School of Chemistry - Research Publications

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    Selenium Nanoparticles as Potential Drug-Delivery Systems for the Treatment of Parkinson's Disease
    Kalcec, N ; Peranic, N ; Mamic, I ; Beus, M ; Hall, CR ; Smith, TA ; Sani, MA ; Turcic, P ; Separovic, F ; Vrcek, IV (AMER CHEMICAL SOC, 2023-09-20)
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    The membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent
    Sani, M-A ; Le Brun, AP ; Rajput, S ; Attard, T ; Separovic, F (CELL PRESS, 2023-03-21)
    Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions.
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    Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain
    Boland, MP ; Hatty, CR ; Separovic, F ; Hill, AF ; Tew, DJ ; Barnham, KJ ; Haigh, CL ; James, M ; Masters, CL ; Collins, SJ (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-10-15)
    Although the N terminus of the prion protein (PrP(C)) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23-110) and N2 (23-89) fragments derived from constitutive processing of PrP(C) and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.
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    In-cell DNP NMR reveals multiple targeting effect of antimicrobial peptide
    Separovic, F ; Hofferek, V ; Duff, AP ; McConville, MJ ; Sani, M-A (ELSEVIER, 2022)
    Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on E. coli cells. The enhanced 15N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on E. coli cells. DNP-enhanced 15N-observed 31P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main E. coli lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using E. coli PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides in situ will provide greater insight into their mode of action.
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    Ultrasound-induced protein restructuring and ordered aggregation to form amyloid crystals
    Pathak, R ; Bhangu, SK ; Martin, GJO ; Separovic, F ; Ashokkumar, M (SPRINGER, 2022-07)
    Amyloid crystals, a form of ordered protein aggregates documented relatively recently, have not been studied as extensively as amyloid fibres. This study investigates the formation of amyloid crystals with low frequency ultrasound (20 kHz) using β-lactoglobulin, as a model protein for amyloid synthesis. Acoustic cavitation generates localised zones of intense shear, with extreme heat and pressure that could potentially drive the formation of amyloid structures at ambient bulk fluid temperatures (20 ± 1 °C). Thioflavin T fluorescence and electron microscopy showed that low-frequency ultrasound at 20 W/cm3 input power induced β-stacking to produce amyloid crystals in the mesoscopic size range, with a mean length of approximately 22 µm. FTIR spectroscopy indicated a shift towards increased intermolecular antiparallel β-sheet content. An increase in sonication time (0-60 min) and input power (4-24 W/cm3) increased the mean crystal length, but this increase was not linearly proportional to sonication time and input power due to the delayed onset of crystal growth. We propose that acoustic cavitation causes protein unfolding and aggregation and imparts energy to aggregates to cross the torsion barrier, to achieve their lowest energy state as amyloid crystals. The study contributes to a further understanding of protein chemistry relating to the energy landscape of folding and aggregation. Ultrasound presents opportunities for practical applications of amyloid structures, presenting a more adaptable and scalable approach for synthesis.
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    C-terminus amidation influences biological activity and membrane interaction of maculatin 1.1
    Zhu, S ; Li, W ; O'Brien-Simpson, N ; Separovic, F ; Sani, M-A (SPRINGER WIEN, 2021-05)
    Cationic antimicrobial peptides have been investigated for their potential use in combating infections by targeting the cell membrane of microbes. Their unique chemical structure has been investigated to understand their mode of action and optimize their dose-response by rationale design. One common feature among cationic AMPs is an amidated C-terminus that provides greater stability against in vivo degradation. This chemical modification also likely modulates the interaction with the cell membrane of bacteria yet few studies have been performed comparing the effect of the capping groups. We used maculatin 1.1 (Mac1) to assess the role of the capping groups in modulating the peptide bacterial efficiency, stability and interactions with lipid membranes. Circular dichroism results showed that C-terminus amidation maintains the structural stability of the peptide (α-helix) in contact with micelles. Dye leakage experiments revealed that amidation of the C-terminus resulted in higher membrane disruptive ability while bacteria and cell viability assays revealed that the amidated form displayed higher antibacterial ability and cytotoxicity compared to the acidic form of Mac1. Furthermore, 31P and 2H solid-state NMR showed that C-terminus amidation played a greater role in disturbance of the phospholipid headgroup but had little effect on the lipid tails. This study paves the way to better understand how membrane-active AMPs act in live bacteria.
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    Multi-Omic Analysis to Characterize Metabolic Adaptation of the E. coli Lipidome in Response to Environmental Stress
    Kralj, T ; Nuske, M ; Hofferek, V ; Sani, M-A ; Lee, T-H ; Separovic, F ; Aguilar, M-I ; Reid, GE (MDPI, 2022-02)
    As an adaptive survival response to exogenous stress, bacteria undergo dynamic remodelling of their lipid metabolism pathways to alter the composition of their cellular membranes. Here, using Escherichia coli as a well characterised model system, we report the development and application of a 'multi-omics' strategy for comprehensive quantitative analysis of the temporal changes in the lipidome and proteome profiles that occur under exponential growth phase versus stationary growth phase conditions i.e., nutrient depletion stress. Lipidome analysis performed using 'shotgun' direct infusion-based ultra-high resolution accurate mass spectrometry revealed a quantitative decrease in total lipid content under stationary growth phase conditions, along with a significant increase in the mol% composition of total cardiolipin, and an increase in 'odd-numbered' acyl-chain length containing glycerophospholipids. The inclusion of field asymmetry ion mobility spectrometry was shown to enable the enrichment and improved depth of coverage of low-abundance cardiolipins, while ultraviolet photodissociation-tandem mass spectrometry facilitated more complete lipid structural characterisation compared with conventional collision-induced dissociation, including unambiguous assignment of the odd-numbered acyl-chains as containing cyclopropyl modifications. Proteome analysis using data-dependent acquisition nano-liquid chromatography mass spectrometry and tandem mass spectrometry analysis identified 83% of the predicted E. coli lipid metabolism enzymes, which enabled the temporal dependence associated with the expression of key enzymes responsible for the observed adaptive lipid metabolism to be determined, including those involved in phospholipid metabolism (e.g., ClsB and Cfa), fatty acid synthesis (e.g., FabH) and degradation (e.g., FadA/B,D,E,I,J and M), and proteins involved in the oxidative stress response resulting from the generation of reactive oxygen species during β-oxidation or lipid degradation.
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    Enhancing proline-rich antimicrobial peptide action by homodimerization: influence of bifunctional linker
    Li, W ; Lin, F ; Hung, A ; Barlow, A ; Sani, M-A ; Paolini, R ; Singleton, W ; Holden, J ; Hossain, MA ; Separovic, F ; O'Brien-Simpson, NM ; Wade, JD (ROYAL SOC CHEMISTRY, 2022-02-23)
    Antimicrobial peptides (AMPs) are host defense peptides, and unlike conventional antibiotics, they possess potent broad spectrum activities and, induce little or no antimicrobial resistance. They are attractive lead molecules for rational development to improve their therapeutic index. Our current studies examined dimerization of the de novo designed proline-rich AMP (PrAMP), Chex1-Arg20 hydrazide, via C-terminal thiol addition to a series of bifunctional benzene or phenyl tethers to determine the effect of orientation of the peptides and linker length on antimicrobial activity. Antibacterial assays confirmed that dimerization per se significantly enhances Chex1-Arg20 hydrazide action. Greatest advantage was conferred using perfluoroaromatic linkers (tetrafluorobenzene and octofluorobiphenyl) with the resulting dimeric peptides 6 and 7 exhibiting potent action against Gram-negative bacteria, especially the World Health Organization's critical priority-listed multidrug-resistant (MDR)/extensively drug-resistant (XDR) Acinetobacter baumannii as well as preformed biofilms. Mode of action studies indicated these lead PrAMPs can interact with both outer and inner bacterial membranes to affect the membrane potential and stress response. Additionally, 6 and 7 possess potent immunomodulatory activity and neutralise inflammation via nitric oxide production in macrophages. Molecular dynamics simulations of adsorption and permeation mechanisms of the PrAMP on a mixed lipid membrane bilayer showed that a rigid, planar tethered dimer orientation, together with the presence of fluorine atoms that provide increased bacterial membrane interaction, is critical for enhanced dimer activity. These findings highlight the advantages of use of such bifunctional tethers to produce first-in-class, potent PrAMP dimers against MDR/XDR bacterial infections.
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    NMR measurement of biomolecular translational and rotational motion for evaluating changes of protein oligomeric state in solution
    Yao, S ; Keizer, DW ; Babon, JJ ; Separovic, F (SPRINGER, 2022-04)
    Defining protein oligomeric state and/or its changes in solution is of significant interest for many biophysical studies carried out in vitro, especially when the nature of the oligomeric state is crucial in the subsequent interpretation of experimental results and their biological relevance. Nuclear magnetic resonance (NMR) is a well-established methodology for the characterization of protein structure, dynamics, and interactions at the atomic level. As a spectroscopic method, NMR also provides a compelling means for probing both molecular translational and rotational motion, two predominant measures of effective molecular size in solution, under identical conditions as employed for structural, dynamic and interaction studies. Protein translational diffusion is readily measurable by pulse gradient spin echo (PGSE) NMR, whereas its rotational correlation time, or rotational diffusion tensor when its 3D structure is known, can also be quantified from NMR relaxation parameters, such as 15N relaxation parameters of backbone amides which are frequently employed for probing residue-specific protein backbone dynamics. In this article, we present an introductory overview to the NMR measurement of bimolecular translational and rotational motion for assessing changes of protein oligomeric state in aqueous solution, via translational diffusion coefficients measured by PGSE NMR and rotational correlation times derived from composite 15N relaxation parameters of backbone amides, without need for the protein structure being available.
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    Antimicrobial Peptide Structures: From Model Membranes to Live Cells
    Sani, M-A ; Separovic, F (WILEY-V C H VERLAG GMBH, 2018-01-09)
    The rise in antibiotic resistance has led to a renewed interest in antimicrobial peptides (AMPs) that target membranes. The mode of action of AMPs involves the disruption of the lipid bilayer and leads to growth inhibition and death of the bacteria. However, details at the molecular level of how these peptides kill bacteria and the reasons for the observed differences in selectivity remain unclear. Structural information is crucial for defining the molecular mechanism by which these peptides recognize, self-assemble and interact with a particular lipid membrane. Solid-state NMR is a non-invasive technique that allows the study of the structural details of lipid-peptide and peptide-peptide interactions. Following on from studies of antibiotic and lytic peptides, gramicidin A and melittin, respectively, we investigated maculatin 1.1, an AMP from the skin of Australian tree frogs that acts against Gram-positive bacteria. By using perdeuterated phospholipids and specifically labelled peptides, 2 H, 31 P and {31 P}15 N REDOR solid-state NMR experiments have been used to localize, maculatin 1.1 in neutral and anionic model membranes. However, the structure, location and activity depend on the composition of the model membrane and current advances in solid-state NMR spectroscopy now allow structure determination of AMPs in live bacteria.