22 research outputs found
Site-selective tagging of proteins by pnictogen-mediated self-assembly
Site-selective chemical protein modification is achieved by self-assembly of a specific di-cysteine motif, trivalent pnictogens (As, Sb or Bi) and an aromatic mercaptomethyl-based probe. The strategy is demonstrated with a quaternary complex involving Zika virus protease and a lanthanide ion, enabling paramagnetic nuclear magnetic resonance spectroscopy and luminescence measurements.Financial support by the Australian Research Council is gratefully acknowledge
Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease
The Zika virus presents a serious risk for global health. Crystal structures of different constructs of the Zika virus NS2B-NS3 protease (NS2B-NS3pro) have been determined with the aim to provide a basis for rational drug discovery. In these structures, the C-terminal β-hairpin of NS2B, NS2Bc, was observed to be either disordered (open conformation) or bound to NS3pro complementing the substrate binding site (closed conformation). Enzymatically active constructs of flaviviral NS2B-NS3 proteases commonly used for inhibitor testing contain a covalent peptide linker between NS2B and NS3pro. Using a linked construct of Zika virus NS2B-NS3pro, we studied the location of NS2Bc relative to NS3pro in solution by pseudocontact shifts generated by a paramagnetic lanthanide tag attached to NS3pro. Both closed and open conformations were observed with different inhibitors. As the NS2B co-factor is involved in substrate binding of flaviviral NS2B-NS3 proteases, the destabilization of the closed conformation in the linked construct makes it an attractive tool to search for inhibitors that interfere with the formation of the enzymatically active, closed conformation.C.N. thanks the Alexander vonHumbolt Foundation for a Feodor-Lynen fellowship. Financialsupport by the Australian Research Council is gratefully acknowl-edged. C.K. acknowledges support by the Deutsche For-schungsgemeinschaft, grant KL 1356/3
De Novo Discovery of Nonstandard Macrocyclic Peptides as Noncompetitive Inhibitors of the Zika Virus NS2B-NS3 Protease
The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.Open AccessFinancial support by the
Australian Research Council, including a Laureate Fellowship
for G.O. is gratefully acknowledged. This work was also
partially supported by CREST for Molecular Technologies,
JST, and JSPS KAKENHI (16H06444 and 26220204) to H.S.
C.K. acknowledges support by the Deutsche Forschungsgemeinschaft (KL-1356/3-2). We thank Mrs. Natascha Stefan for
technical support
Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with α-cysteine, penicillamine or β-cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with Gd3+ ions.This research has been supported by the Australian Research Council (grant no. FL170100019 and DP210100088), the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (grant no. CE200100012) and the European Regional Development Fund (ERDF; PostDoc grant no. 1.1.1.2/VIAA/2/18/381)
Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data
We present Farseer-NMR (https://git.io/vAueU), a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution. These advances have created a growing need to analyse those systems’ responses to multiple variables. The combined analysis of NMR peaklists from large and multivariable datasets has become a new bottleneck in the NMR analysis pipeline, whereby information-rich NMR-derived parameters have to be manually generated, which can be tedious, repetitive and prone to human error, or even unfeasible for very large datasets. There is a persistent gap in the development and distribution of software focused on peaklist treatment, analysis and representation, and specifically able to handle large multivariable datasets, which are becoming more commonplace. In this regard, Farseer-NMR aims to close this longstanding gap in the automated NMR user pipeline and, altogether, reduce the time burden of analysis of large sets of peaklists from days/weeks to seconds/minutes. We have implemented some of the most common, as well as new, routines for calculation of NMR parameters and several publication-quality plotting templates to improve NMR data representation. Farseer-NMR has been written entirely in Python and its modular code base enables facile extension
Assessment of Mangrove Restoration Potential in the North Western Province of Sri Lanka for Climate Change Mitigation
Over the geological time, the planet's climate has been changing constantly, with large variations in global average temperatures. Millions of people are already suffering as a result of natural disasters worsened by climate change. The Paris Agreement is an international climate change treaty, and countries will present their climate action plans, referred to as Nationally Determined Contributions (NDCs). In response to the Paris Agreement, Sri Lanka presented NDCs in 2016. In which the coastal and marine sector has a restoration target of 10,000 ha of mangroves as a coastal greenbelt with mitigation co-benefits. However, identification of potential lands for such restoration of mangroves has not been done. Therefore, identification of potential areas for mangrove restoration is most important because mangroves cannot be restored where they were not previously existed. The main objective of this research therefore was to identify potential areas for mangrove restoration within the North-Western Province. Identification of potential lands in North-Western Province were done by using Google Earth Pro and ArcGIS 10.2.2. Most important physicochemical parameters such as salinity, pH, soil organic matter content, soil water holding capacity were analyzed under laboratory conditions, samples were obtained by different sites of mangrove habitats. Soil salinity, pH were measured by using calibrated multiparameter. The loss of ignition method for soil organic matter content and a customized method for soil water holding capacity were used. All parameters were subjected to two-way ANOVA in MINITAB 14 after following Anderson Darling Normality test. The mean of soil salinity, pH, soil organic matter content, and soil water holding capacity were ranged respectively; 4.26% to 5.93%, 6.07 to 8.62, 3.40% to 9.31%, and 30.39% to 46.38%. The potential lands for restoring mangroves in North-Western Province were identified in Chilaw, Kalpitiya, Mundel, Panirendawa, Puttalam, Vanathawilluwa and Vennappuwa Divisional Secretariant Divisions. Among them, availability for historical images in Google Earth Pro there were specially chosen areas to restore the mangroves. There is no any significant difference of physicochemical parameters among mangrove habitats and an abandoned shrimp farm where there were previously mangrove plants existed. Hence, abandoned shrimp farms and salterns can be used to restore the mangrove plants and the assisted natural regeneration of mangroves in suitable abandoned shrimp farms by way of facilitating hydrology to be explored as the best option for mangrove restoration.
Keywords: Mangrove restoration, Nationally Determined Contributions (NDCs), Assisted natural regeneration, Areas suitable for mangrove restoratio
Organoarsenic probes to study proteins by NMR spectroscopy
Arsenical probes enable structural studies of proteins. We report the first organoarsenic probes for nuclear magnetic resonance (NMR) spectroscopy to study proteins in solutions. These probes can be attached to irregular loop regions. A lanthanide-binding tag induces sizable pseudocontact shifts in protein NMR spectra of a magnitude never observed for small paramagnetic probes before
Organoarsenic probes to study proteins by NMR spectroscopy
Arsenical probes enable structural studies of proteins. We report the first organoarsenic probes for nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to study proteins in solutions. These probes can be attached to irregular loop regions. A lanthanide-binding tag induces sizable pseudocontact shifts in protein NMR spectra of a magnitude never observed for small paramagnetic probes before.C. N. thanks the Alexander von
Humboldt Foundation for a Feodor Lynen fellowship and the
Australian Research Council (ARC) for a Discovery Early Career
Research Award (DE190100015). G. O. gratefully acknowledges an
ARC Laureate Fellowship (project FL170100019) and research support (project CE200100012). Financial support by the Australian
Research Council for project funding (DP200100348) is gratefully
acknowledged
Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy
Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling
proteins site-specifically with a single lanthanide ion remains an ongoing
challenge, especially for proteins that are not suitable for ligation with
cysteine-reactive lanthanide complexes. We show that a specific lanthanide-binding site can be installed on proteins by incorporation of phosphoserine
in conjunction with other negatively charged residues, such as aspartate,
glutamate or a second phosphoserine residue. The close proximity of the
binding sites to the protein backbone leads to good immobilization of the
lanthanide ion, as evidenced by the excellent quality of fits between
experimental PCSs and PCSs calculated with a single magnetic susceptibility
anisotropy (Δχ) tensor. An improved two-plasmid system was
designed to enhance the yields of proteins with genetically encoded
phosphoserine, and good lanthanide ion affinities were obtained when the side chains of the phosphoserine and aspartate residues are not engaged in salt
bridges, although the presence of too many negatively charged residues in
close proximity can also lead to unfolding of the protein. In view of the
quality of the Δχ tensors that can be obtained from lanthanide-binding sites generated by site-specific incorporation of phosphoserine,
this method presents an attractive tool for generating PCSs in stable
proteins, particularly as it is independent of cysteine residues.</p