ORCID Profile
0000-0001-6560-8410
Current Organisation
University of Sydney
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Plant cell and molecular biology | Plant biochemistry | Proteins and Peptides | Biochemistry and cell biology | Synthetic biology | Macromolecular and Materials Chemistry | Nanochemistry and Supramolecular Chemistry
Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Biological Sciences |
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1MD00098E
Abstract: There are remarkably few ex les of designing cyclic constraints for peptides which adopt an irregular conformation in their bioactive state. We discuss the design strategies, opportunities and challenges for this underexplored class of inhibitors.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4CS00246F
Abstract: This review analyses how the choice of macrocyclisation chemistry can influence the biological properties and applications of stapled peptides.
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9OB01745C
Abstract: The Sondheimer dialkyne is extensively used in double strain-promoted azide–alkyne cycloadditions.
Publisher: Oxford University Press (OUP)
Date: 12-05-2017
DOI: 10.1093/NAR/GKX415
Publisher: Wiley
Date: 16-03-2016
Publisher: Wiley
Date: 08-02-2011
Abstract: A cyclam-based fluorescent sensor featuring a novel triazole pendant arm has been synthesised using click chemistry. The sensor is highly responsive to both Cu(II) and Hg(II) in neutral aqueous solution and displays excellent selectivity in the presence of various competing metal ions in 50-fold excess. The addition of specific anions such as I(-) and S(2)O(3)(2-) causes a complete revival of fluorescence only in the case of Hg(II), providing a simple and effective method for distinguishing solutions containing Cu(II), Hg(II) or a mixture of both ions, even in doped seawater s les. X-ray crystal structures of both the Hg(II) sensor complex and a model Cu(II) complex show that pendant triazole coordination occurs through the central nitrogen atom (N2), providing to the best of our knowledge the first reported ex les of this unusual coordination mode in macrocycles. Fluorescence, mass spectrometry and (1)H NMR experiments reveal that the mechanism of anion-induced fluorescence revival involves either displacement of pendant coordination or complete removal of the Hg(II) from the macrocycle, depending on the anion.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3TB00991B
Abstract: There are many native and engineered capsid-forming proteins which can self-assemble into different non-canonical structures. In this review, we categorise ex les of structural polymorphism by their method of formation.
Publisher: CSIRO Publishing
Date: 22-08-2023
DOI: 10.1071/CH23102
Publisher: Elsevier BV
Date: 02-2011
Publisher: Wiley
Date: 27-12-2017
Abstract: Molecular switches have many potential applications in nanoscience and biomedicine. Transition metal complexes that can be switched from an inert, unreactive state to a catalytically active one by a simple change in conditions (e.g. pH shift) or by binding to a specific biomolecular target-so-called target-activated metal complexes (TAMCs)-hold particular allure as a means of harnessing the potent but at times indiscriminate reactivity of metal-based drugs. Towards this goal, we have prepared a series of ten structurally related ligands, each of which bears a different pendant side-arm functional group appended to a common macrocyclic core, along with copper(II) and nickel(II) complexes of these cyclam-based "molecular scorpionands". X-ray crystal structures reveal a variety of binding modes between pendant side-arm and metal centre that depend on the constituent donor atoms. To investigate the switchability of side-arm coordination in solution, spectrophotometric pH titrations were carried out for all 20 metal complexes. The majority of the complexes undergo spectroscopic changes that are consistent with a switch in pendant coordination state at a specific pH. This ligand series represents a comprehensive model platform from which to build pH-switchable metal complexes for applications in nanoscience and biomedicine.
Publisher: American Chemical Society (ACS)
Date: 07-02-2017
DOI: 10.1021/JACS.6B10234
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4OB00742E
Abstract: Evaluating the influence of staple position, azido amino acid side-chain length and point mutation on the activity of ‘double-click’ stapled p53 peptides.
Publisher: American Chemical Society (ACS)
Date: 30-09-2021
Publisher: Cold Spring Harbor Laboratory
Date: 27-01-2021
DOI: 10.1101/2021.01.27.428512
Abstract: Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. While pores lined with negatively charged residues resulted in more robust assemblies than their corresponding positively charged variants, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures of pore variants elucidated at resolutions between 2.5-3.6 Å. Alongside these structures, molecular dynamics simulations and stopped flow kinetics experiments reveal the importance of considering both pore size and surface charge, together with flexibility and rate determining steps, when designing protein cages for controlling molecular flux.
Publisher: American Chemical Society (ACS)
Date: 07-01-2020
Publisher: Wiley
Date: 06-12-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C0CS00143K
Abstract: Since the advent of click chemistry in 2001, the 1,4-disubstituted triazole has become an increasingly common motif in chemical sensors. Although these click-derived triazoles are generally used as a convenient method of ligation, their prevalence in chemosensors can be attributed to their ability to bind both cations and anions. In this critical review, we present an overview of the wide range of chemosensors that contain click-derived triazoles, with a particular focus on those cases where the triazole plays a functional, rather than merely a structural, role. Ex les are categorised based on method of detection and key structural features, providing a complete picture of the current state of click-based chemosensors, as well as potential future directions for sensor design. (140 references).
Publisher: Wiley
Date: 06-12-2021
Abstract: The front cover artwork is provided by the Lau Group from the University of Sydney. The image shows an artistic ex le of a catalytic system depicting cross‐talk between enzymes displayed on the surface and sequestered within the compartment. Read the full text of the Review at 10.1002/syst.202100025 .
Publisher: Wiley
Date: 02-11-2015
Abstract: Peptide stapling is a method for designing macrocyclic alpha‐helical inhibitors of protein–protein interactions. However, obtaining a cell‐active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain‐promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell‐active stapled peptides. As a proof of concept, MDM2‐binding peptides were stapled in parallel, directly in cell culture medium in 96‐well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α‐Helicity was confirmed by a crystal structure of the MDM2‐peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high‐throughput biological applications.
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4SC00045E
Abstract: A ergent synthetic strategy for generating helical p53 peptides bearing functionalised staple linkages, allowing for efficient optimisation of cellular activity.
Publisher: Wiley
Date: 07-09-2017
Abstract: The exploitation of synthetic lethality by small-molecule targeting of pathways that maintain genomic stability is an attractive chemotherapeutic approach. The Ctf4/AND-1 protein hub, which links DNA replication, repair, and chromosome segregation, represents a novel target for the synthetic lethality approach. Herein, we report the design, optimization, and validation of double-click stapled peptides encoding the Ctf4-interacting peptide (CIP) of the replicative helicase subunit Sld5. By screening stapling positions in the Sld5 CIP, we identified an unorthodox i,i+6 stapled peptide with improved, submicromolar binding to Ctf4. The mode of interaction with Ctf4 was confirmed by a crystal structure of the stapled Sld5 peptide bound to Ctf4. The stapled Sld5 peptide was able to displace the Ctf4 partner DNA polymerase α from the replisome in yeast extracts. Our study provides proof-of-principle evidence for the development of small-molecule inhibitors of the human CTF4 orthologue AND-1.
Publisher: Wiley
Date: 13-07-2021
Abstract: The design of biomimetic systems in the laboratory is a long‐sought goal for systems chemists and synthetic biologists alike. Fundamental to this design is the generation of self‐assembled structures capable of mimicking compartmentalisation, which includes the encapsulation of molecular cargo as well as the display of molecules on the exterior. Protein nanocompartments are fast becoming popular scaffolds for these systems due to their robust self‐assembly, ability to encapsulate non‐native cargo, and amenability to surface modifications. In this Review, we discuss the primary methods for displaying a wide array of molecular motifs on compartment surfaces. We discuss benefits and drawbacks of each type of display and examine three recent case studies wherein molecular display was a critical design element in the construction of multi‐enzyme chemical systems. The analyses and case studies presented in this Review aim to provide a critical summary of the technologies currently used for molecular display to add another dimension to the design of chemical systems and nanoreactors.
Publisher: Wiley
Date: 29-10-2014
Abstract: We investigated linear aliphatic dialkynes as a new structural class of i,i+7 linkers for the double-click stapling of p53-based peptides. The optimal combination of azido amino acids and dialkynyl linker length for MDM2 binding was determined. In a direct comparison between aliphatic and aromatic staple scaffolds, the aliphatic staples resulted in superior binding to MDM2 in vitro and superior p53-activating capability in cells when using a diazidopeptide derived from phage display. This work demonstrates that the nature of the staple scaffold is an important factor that can affect peptide bioactivity in cells.
Publisher: Springer Science and Business Media LLC
Date: 05-10-2017
Publisher: American Association for the Advancement of Science (AAAS)
Date: 04-02-2022
Abstract: Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the Thermotoga maritima encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.
Publisher: American Chemical Society (ACS)
Date: 31-01-2019
DOI: 10.1021/ACSCHEMBIO.9B00063
Abstract: Stapled peptides have great potential as modulators of protein-protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of ersified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure-activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.
Publisher: Royal Society of Chemistry
Date: 2018
Publisher: Springer Science and Business Media LLC
Date: 03-04-2018
DOI: 10.1038/S41467-018-03768-X
Abstract: Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae , based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments afford protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.
Publisher: American Chemical Society (ACS)
Date: 18-10-2023
Publisher: American Chemical Society (ACS)
Date: 18-05-2022
Abstract: Self-assembling proteins can form porous compartments that adopt well-defined architectures at the nanoscale. In nature, protein compartments act as semipermeable barriers to enable spatial separation and organization of complex biochemical processes. The compartment pores play a key role in their overall function by selectively controlling the influx and efflux of important biomolecular species. By engineering the pores, the functionality of compartments can be tuned to facilitate non-native applications, such as artificial nanoreactors for catalysis. In this review, we analyze how protein structure determines the porosity and impacts the function of both native and engineered compartments, highlighting the wealth of structural data recently obtained by cryo-EM and X-ray crystallography. Through this analysis, we offer perspectives on how current structural insights can inform future studies into the design of artificial protein compartments as nanoreactors with tunable porosity and function.
Publisher: Springer Science and Business Media LLC
Date: 12-03-2015
Abstract: Peptide cyclization is a useful strategy for the stabilization of short flexible peptides into well-defined bioactive conformations, thereby enhancing their ability to interact with proteins and other important biomolecules. We present an optimized procedure for the stabilization of linear diazido peptides in an α-helical conformation upon reaction with dialkynyl linkers under Cu(I) catalysis. As this procedure generates side chain-cyclized peptides bearing a bis-triazole linkage, it is referred to as 'double-click' stapling. Double-click stapling can enhance the binding affinity, proteolytic stability and cellular activity of a peptide inhibitor. A distinguishing feature of double-click stapling is the efficiency with which peptides bearing different staple linkages can be synthesized, thus allowing for modular control over peptide bioactivity. This protocol describes the double-click reaction between a 1,3-dialkynylbenzene linker and peptides that contain azidoornithine. Subsequent peptide purification and confirmation steps are also described. The entire double-click stapling protocol can be completed in ∼48 h, including two overnight lyophilization steps.
Publisher: Cold Spring Harbor Laboratory
Date: 31-01-2021
DOI: 10.1101/2021.01.30.428974
Abstract: Metabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme athway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast ( Saccharomyces cerevisiae ) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing ‘free’ MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.
Publisher: Georg Thieme Verlag KG
Date: 14-07-2011
Publisher: American Chemical Society (ACS)
Date: 09-08-2023
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2019
End Date: 12-2021
Amount: $422,556.00
Funder: Australian Research Council
View Funded ActivityStart Date: 08-2023
End Date: 08-2026
Amount: $691,716.00
Funder: Australian Research Council
View Funded Activity