School of Chemistry - Research Publications

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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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    Water diffusion in complex systems measured by PGSE NMR using chemical shift selective stimulated echo: Elimination of magnetization exchange effects
    Meikle, T ; Keizer, DW ; Separovic, F ; Yao, S (AIP Publishing, 2021-12-14)
    The interpretation of molecular translational diffusion as measured by pulsed gradient spin-echo NMR (PGSE NMR) can be complicated by the presence of chemical exchange and/or dipolar cross-relaxation (including relayed cross-relaxation via spin diffusion). The magnitude of influence depends on the kinetics of exchange and/or dipolar cross-relaxation present within the system as well as the PGSE NMR sequences chosen for measurements. First, we present an exchange induced zero-crossing phenomenon for signal attenuation of water in lipidic cubic phases (formed by a mixture of monoolein and water) in the presence of pulsed gradients observed using a standard STimulated Echo (STE) sequence. This magnetization exchange induced zero-crossing phenomenon, a pseudo-negative diffraction-like feature, resembles that reported previously for restricted diffusion when locally anisotropic pores are polydisperse or randomly oriented. We then demonstrate the elimination of these exchange and/or dipolar cross-relaxation induced effects with the use of a chemical shift selective STE (CHESS-STE) sequence, adapted from the previously reported band-selective short transient STE sequence, along with results obtained from the bipolar pulse pair STE sequence for comparison. The CHESS-STE sequence introduced here represents a generic form of PGSE NMR sequences for obtaining water diffusion coefficients free from the influence of exchange and/or dipolar cross-relaxation in complex systems. It has potential applications in measuring translational diffusion of water in biopolymer mixtures as well as probing the microscopic structure in materials via water restricted diffusion measured by PGSE NMR, particularly when the potential presence of exchange/cross-relaxation is of concern.
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    Utilizing magnetic resonance techniques to study membrane interactions of amyloid peptides
    Rajput, S ; Sani, M-A ; Keizer, DW ; Separovic, F (PORTLAND PRESS LTD, 2021-06)
    Alzheimer's disease (AD) is a common neurodegenerative condition that involves the extracellular accumulation of amyloid plaques predominantly consisting of Aβ peptide aggregates. The amyloid plaques and soluble oligomeric species of Aβ are believed to be the major cause of synaptic dysfunction in AD brain and their cytotoxic mechanisms have been proposed to involve interactions with cell membranes. In this review, we discuss our solid-state nuclear magnetic resonance (ssNMR) studies of Aβ interactions with model membranes.
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    Prospects for nuclear spin hyperpolarization of molecular samples using nitrogen-vacancy centers in diamond
    Tetienne, J-P ; Hall, LT ; Healey, AJ ; White, GAL ; Sani, M-A ; Separovic, F ; Hollenberg, LCL (AMER PHYSICAL SOC, 2021-01-21)
    After initial proof-of-principle demonstrations, optically pumped nitrogen-vacancy (NV) centers in diamond have been proposed as a noninvasive platform to achieve hyperpolarization of nuclear spins in molecular samples over macroscopic volumes and enhance the sensitivity in nuclear magnetic resonance (NMR) experiments. In this work we model the process of polarization of external samples by NV centers and theoretically evaluate their performance in a range of scenarios. We find that average nuclear spin polarizations exceeding 10% can in principle be generated over macroscopic sample volumes (≤μl) with a careful engineering of the system's geometry to maximize the diamond-sample contact area. The fabrication requirements and other practical challenges are discussed. We then explore the possibility of exploiting local polarization enhancements in nano/micro-NMR experiments based on NV centers. For micro-NMR we find that modest signal enhancements over thermal polarization (by 1-2 orders of magnitude) can in essence be achieved with existing technology, with larger enhancements achievable via microstructuring of the sample/substrate interface. However, there is generally no benefit for nano-NMR where the detection of statistical polarization provides the largest signal-to-noise ratio. This work will guide future experimental efforts to integrate NV-based hyperpolarization to NMR systems.
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    Expression and purification of the native C-amidated antimicrobial peptide maculatin 1.1
    Zhu, S ; Weber, DK ; Separovic, F ; Sani, M-A (WILEY, 2021-08)
    Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from an Australian tree frog and exhibits low micromolar activity against Gram-positive bacteria. The antimicrobial properties of Mac1 are linked to its disruption of bacterial lipid membranes, which has been studied extensively by in vitro nuclear magnetic resonance (NMR) spectroscopy and biophysical approaches. Although in vivo NMR has recently proven effective in probing peptide-lipid interplay in live bacterial cells, direct structural characterisation of AMPs has been prohibited by low sensitivity and overwhelming background noise. To overcome this issue, we report a recombinant expression protocol to produce isotopically enriched Mac1. We utilized a double-fusion construct to alleviate toxicity against the Escherichia coli host and generate the native N-free and C-amidated termini Mac1 peptide. The SUMO and intein tags allowed native N-terminus and C-terminal amidation, respectively, to be achieved in a one-pot reaction. The protocol yielded 0.1 mg/L of native, uniformly 15 N-labelled, Mac1, which possessed identical structure and activity to peptide obtained by solid-phase peptide synthesis.