Tara Pukala

Chemical Cross-linking and Mass Spectrometry for the Structural Analysis of Protein Assemblies

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13164

Abstract: Cellular functions are performed and regulated at a molecular level by the coordinated action of intricate protein assemblies, and hence the study of protein folding, structure, and interactions is vital to the appreciation and understanding of complex biological problems. In the past decade, continued development of chemical cross-linking methodologies combined with mass spectrometry has seen this approach develop to enable detailed structural information to be elucidated for protein assemblies often intractable by traditional structural biology methods. In this review article, we describe recent advances in reagent design, cross-linking protocols, mass spectrometric analysis, and incorporation of cross-linking constraints into structural models, which are contributing to overcoming the intrinsic challenges of the cross-linking method. We also highlight pioneering applications of chemical cross-linking mass spectrometry approaches to the study of structure and function of protein assemblies.

Ion Mobility Mass Spectrometry Studies of the Inhibition of Alpha Synuclein Amyloid Fibril Formation by ( - )-Epigallocatechin-3-Gallate

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/CH10334

Abstract: Ion mobility-mass spectrometry (IM-MS) is emerging as an important biophysical technique for the structural analysis of proteins and their assemblies, in particular for structurally heterogeneous systems such as those on the protein misfolding and aggregation pathway. Using IM-MS we have monitored amyloid fibril formation of A53T α-synuclein, a mutant synuclein protein associated with Parkinson’s disease, and identified that a conformational change towards a more compact structure occurs during the initial stages of aggregation. Binding of A53T α-synuclein to a flavenoid based amyloid fibril inhibitor, (–)-epigallocatechin-3-gallate, has been observed with a 1:1 stoichiometry. By analysis of ion collision cross-sections, we show epigallocatechin gallate binding prevents protein conformational change, and in turn decreases the formation of fibrillar aggregates.

Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.JBC.2022.102392

Rationally designed peptide-based inhibitor of Aβ42 fibril formation and toxicity: a potential therapeutic strategy for Alzheimer's disease

Publisher: Portland Press Ltd.

Date: 10-06-2020

DOI: 10.1042/BCJ20200290

Abstract: Amyloid beta peptide (Aβ42) aggregation in the brain is thought to be responsible for the onset of Alzheimer's disease, an insidious condition without an effective treatment or cure. Hence, a strategy to prevent aggregation and subsequent toxicity is crucial. Bio-inspired peptide-based molecules are ideal candidates for the inhibition of Aβ42 aggregation, and are currently deemed to be a promising option for drug design. In this study, a hexapeptide containing a self-recognition component unique to Aβ42 was designed to mimic the β-strand hydrophobic core region of the Aβ peptide. The peptide is comprised exclusively of D-amino acids to enhance specificity towards Aβ42, in conjunction with a C-terminal disruption element to block the recruitment of Aβ42 monomers on to fibrils. The peptide was rationally designed to exploit the synergy between the recognition and disruption components, and incorporates features such as hydrophobicity, β-sheet propensity, and charge, that all play a critical role in the aggregation process. Fluorescence assays, native ion-mobility mass spectrometry (IM-MS) and cell viability assays were used to demonstrate that the peptide interacts with Aβ42 monomers and oligomers with high specificity, leading to almost complete inhibition of fibril formation, with essentially no cytotoxic effects. These data define the peptide-based inhibitor as a potentially potent anti-amyloid drug candidate for this hitherto incurable disease.

Differential proteome analysis of the leaves of lead hyperaccumulator, Rhoeo discolor (L. Her.) Hance

Publisher: Wiley

Date: 27-11-2020

DOI: 10.1002/JMS.4689

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Publisher: American Chemical Society (ACS)

Date: 30-05-2012

DOI: 10.1007/S13361-012-0407-X

Abstract: Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.

Structural insights into the antifungal drug target guanosine monophosphate synthase from Aspergillus fumigatus

Publisher: International Union of Crystallography (IUCr)

Date: 26-01-2022

DOI: 10.1107/S2059798321012031

Abstract: Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus , Candida albicans and Cryptococcus neoformans . However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Å resolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC 50 = 16.6 ± 2.4 µ M ) and 6-diazo-5-oxo-L-norleucine (IC 50 = 29.6 ± 5.6 µ M ) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug-discovery endeavours.

Polyphenol characterisation and diverse bioactivities of native Australian lilly pilly (Syzygium paniculatum) extract

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2FO01305C

Abstract: The magenta lilly pilly ( Syzygium paniculatum ) has been characterised as a high-yielding source of phenolic compounds, particularly anthocyanins, with the berry fruit extracts shown to have broad bioactivity of potential benefit to human health.

DNA triplex structure, thermodynamics, and destabilisation: insight from molecular simulations

Publisher: Royal Society of Chemistry (RSC)

Date: 2018

DOI: 10.1039/C8CP02385A

Abstract: Molecular dynamics simulations are used to elucidate the structure and thermodynamics of DNA triplexes associated with the neurodegenerative disease Friedreich's ataxia (FRDA), as well as complexes of these triplexes with the small molecule netropsin, which is known to destabilise triplexes.

Reduction of Copper(II) to Copper(I) in the Copper-Curcumin Complex Induces Decomposition of Curcumin

Publisher: CSIRO Publishing

Date: 2012

DOI: 10.1071/CH12081

Abstract: We report the decomposition of curcumin due to reduction of Cu(ii) to Cu(i). Cu(ii) binds tightly with curcumin to form a complex which exhibits a high stability in methanol, but it decomposes readily in acetonitrile and in SDS micelles in the presence of ascorbic acid, coincident with reduction of Cu(ii) to Cu(i). In this study, the UV-Vis absorption of the Cu-curcumin complex shows a monotonic decrease as a function of time, consistent with the decomposition of curcumin. At a high copper : curcumin molar ratio of 10 : 1, the UV-Vis absorption spectrum of the Cu(ii)-curcumin complex in acetonitrile exhibits a substantial blue shift of the absorption maximum from 420 nm to 350 nm, which is indicative of a significant decrease in conjugation length of curcumin in the presence of Cu(ii). Time-dependent mass spectrometry and high performance liquid chromatography (HPLC) data are also consistent with the decomposition of curcumin as a consequence of reduction of Cu(ii) to Cu(i).

Preliminary Development and Testing of C595 Radioimmunoconjugates for Targeting MUC1 Cancer Epitopes in Pancreatic Ductal Adenocarcinoma

Publisher: MDPI AG

Date: 24-09-2022

DOI: 10.3390/CELLS11192983

Abstract: Mucin 1 is a transmembrane glycoprotein which overexpresses cancer-specific epitopes (MUC1-CE) on pancreatic ductal adenocarcinoma (PDAC) cells. As PDAC is a low survival and highly aggressive malignancy, developing radioimmunoconjugates capable of targeting MUC1-CE could lead to improvements in PDAC outcomes. The aim of this study was to develop and perform preliminary testing of diagnostic and therapeutic radioimmunoconjugates for PDAC using an anti-MUC1 antibody, C595. Firstly, p-SCN-Bn-DOTA was conjugated to the C595 antibody to form a DOTA-C595 immunoconjugate. The stability and binding affinity of the DOTA-C595 conjugate was evaluated using mass spectrometry and ELISA. DOTA-C595 was radiolabelled to Copper-64, Lutetium-177, Gallium-68 and Technetium-99m to form novel radioimmunoconjugates. Cell binding assays were performed in PANC-1 (strong MUC1-CE expression) and AsPC-1 (weak MUC1-CE expression) cell lines using 64Cu-DOTA-C595 and 177Lu-DOTA-C595. An optimal molar ratio of 4:1 DOTA groups per C595 molecule was obtained from the conjugation process. DOTA-C595 labelled to Copper-64, Lutetium-177, and Technetium-99m with high efficiency, although the Gallium-68 labelling was low. 177Lu-DOTA-C595 demonstrated high cellular binding to the PANC-1 cell lines which was significantly greater than AsPC-1 binding at concentrations exceeding 100 nM (p 0.05). 64Cu-DOTA-C595 showed similar binding to the PANC-1 and AsPC-1 cells with no significant differences observed between cell lines (p 0.05). The high cellular binding of 177Lu-DOTA-C595 to MUC1-CE positive cell lines suggests promise as a therapeutic radioimmunoconjugate against PDAC while further work is required to harness the potential of 64Cu-DOTA-C595 as a diagnostic radioimmunoconjugate.

Retro Diels–Alder Fragmentation of Fulvene–Maleimide Bioconjugates for Mass Spectrometric Detection of Biomolecules

Publisher: American Chemical Society (ACS)

Date: 31-08-2021

DOI: 10.1021/ACS.ANALCHEM.1C00193

Structural Analysis of Calmodulin Binding by nNOS Inhibitory Amphibian Peptides

Publisher: American Chemical Society (ACS)

Date: 30-12-2014

DOI: 10.1021/BI5004124

Abstract: Calmodulin (CaM) is a ubiquitous protein in nature and plays a regulatory role in numerous biological processes, including the upregulation of nitric oxide (NO) synthesis in vivo. Several peptides that prevent NO production by interacting with CaM have been isolated in the cutaneous secretions of Australian hibians, and are thought to serve as a defense mechanism against predators. In this work, we probe the mechanism by which three of these peptides, namely, caerin 1.8, dahlein 5.6, and a synthetic modification of citropin 1.1, interact with CaM to inhibit NO signaling. Isothermal titration calorimetry was used to determine thermodynamic parameters of the binding interactions and revealed that all the peptides bind to CaM in a similar fashion, with the peptide encapsulated between the two lobes of CaM. Ion mobility-mass spectrometry was used to investigate the changes in collision cross section that occur as a result of complexation, providing additional evidence for this binding mode. Finally, nuclear magnetic resonance spectroscopy was used to track chemical shift changes upon binding. The results obtained confirm that these complexes adopt canonical collapsed structures and demonstrate the strength of the interaction between the peptides and CaM. An understanding of these molecular recognition events provides insights into the underlying mechanism of the hibian host-defense system.

Ecklonia radiata extract containing eckol protects neuronal cells against Aβ_1–42 evoked toxicity and reduces aggregate density

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0FO01438A

Abstract: Ecklonia radiata extract and phlorotannin fraction containing predominantly eckol-type phlorotannins inhibited the cytotoxicity of Aβ 1–42 in neuronal PC-12 cells and evoked a reduction in the density of Aβ 1–42 aggregates.

Binding studies of nNOS-active amphibian peptides and Ca²⁺calmodulin, using negative ion electrospray ionisation mass spectrometry

Publisher: Wiley

Date: 13-10-2008

DOI: 10.1002/RCM.3757

Abstract: Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin eptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the hibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied hibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the hibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.

Electron transfer ferredoxins with unusual cluster binding motifs support secondary metabolism in many bacteria

Publisher: Royal Society of Chemistry (RSC)

Date: 2018

DOI: 10.1039/C8SC01286E

Abstract: Unusual ferredoxins with different iron–sulfur cluster binding motifs support natural product biosynthesis in a wide range of bacteria.

Structural and mechanistic insights into amyloid‐β and α‐synuclein fibril formation and polyphenol inhibitor efficacy in phospholipid bilayers

Publisher: Wiley

Date: 26-07-2021

DOI: 10.1111/FEBS.16122

Abstract: Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid‐β 40 and α‐synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well‐known polyphenol fibril inhibitors, (‐)‐epigallocatechin gallate (EGCG) and resveratrol however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off‐pathway structures however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.

Chemical Synthesis of a Fluorescent IGF-II Analogue

Publisher: Springer Science and Business Media LLC

Date: 07-12-2012

DOI: 10.1007/S10989-012-9339-3

The Unusual Metalloprotease-Rich Venom Proteome of the Australian Elapid Snake Hoplocephalus stephensii

Publisher: MDPI AG

Date: 28-04-2022

DOI: 10.3390/TOXINS14050314

Abstract: The Australasian region is home to the most erse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents however, their venom compositions are poorly known. The Australian elapid Hoplocephalus stephensii (Stephen’s banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of H. stephensii. 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP 36.7% three identified toxins), phospholipase A2 (PLA2 24.0% five identified toxins), three-finger toxin (3FTx 10.2% two toxins) and snake venom serine protease (SVSP 5.9% one toxin Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO 10.8% one toxin), natriuretic peptide (NP 0.8% two toxins), cysteine-rich secretory protein (CRiSP 1.7% two toxins), c-type lectin (CTL 1.1% one toxin), and one minor protein family, nerve growth factor (NGF 0.8% one toxin). The venom composition of H. stephensii differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. H. stephensii venom appeared to have less toxin ersity than other elapids, with only 18 toxins making up three-quarters of the venom.

In-plane implanting carbon rings into carbon nitride to intrigue nonradical photodegradation

Publisher: Elsevier BV

Date: 03-2024

DOI: 10.1016/J.APCATB.2023.123363

The Human Amyloid Precursor Protein Binds Copper Ions Dominated by a Picomolar-Affinity Site in the Helix-Rich E2 Domain

Publisher: American Chemical Society (ACS)

Date: 12-06-2018

DOI: 10.1021/ACS.BIOCHEM.8B00572

Abstract: A manifestation of Alzheimer's disease (AD) is the aggregation in the brain of amyloid β (Aβ) peptides derived from the amyloid precursor protein (APP). APP has been linked to modulation of normal copper homeostasis, while dysregulation of Aβ production and clearance has been associated with disruption of copper balance. However, quantitative copper chemistry on APP is lacking, in contrast to the plethora of copper chemistry available for Aβ peptides. The soluble extracellular protein domain sAPPα (molar mass including post-translational modifications of ∼100 kDa) has now been isolated in good yield and high quality. It is known to feature several copper binding sites with different affinities. However, under Cu-limiting conditions, it binds either Cu(I) or Cu(II) with picomolar affinity at a single site (labeled M1) that is located within the APP E2 subdomain. M1 in E2 was identified previously by X-ray crystallography as a Cu(II) site that features four histidine side chains (H313, H386, H432, and H436) as ligands. The presence of Cu

Host-defence peptides from the glandular secretions of amphibians: structure and activity

Publisher: Royal Society of Chemistry (RSC)

Date: 2006

DOI: 10.1039/B512118N

Fabrication of Piperazine Functionalized Polymeric Monolithic Tip for Rapid Enrichment of Glycopeptides/Glycans

Publisher: American Chemical Society (ACS)

Date: 16-12-2019

DOI: 10.1021/ACS.ANALCHEM.9B02068

Abstract: Enrichment strategies are designed for the pretreatment of low-abundance glycans and glycopeptides prior to mass spectrometric (MS) analysis. Here, a tip-based strategy is being reported for the enrichment of glycopeptides and glycans using a piperazine modified polymeric monolithic tip. The tip is fabricated using the free radical polymerization. Fast separation (2 min) is achieved under optimized conditions with 20 cycles per step of loading, incubation, washing, and elution followed by MALDI-MS analysis. A total of 25, 22, and 34 glycopeptides covering all glycosylation sites are enriched by the modified tips from tryptic digests of horse radish peroxidase, chicken avidin, and human immunoglobulin G, respectively. Piperazine exhibits high selectivity 1:400 horse radish peroxidase/bovine serum albumin, sensitivity to 100 attomoles, recovery 89.51%, and batch to batch reproducibility (RSD > 1) in glycopeptides enrichment. Piperazine tips also enrich glycans from ovalbumin and human immunoglobulin G. High selectivity (1:1200, ovalbumin/BSA) and detection limit of 100 attomole is attained for glycans and furthermore 58 glycans are enriched from human serum. Thus, piperazine tips can be used as an enrichment tool for swift, cost-effective routine analysis of biological s les for separation of glycopeptides and glycans.

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.PEPTIDES.2004.03.006

Norbornene probes for the study of cysteine oxidation

Publisher: Elsevier BV

Date: 03-2018

DOI: 10.1016/J.TET.2017.11.011

Advanced resistance studies identify two discrete mechanisms in Staphylococcus aureus to overcome antibacterial compounds that target biotin protein ligase

Publisher: MDPI AG

Date: 06-04-2020

DOI: 10.3390/ANTIBIOTICS9040165

Abstract: Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound ( −9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici

Publisher: Elsevier BV

Date: 09-2020

DOI: 10.1016/J.ABB.2020.108509

Polyphenol Honokiol and Flavone 2′,3′,4′-Trihydroxyflavone Differentially Interact with α-Synuclein at Distinct Phases of Aggregation

Publisher: American Chemical Society (ACS)

Date: 13-11-2020

DOI: 10.1021/ACSCHEMNEURO.0C00654

Hemin as a generic and potent protein misfolding inhibitor

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.BBRC.2014.10.062

Abstract: Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer's disease, Parkinson's disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

Conditions for Analysis of Native Protein Structures Using Uniform Field Drift Tube Ion Mobility Mass Spectrometry and Characterization of Stable Calibrants for TWIM-MS

Publisher: American Chemical Society (ACS)

Date: 15-10-2018

DOI: 10.1007/S13361-018-2074-Z

Abstract: Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A

Disulfide-containing peptides from the glandular skin secretions of froglets of the genus Crinia: Structure, activity and evolutionary trends

Publisher: Elsevier BV

Date: 11-2008

DOI: 10.1016/J.REGPEP.2008.06.004

Abstract: The skin secretions of Crinia signifera, C. riparia and C. deserticola contain bioactive disulfide-containing peptides. Signiferin 1 (RLCIPYIIPC-OH) from C. signifera and C. deserticola) contracts smooth muscle at a concentration of 10(-9) M, and effects proliferation of lymphocytes at 10(-6) M. In contrast, riparin 1.1 (RLCIPVIFC-OH) and riparin 1.2 (FLPPCAYKGTC-OH) from C. riparia show lymphocyte activity but do not contract smooth muscle. The lymphocyte and smooth muscle activities involve CCK2R. 3D structures of signiferin 1 and riparin 1.1 have been established using 2D NMR methods: these studies show significant differences in the shapes of the disulfide rings and with the orientations of the N-terminal residues. cDNA cloning establishes that the pre sections of the precursor pre-pro-riparin 1.4-1.6 peptides are different from the conserved pre regions of disulfide-containing antimicrobial peptides from species of the genus Rana found in the northern hemisphere and caerin antimicrobial peptides isolated from Australian tree frogs of the genus Litoria. This suggests that (i) either that riparins 1 have converged to similar structure and function to the ranid and hyloid prepropeptides which were lost initially from the myobatrachid lineage, or (ii) the prepropeptides in all three groups were derived from a single ancestral form that has remained relatively conserved in the hyloid and ranoid lineages but has undergone substantial ergent evolution in the myobatrachids.

Interrogating the higher order structures of snake venom proteins using an integrated mass spectrometric approach

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.JPROT.2020.103680

PPARγ Corepression Involves Alternate Ligand Conformation and Inflation of H12 Ensembles

Publisher: American Chemical Society (ACS)

Date: 05-05-2023

DOI: 10.1021/ACSCHEMBIO.2C00917

Native mass spectrometry identifies an alternative DNA-binding pathway for BirA from Staphylococcus aureus

Publisher: Springer Science and Business Media LLC

Date: 26-02-2019

DOI: 10.1038/S41598-019-39398-6

Abstract: An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus . The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation. The mechanism of BirA regulated transcription has been extensively characterized in Escherichia coli , but less so in other bacteria. Biotin-induced homodimerization of E. coli BirA ( Ec BirA) is a necessary prerequisite for stable DNA binding and transcriptional repression. Here, we employ a combination of native mass spectrometry, in vivo gene expression assays, site-directed mutagenesis and electrophoretic mobility shift assays to elucidate the DNA binding pathway for S. aureus BirA ( Sa BirA). We identify a mechanism that differs from that of Ec BirA, wherein Sa BirA is competent to bind DNA as a monomer both in the presence and absence of biotin and/or MgATP, allowing homodimerization on the DNA. Bioinformatic analysis demonstrated the Sa BirA sequence used here is highly conserved amongst other S. aureus strains, implying this DNA-binding mechanism is widely employed.

Fractional Deletion of Compound Kushen Injection Indicates Cytokine Signaling Pathways are Critical for its Perturbation of the Cell Cycle

Publisher: Springer Science and Business Media LLC

Date: 02-10-2019

DOI: 10.1038/S41598-019-50271-4

Abstract: We used computational and experimental biology approaches to identify candidate mechanisms of action of aTraditional Chinese Medicine, Compound Kushen Injection (CKI), in a breast cancer cell line (MDA-MB-231). Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis. The initial fractionation separated major from minor compounds, and it showed that major compounds accounted for little of the activity of CKI. Furthermore, removal of no single major compound altered the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical with respect to CKI activity. Transcriptome analysis was used to correlate compound removal with gene expression and phenotype data. Many compounds in CKI are required to trigger apoptosis but significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.

Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase

Publisher: Wiley

Date: 22-02-2007

DOI: 10.1111/J.1742-4658.2007.05726.X

Abstract: Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Utility of an improved model of amyloid-beta (Aβ1-42) toxicity in Caenorhabditis elegans for drug screening for Alzheimer’s disease

Publisher: Springer Science and Business Media LLC

Date: 2012

DOI: 10.1186/1750-1326-7-57

From biomolecules to biogeochemistry: Exploring the interaction of an indigenous bacterium with gold

Publisher: Elsevier BV

Date: 10-2023

DOI: 10.1016/J.CHEMOSPHERE.2023.139657

Glycosylation heterogeneity and low abundant serum glycoproteins MS analysis by boronic acid immobilized Fe3O4@1,2-Epoxy-5-Hexene/DVB magnetic core shell nanoparticles

Publisher: Elsevier BV

Date: 12-2020

DOI: 10.1016/J.MICROC.2020.105351

Nucleoside selectivity of Aspergillus fumigatus nucleoside‐diphosphate kinase

Publisher: Wiley

Date: 06-11-2020

DOI: 10.1111/FEBS.15607

PPARγ in Complex with an Antagonist and Inverse Agonist: a Tumble and Trap Mechanism of the Activation Helix

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.ISCI.2018.06.012

Ion Mobility-Mass Spectrometry Reveals Details of Formation and Structure for GAA·TCC DNA and RNA Triplexes

Publisher: American Chemical Society (ACS)

Date: 19-10-2018

DOI: 10.1007/S13361-018-2077-9

Abstract: DNA and RNA triplexes are thought to play key roles in a range of cellular processes such as gene regulation and epigenetic remodeling and have been implicated in human disease such as Friedreich's ataxia. In this work, ion mobility-mass spectrometry (IM-MS) is used with supporting UV-visible spectroscopy to investigate DNA triplex assembly, considering stability and specificity, for GAA·TTC oligonucleotide sequences of relevance to Friedreich's ataxia. We demonstrate that, contrary to other ex les, parallel triplex structures are favored for these sequences and that stability is enhanced by increasing oligonucleotide length and decreasing pH. We also provide evidence for the self-association of these triplexes, consistent with a proposed model of higher order DNA structures formed in Friedreich's ataxia. By comparing triplex assembly using DNA- and RNA-based triplex-forming oligonucleotides, we demonstrate more favorable formation of RNA triplexes, suggesting a role for their formation in vivo. Finally, we interrogate the binding properties of netropsin, a known polyamide triplex destabilizer, with RNA-DNA hybrid triplexes, where preference for duplex binding is evident. We show that IM-MS is able to report on relevant solution-phase populations of triplex DNA structures, thereby further highlighting the utility of this technology in structural biology. Our data therefore provides new insights into the possible DNA and RNA assemblies that may form as a result of GAA triplet repeats. Graphical Abstract ᅟ.

Characterisation of Calmodulin Structural Transitions by Ion Mobility Mass Spectrometry

Publisher: CSIRO Publishing

Date: 2012

DOI: 10.1071/CH12047

Abstract: This study demonstrates the ability of travelling wave ion mobility-mass spectrometry to measure collision cross-sections of ions in the negative mode, using a calibration based approach. Here, negative mode ion mobility-mass spectrometry was utilised to understand structural transitions of calmodulin upon Ca2+ binding and complexation with model peptides melittin and the plasma membrane Ca2+ pump C20W peptide. Coexisting calmodulin conformers were distinguished on the basis of their mass and cross-section, and identified as relatively folded and unfolded populations, with good agreement in collision cross-section to known calmodulin geometries. Titration of calcium tartrate to physiologically relevant Ca2+ levels provided evidence for intermediately metalated species during the transition from apo- to holo-calmodulin, with collision cross-section measurements indicating that higher Ca2+ occupancy is correlated with more compact structures. The binding of two representative peptides which exemplify canonical compact (melittin) and extended (C20W) peptide-calmodulin binding models has also been interrogated by ion mobility mass spectrometry. Peptide binding to calmodulin involves intermediates with metalation states from 1–4 Ca2+, which demonstrate relatively collapsed structures, suggesting neither the existence of holo-calmodulin or a pre-folded calmodulin conformation is a prerequisite for binding target peptides or proteins. The biological importance of the different metal unsaturated calmodulin complexes, if any, is yet to be understood.

Bioactive polyphenol interactions with β amyloid: a comparison of binding modelling, effects on fibril and aggregate formation and neuroprotective capacity

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C5FO01281C

Abstract: Plant polyphenols such as the lignin honokiol pictured are able to bind to specific regions in the amyloid β oligomer and this may be associated with neuroprotective effects in neuronal cells.

Norbornene Probes for the Detection of Cysteine Sulfenic Acid in Cells.

Publisher: American Chemical Society (ACS)

Date: 20-03-2019

DOI: 10.1021/ACSCHEMBIO.8B01104

Abstract: Norbornene derivatives were validated as probes for cysteine sulfenic acid on proteins and in live cells. Trapping sulfenic acids with norbornene probes is highly selective and revealed a different reactivity profile than the traditional dimedone reagent. The norbornene probe also revealed a superior chemoselectivity when compared to a commonly used dimedone probe. Together, these results advance the study of cysteine oxidation in biological systems.

Host-defence peptide profiles of the skin secretions of interspecific hybrid tree frogs and their parents, female Litoria splendida and male Litoria caerulea

Publisher: Wiley

Date: 17-07-2006

DOI: 10.1111/J.1742-4658.2006.05358.X

Abstract: Five healthy adult female first-generation hybrid tree frogs were produced by interspecific breeding of closely related tree frogs Litoria splendida and L. caerulea in a cage containing large numbers of males and females of both species. Phylogenetic analysis of mitochondrial DNA sequences established the female parent to be L. splendida. The peptide profile of the hybrid frogs included the neuropeptide caerulein, four antibiotics of the caerin 1 family and several neuronal nitric oxide synthase inhibitors of the caerin 1 and 2 classes of peptides. The skin secretions of the hybrids contained some peptides common to only one parent, some produced by both parental species, and four peptides expressed by the hybrids but not the parental species.

Skin peptide and cDNA profiling of Australian anurans: Genus and species identification and evolutionary trends

Publisher: Elsevier BV

Date: 2011

DOI: 10.1016/J.PEPTIDES.2010.09.019

Abstract: Host defense peptides of 35 species of Australian frogs from the hylids Cyclorana and Litoria, and the myobatrachids Crinia, Limnodynastes and Uperoleia have been identified. The biological activities of the majority of these peptides have been determined and include hormones, neuropeptides, opioids, immunomodulators, membrane active peptides [including antimicrobial, anticancer, antiviral (enveloped viruses like HIV and Herpes) and antifungal peptides], neuronal nitric oxide synthase inhibitors, pheromones and in idual peptides with other specific activities. The host defense peptide skin profile can be diagnostic at both the species and higher taxonomic levels for ex le, species of Crinia, Litoria and Uperoleia each produce quite different types of peptides. Species of Cyclorana and Limnodynastes are more difficult to characterize by skin peptides alone: species of both genera produce similar peptides with no apparent activity. The skin peptide profiles of frogs from the genera Crinia, Litoria and Uperoleia may be used together with morphological and cognate methods, to differentiate between sub-species and even different population clusters of the same species. Nucleotide sequencing of cDNAs of precursors (pre-pro peptides) of bioactive peptides from the skin glands of various species of the genus Litoria show that the majority of these peptides originated from a single ancestor gene before the break away of Australia from Gondwana. The exceptions are the caerulein neuropeptides {e.g. caerulein [pEQDY(SO(3)H)TGWMDF(NH(2))]} which have a different origin to that of other Litoria peptides. Disulfide containing peptides from skin glands of species of Crinia show a different evolutionary route to peptides from species of Litoria.

The Amyloid Fibril-Forming Properties of the Amphibian Antimicrobial Peptide Uperin 3.5

Publisher: Wiley

Date: 16-12-2015

DOI: 10.1002/CBIC.201500518

Abstract: The hibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum hibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.

Engineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme

Publisher: American Chemical Society (ACS)

Date: 12-04-2023

DOI: 10.1021/JACS.3C01456

Escherichia coli YgiC and YjfC Possess Peptide─Spermidine Ligase Activity

Publisher: American Chemical Society (ACS)

Date: 06-02-2023

DOI: 10.1021/ACS.BIOCHEM.2C00592

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

Publisher: American Chemical Society (ACS)

Date: 02-05-2019

DOI: 10.1021/ACS.ANALCHEM.9B00658

Negative ion fragmentations of disulfide‐containing cross‐linking reagents are competitive with aspartic acid side‐chain‐induced cleavages

Publisher: Wiley

Date: 12-12-2012

DOI: 10.1002/RCM.6445

Abstract: It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide. Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid.

Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBAPAP.2014.04.013

Abstract: The accumulation of protein aggregates containing amyloid fibrils, with α-synuclein being the main component, is a pathological hallmark of Parkinson's disease (PD). Molecules which prevent the formation of amyloid fibrils or disassociate the toxic aggregates are touted as promising strategies to prevent or treat PD. In the present study, in vitro Thioflavin T fluorescence assays and transmission electron microscopy imaging results showed that gallic acid (GA) potently inhibits the formation of amyloid fibrils by α-synuclein. Ion mobility-mass spectrometry demonstrated that GA stabilises the extended, native structure of α-synuclein, whilst NMR spectroscopy revealed that GA interacts with α-synuclein transiently.

Histidine‐containing host‐defence skin peptides of anurans bind Cu²⁺. An electrospray ionisation mass spectrometry and computational modelling study

Publisher: Wiley

Date: 12-04-2011

DOI: 10.1002/RCM.4981

Abstract: Anuran peptides which contain His, including caerin 1.8 (GLFKVLGSVAKHLLPHVVPVIAEKL-NH(2)), caerin 1.2 (GLLGVLGSVAKHVLPHVVPVIAEHL-NH(2)), Ala(15) maculatin 1.1 (GLFGVLAKVAAHVVAIEHF-NH(2)), fallaxidin 4.1 (GLLSFLPKVIGHLIHPPS-OH), riparin 5.1 (IVSYPDDAGEHAHKMG-NH(2)) and signiferin 2.1 (IIGHLIKTALGMLGL-NH(2)), all form MMet(2+) and (M + Met(2+)-2H(+))(2+) cluster ions (where Met is Cu, Mg and Zn) following electrospray ionisation (ESI) in a Waters QTOF 2 mass spectrometer. Peaks due to Cu(II) complexes are always the most abundant relative to other metal complexes. Information concerning metal(2+) connectivity in a complex has been obtained (at least in part) using b and y fragmentation data from ESI collision-induced dissociation tandem mass spectrometry (CID MS/MS). Theoretical calculations, using AMBER version 10, show that MCu(2+) complexes with the membrane active caerin 1.8, Ala(15) maculatin 1.1 and fallaxidin 4.1 are four-coordinate and approximating square planar, with ligands including His and Lys, together with the carbonyl oxygens of particular backbone amide groups. When binding can occur through two His, or one His and one Lys, the His/Lys ligand structure is the more stable for the studied systems. The three-dimensional (3D) structures of the complexes are always different from the previously determined structures of the uncomplexed model peptides (using 2D nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking solvents like trifluoroethanol/water).

Efficient biocatalytic C–H bond oxidation: an engineered heme-thiolate peroxygenase from a thermostable cytochrome P450

Publisher: Royal Society of Chemistry (RSC)

Date: 2023

DOI: 10.1039/D3CC04626E

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

Publisher: Portland Press Ltd.

Date: 10-06-2020

DOI: 10.1042/BCJ20200297

Abstract: Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC–MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

The molecular chaperone β-casein prevents amorphous and fibrillar aggregation of α-lactalbumin by stabilisation of dynamic disorder

Publisher: Portland Press Ltd.

Date: 11-02-2020

DOI: 10.1042/BCJ20190638

Abstract: Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility–mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone β-casein (β-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between β-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility–mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of β-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein–chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms.

Structural Analysis and Identity Confirmation of Anthocyanins in Brassica oleracea Extracts by Direct Injection Ion Mobility-Mass Spectrometry

Publisher: American Chemical Society (ACS)

Date: 09-02-2023

DOI: 10.1021/ACSMEASURESCIAU.2C00058

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.BMCL.2013.09.071

Abstract: Many protein misfolding diseases, for ex le, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimer's disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

Better late than never: Optimising the proteomic analysis of field-collected octopus

Publisher: Public Library of Science (PLoS)

Date: 12-07-2023

DOI: 10.1371/JOURNAL.PONE.0288084

Abstract: Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the s ling and preparation of animal tissues from the field. Although RNA later is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require s les to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught s les. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNA later can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNA later immediately, 3 h, and 6 h after euthanasia. Processed tissue s les were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of s le treatment. However, nearly 10% additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that s ling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

Establishment Of The Protein Quantitation Centre Of South Australia (PQCSA)

Start Date: 2013

End Date: 2013

Funder: Australian Research Council

Strengthening Merit-based Access And Support At The New National Computing Infrastructure Petascale Supercomputing Facility

Start Date: 2012

End Date: 2012

Funder: Australian Research Council

Discovery Projects - Grant ID: DP170102033

Start Date: 2017

End Date: 06-2020

Amount: $323,500.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1093143

Start Date: 2010

End Date: 12-2013

Amount: $360,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180103974

Start Date: 2018

End Date: 10-2021

Amount: $546,441.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100496

Start Date: 2011

End Date: 06-2017

Amount: $340,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100181

Start Date: 03-2012

End Date: 12-2015

Amount: $650,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200102291

Start Date: 07-2020

End Date: 12-2024

Amount: $450,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE130100199

Start Date: 2013

End Date: 12-2014

Amount: $475,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100183

Start Date: 2020

End Date: 06-2021

Amount: $950,000.00

Funder: Australian Research Council