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Your search keyword '"Timothy J. Nott"' showing total 4 results
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4 results on '"Timothy J. Nott"'

1. UnidecNMR: automatic peak detection for NMR spectra in 1-4 dimensions

Author

Charles Buchanan, Gogulan Karunanithy, Olga Tkachenko, Michael Barber, Michael T. Marty, Timothy J. Nott, Christina Redfield, and Andrew J. Baldwin

Subjects
Science
Abstract

Abstract To extract information from NMR experiments, users need to identify the number of resonances in the spectrum, together with characteristic features such as chemical shifts and intensities. In many applications, particularly those involving biomolecules, this procedure is typically a manual and laborious process. While many algorithms are available to tackle this problem, their performance tends to be inferior to that of an experienced user. Here, we introduce UnidecNMR, which identifies resonances in NMR spectra using deconvolution. We demonstrate its favourable performance on 1 and 2D simulated spectra, strongly overlapped 1D spectra of oligosaccharides and 2D HSQC, 3D HNCO, 3D HNCA and 3/4D methyl-methyl NOE experimental spectra from a range of proteins. UnidecNMR outperforms a number of freely available algorithms and provides results comparable to those generated manually. Introducing additional restraints, such as a 2D peak list when analysing 3 and 4D data and incorporating reflection symmetry in NOE analysis further improves the results. UnidecNMR outputs a back-calculated spectrum and a peak list, both of which can be easily examined using the supplied GUI. The software allows interactive processing using nmrPipe, allowing users to go directly from raw data to processed spectra with picked peak lists.

Published
2025
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2. Ion binding with charge inversion combined with screening modulates DEAD box helicase phase transitions

Author

Michael D. Crabtree, Jack Holland, Arvind S. Pillai, Purnima S. Kompella, Leon Babl, Noah N. Turner, James T. Eaton, Georg K.A. Hochberg, Dirk G.A.L. Aarts, Christina Redfield, Andrew J. Baldwin, and Timothy J. Nott

Subjects
CP: Molecular biology, Biology (General), QH301-705.5
Abstract

Summary: Membraneless organelles, or biomolecular condensates, enable cells to compartmentalize material and processes into unique biochemical environments. While specific, attractive molecular interactions are known to stabilize biomolecular condensates, repulsive interactions, and the balance between these opposing forces, are largely unexplored. Here, we demonstrate that repulsive and attractive electrostatic interactions regulate condensate stability, internal mobility, interfaces, and selective partitioning of molecules both in vitro and in cells. We find that signaling ions, such as calcium, alter repulsions between model Ddx3 and Ddx4 condensate proteins by directly binding to negatively charged amino acid sidechains and effectively inverting their charge, in a manner fundamentally dissimilar to electrostatic screening. Using a polymerization model combined with generalized stickers and spacers, we accurately quantify and predict condensate stability over a wide range of pH, salt concentrations, and amino acid sequences. Our model provides a general quantitative treatment for understanding how charge and ions reversibly control condensate stability.

Published
2023
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3. Effective inhibitors of the essential kinase PknB and their potential as anti-mycobacterial agents

Author

Jasveen Chugh, David Whalley, Timothy J. Nott, Roger S. Buxton, Nathalie Bouloc, Barbara Saxty, Justin S. Bryans, Tim Chapman, Simon A. Osborne, Stephen J. Smerdon, Debra L. Taylor, Dony Patel, Vicky L. Spivey, Kathryn E.A. Lougheed, and Catherine A. Kettleborough

Subjects
Microbiology (medical), Immunology, Kinases, Protein Serine-Threonine Kinases, Microbiology, PknB, Mycobacterium tuberculosis, In vivo, Drug Discovery, Humans, Tuberculosis, Protein Kinase Inhibitors, Cells, Cultured, Protein-Serine-Threonine Kinases, biology, Drug discovery, Kinase, Inhibitors, Macrophages, Autophosphorylation, biology.organism_classification, Anti-Bacterial Agents, Infectious Diseases, Biochemistry, Phosphorylation, Signal transduction, Cell Division, Drug Discovery and Resistance, Signal Transduction
Abstract

Summary PknB is an essential serine/threonine kinase of Mycobacterium tuberculosis with possible roles in a number of signalling pathways involved in cell division and metabolism. We screened a library of >50,000 compounds for inhibitors of the in vitro phosphorylation of GarA (Rv1827) by PknB and identified a number of inhibitors. A program of synthetic medicinal chemistry was subsequently conducted around one class of inhibitors and was successful in generating ATP competitive inhibitors with potency in the nanomolar range. Compounds in this class showed cross-reactivity with the related M. tuberculosis kinase, PknF, but not with PknG in an in vitro autophosphorylation assay. These synthesised inhibitors were able to prevent the growth of M. tuberculosis in an Alamar blue assay and in an intracellular model of infection, but only in the micromolar range. We attempted to determine if cell wall permeability was an explanation for the discrepancy between the potent in vitro compared with relatively poor in vivo activity, but found no evidence that the activity of the inhibitors could be improved by weakening the cell wall. Despite a number of drug discovery efforts attempting to develop inhibitors against PknB, it is yet to be reported that any such inhibitors prevent mycobacterial growth at submicromolar concentrations.

Published
2011

4. Versatility in Phospho-dependent Molecular Recognition of the XRCC1 and XRCC4 DNA-damage Scaffolds by Aprataxin-family FHA Domains

Author

Keith W. Caldecott, Geoffrey Kelly, Timothy J. Nott, Stephen J. Smerdon, Stuart L. Rulten, and A.L. Cherry

Subjects
ZF, zinc finger, DNA Repair, BRCT, BRCA1 C-terminal, APLF, aprataxin- and PNK-like factor, Crystallography, X-Ray, Biochemistry, APTX, aprataxin, XRCC1, DNA-PKcs, catalytic subunit of DNA-dependent protein kinase, DNA-(Apurinic or Apyrimidinic Site) Lyase, Poly-ADP-Ribose Binding Proteins, Phosphorylation, Casein Kinase II, PARP-1, poly(ADP-ribose)polymerase 1, ITC, isothermal titration calorimetry, Nuclear Proteins, 3. Good health, DNA-Binding Proteins, NHEJ, non-homologous end-joining, HIT, Histidine triad, Phosphotransferases (Alcohol Group Acceptor), DNA break repair, FHA, forkhead-associated, FHA domain, Casein kinase 1, Casein kinase 2, XRCC1, X-ray repair cross-complementing protein 1, DNA repair, Molecular Sequence Data, DNA-damage signalling, Biology, DNA-binding protein, Article, CC, coiled-coil, QH301, PNKP, polynucleotide kinase phosphatase, Humans, Amino Acid Sequence, NTD, N-terminal domain, Binding site, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, Aprataxin, Binding Sites, XRCC4, X-ray repair cross-complementing protein 4, Cell Biology, QP, Protein Structure, Tertiary, CK2, casein kinase 2, DNA Repair Enzymes, X-ray Repair Cross Complementing Protein 1, DNA Damage
Abstract

Highlights • Aprataxin and APLF FHA domains show distinct specificities for XRCC4 and XRCC1. • Specificity is modulated by multi-site phosphorylation. • The crystal structure of APLF FHA and triphosphorylated XRCC4 peptide reveals a conserved mode of core pSpT motif binding. • Overall phospho-dependent binding versatility of Aptx-family FHA domain proteins is provided by FHA surface basicity., Aprataxin, aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.

Published
2015

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