23 research outputs found
Biophysical Studies of Asymmetric Homodimerisation of the microRNA Biogenesis Cofactors PACT and TRBP
No full textProcessing of precursor microRNAs by Dicer is a key step in microRNA biogenesis. This process is assisted by the homologous proteins PACT and TRBP, which bind to the helicase domain of Dicer. The mechanism by which they assist microRNA biogenesis is poorly understood, but could include facilitating substrate positioning, assisting Argonaute loading, or discriminating between different classes of pre-miRNA.
PACT also regulates innate immune pathways that respond to viral double-stranded RNA, including via the kinase PKR. Mutations in PACT lead to early onset dystonia in humans, while depletion of PACT in mice results in both growth and fertility defects: both have been linked to inappropriate or altered activation of PKR. Homodimerisation of PACT via its C-terminal domain (PACT-D3) is thought to be necessary for it to induce PKR activation.
Homodimerisation of wild-type and mutant constructs of PACT-D3 was assayed using biophysical techniques. SEC-MALLS and analytical ultracentrifugation data demonstrate that PACT-D3 homodimerises via a different mechanism to a previously reported dsRBD homodimer, dsRBD-5 of Staufen1. Instead, NMR analyses show that PACT-D3 forms an asymmetric homodimer similar to that observed in the Drosophila melanogaster homologue Loquacious. Dimerisation could be abolished by the L273R mutation, while phospho-mimic mutations did not appear to significantly affect dimerisation. TRBP domain 3 also forms asymmetric dimers, but with weaker affinity due to sequence differences in its C- terminal -helix. Asymmetry is caused by a register shift between intermolecular parallel beta-strands, but the functional significance of asymmetric homodimerisation remains unclear.
The data presented in this thesis supports a model in which the homodimerisation interface of PACT-D3 overlaps with the surface that binds to Dicer, and suggests that PACT homodimerisation and the formation of a Dicer-PACT complex are incompatible
Profiling substrate promiscuity of wild-type sugar kinases for multifluorinated monosaccharides
No full textFluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluoro galactose, giving insight into changes in the active site that may account for the specificity of the enzyme towards certain substrate analogues
Gallium exchange in Fe-PQS complexes
No full textDFT structures from Gallium-For Iron substitutions within PQS complexes. Raw NMR data for all PQS, Fe-PQS and Ga-PQS complexe
Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides
No full textFluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs
Modern triple resonance protein NMR backbone assignment, using AlphaFold and unlabelling to drive chemical shifts assignment in proteins
No full textNMR is a powerful technique to study the structure, dynamics and interactions of proteins. However, to obtain atomic resolution data, NMR signals must first be correlated with specific chemical groups – a problem called assignment. The software AlphaFold has shown to be a great advancement in modern-day science. Until now, structural analysis of proteins had been bottlenecked by months/ years’ worth of slower techniques that were traditionally used to determine a protein structure In this project we have designed and applied a semi-automated assignment program called SNAPS (Simple NMR Assignment using Predicted Shifts). This allows the user to go from a set of NMR spectra of a protein with a known 3D AlphaFold or X-ray crystallography structure to a fully assigned chemical shifts of the backbone resonances. In addition, unlabelling experimental data can be incorporated into the use of the program to generate more reliable assignment data by helping the program along in the mapping of the amino acids for assignment by providing places in the assignment where the amino acid it is known. The program was largely written by Dr Alex Heyam from the university of Leeds but testing scripts and well as a NEF importer was written to ensure the program was user-friendly and ensured rigid, fool-proof datasets being imported for backbone assignment. The program also underwent large-scale testing on roughly 150 proteins with known 3D crystal structures as well as 15N unlabelleing data being implemented to improve assignment. AlphaFold structures could also be implemented for use in the program also. Assignment was as good as 86-88% dependent upon parameters, with the unlabelling being slightly more effective with assignment (for PDB crystal structures)
Correction to:1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal β-glucosidase from Trichoderma reesei (Biomolecular NMR Assignments, (2020), 10.1007/s12104-020-09959-2)
No full textIn the original publication of the article, the name of one of the authors is incorrect. The author's name is Eiso AB, but was modified to A. B. Eiso. The correct name is given in this Correction
1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal β-glucosidase from Trichoderma reesei
No full textβ-glucosidases have received considerable attention due to their essential role in bioethanol production from lignocellulosic biomass. β-glucosidase can hydrolyse cellobiose in cellulose degradation and its low activity has been considered as one of the main limiting steps in the process. Large-scale conversions of cellulose therefore require high enzyme concentration which increases the cost. β-glucosidases with improved activity and thermostability are therefore of great commercial interest. The fungus Trichoderma reseei expresses thermostable cellulolytic enzymes which have been widely studied as attractive targets for industrial applications. Genetically modified β-glucosidases from Trichoderma reseei have been recently commercialised. We have developed an approach in which screening of low molecular weight molecules (fragments) identifies compounds that increase enzyme activity and are currently characterizing fragment-based activators of TrBgl2. A structural analysis of the 55 kDa apo form of TrBgl2 revealed a classical (α/β)8-TIM barrel fold. In the present study we present a partial assignment of backbone chemical shifts, along with those of the Ile (I)-Val (V)-Leu (L) methyl groups of TrBgl2. These data will be used to characterize the interaction of TrBgl2 with the small molecule activators
Nuclear Magnetic Resonance and Computational Study of trans-(2:2,21,3-Butadiene)bis(trichloroplatinate(II))
No full textFragment-derived modulators of an industrial β-glucosidase
No full textA fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate binding site, generating a productive conformation for the enzyme-substrate complex
