School of Chemistry - Research Publications
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ItemAntimicrobial Peptide Structures: From Model Membranes to Live Cells(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.
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ItemA QCM-D and SAXS Study of the Interaction of Functionalised Lyotropic Liquid Crystalline Lipid Nanoparticles with siRNA(Wiley, 2017-05-18)Biophysical studies were undertaken to investigate the binding and release of short interfering ribonucleic acid (siRNA) from lyotropic liquid crystalline lipid nanoparticles (LNPs) by using a quartz crystal microbalance (QCM). These carriers are based on phytantriol (Phy) and the cationic lipid DOTAP (1,2‐dioleoyloxy‐3‐(trimethylammonium)propane). The nonlamellar phase LNPs were tethered to the surface of the QCM chip for analysis based on biotin–neutravidin binding, which enabled the controlled deposition of siRNA–LNP complexes with different lipid/siRNA charge ratios on a QCM‐D crystal sensor. The binding and release of biomolecules such as siRNA from LNPs was demonstrated to be reliably characterised by this technique. Essential physicochemical parameters of the cationic LNP/siRNA lipoplexes—such as particle size, lyotropic phase behaviour, cytotoxicity, gene silencing and uptake efficiency—were also assessed. The SAXS data show that when the pH was lowered to 5.5 the structure of the lipoplexes did not change, thus indicating that the acidic conditions of the endosome were not a significant factor in the release of siRNA from the cationic lipidic carriers.
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ItemRelaxin family peptides: structure-activity relationship studies(WILEY, 2017-05)UNLABELLED: The human relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through relaxin family peptide receptors, RXFP1-4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure-activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5). LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
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ItemC-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide(WILEY-V C H VERLAG GMBH, 2017-01-05)Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.
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ItemListeriolysinO Binding Affects Cholesterol and Phospholipid Acyl Chain Dynamics in Fluid Cholesterol-Rich Bilayers(WILEY-V C H VERLAG GMBH, 2018-09-20)Listeriolysin O (LLO) is a pore-forming toxin that enables survival and cell-to-cell spread of foodborne bacterial pathogen Listeria monocytogenes, which is responsible for the life-threatening disease, listeriosis. LLO-membrane interactions are crucial for pathogenicity of Listeria, but remained unexplained in detail at the molecular level. Here we addressed them by means of 2 H, 31 P, 13 C and 19 F solid-state NMR spectroscopy. Different fluid and ordered cholesterol-rich membrane lipid bilayer systems were prepared and checked for the integrity and properties in the presence of LLO. LLO has significantly changed dynamics of phospholipid acyl chains of more fluid cholesterol-rich bilayers, whereas the lipid bilayer organization was not affected. LLO has also affected cholesterol dynamics by increasing the intensity of low frequency motions, indicating direct interactions of LLO with cholesterol. Additionally, the LLO protein was shown to interact differently with lipid membranes, depending on the properties of cholesterol-rich membranes. The presented results, therefore, provide new insights into the interactions of the bacterial toxin LLO with cholesterol-rich membrane systems.
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ItemMeasuring translational diffusion of 15N-enriched biomolecules in complex solutions with a simplified 1H-15N HMQC-filtered BEST sequence(SPRINGER, 2018-12)Pulsed-field gradient nuclear magnetic resonance has seen an increase in applications spanning a broad range of disciplines where molecular translational diffusion properties are of interest. The current study introduces and experimentally evaluates the measurement of translational diffusion coefficients of 15N-enriched biomolecules using a 1H-15N HMQC-filtered band-selective excitation short transient (BEST) sequence as an alternative to the previously described SOFAST-XSTE sequence. The results demonstrate that accurate translational diffusion coefficients of 15N-labelled peptides and proteins can be obtained using this alternative 1H-15N HMQC-filtered BEST sequence which is implementable on NMR spectrometers equipped with probes fitted with a single-axis field gradient, including most cryoprobes dedicated to bio-NMR. The sequence is of potential use for direct quantification of protein or peptide translational diffusion within complex systems, such as in mixtures of macromolecules, crowded solutions, membrane-mimicking media and in bicontinuous cubic phases, where conventional sequences may not be readily applicable due to the presence of intense signals arising from sources other than the protein or peptide under investigation.
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ItemOne pathogen two stones: are Australian tree frog antimicrobial peptides synergistic against human pathogens?(SPRINGER, 2017-10)Antimicrobial peptides (AMPs) may act by targeting the lipid membranes and disrupting the bilayer structure. In this study, three AMPs from the skin of Australian tree frogs, aurein 1.2, maculatin 1.1 and caerin 1.1, were investigated against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and vesicles that mimic their lipid compositions. Furthermore, equimolar mixtures of the peptides were tested to identify any synergistic interactions in antimicrobial activity. Minimum inhibition concentration and minimum bactericidal concentration assays showed significant activity against S. aureus but not against E. coli. Aurein was the least active while maculatin was the most active peptide and some synergistic effects were observed against S. aureus. Circular dichroism experiments showed that, in the presence of phospholipid vesicles, the peptides transitioned from an unstructured to a predominantly helical conformation (>50%), with greater helicity for POPG/TOCL compared to POPE/POPG vesicles. The helical content, however, was less in the presence of live E. coli and S. aureus, 25 and 5%, respectively. Equimolar concentrations of the peptides did not appear to form greater supramolecular structures. Dye release assays showed that aurein required greater concentration than caerin and maculatin to disrupt the lipid bilayers, and mixtures of the peptides did not cooperate to enhance their lytic activity. Overall, aurein, maculatin, and caerin showed moderate synergy in antimicrobial activity against S. aureus without becoming more structured or enhancement of their membrane-disrupting activity in phospholipid vesicles.
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ItemThe efficient synthesis and purification of amyloid-β(1-42) using an oligoethylene glycol-containing photocleavable lysine tag(ROYAL SOC CHEMISTRY, 2017-06-28)A short, monodisperse oligoethylene glycol-containing photocleavable lysine tag was developed to facilitate the efficient purification of hydrophobic and fibril-forming peptides. This new tag was used to prepare a modified Aβ42 peptide with increased solubility and decreased propensity to aggregate in aqueous media. The solubilising tag was readily removed by irradiation with UV light and permitted the preparation and isolation of Aβ42 in high purity and yield.
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ItemIn Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR(MDPI, 2019-01-01)In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.
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ItemA nanomechanical study of the effects of colistin on the Klebsiella pneumoniae AJ218 capsule(SPRINGER, 2017-05)Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 μM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin; genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the divalent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking divalent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.