred binding site for both metal oxidation states is a (His)4 centre of picomolar affinity in the α-helical E2 domain. Interestingly, copper coordination leads to conformational changes in this flexible subdomain which could be significant in a cellular context. Homo- and hetero-dimeric interactions between APP and its ‘amyloid precursor-like protein’ homologues (APLP1 and APLP2) have been observed in many previous in vivo studies. These interactions are promoted by the binding of heparins (glycosaminoglycan chains abundant in the extracellular space). The dimers are thought to play a critical role in the normal functions of this protein family including trans-cellular adhesion and synaptogenesis. Detailed structural studies of these dimeric assemblies have not previously been performed. This is largely due to the size and heterogeneity of these complexes which present challenges for high-resolution techniques such as NMR and X-ray crystallography. However, homo-dimeric structures have been solved for two isolated extracellular domains (E1 and E2) which each contain heparin-binding regions. In this work, a chemical cross-linking mass spectrometry approach is used to provide structural information about the dimeric assembly of the complete secreted ectodomain sAPPα. The results offer direct proof of a parallel dimeric interaction between two sAPPα monomers. Additionally, this provides a means to evaluate E1 and E2 homo-dimeric models within the context of the overall ectodomain structure. This preliminary study provides a basis for further investigation of homo- and hetero-dimeric interactions involving APP homologues.