David George Norman

Publications (68) View all

• Article: The Architecture of EssB, an Integral Membrane Component of the Type VII Secretion System.

  Martin Zoltner, David G Norman, Paul K Fyfe, Hassane El Mkami, Tracy Palmer, William N Hunter
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  ABSTRACT: The membrane-bound EssB is an integral and essential component of the bacterial type VII secretion system that can contribute to pathogenicity. The architecture of Geobacillus thermodenitrificans EssB has been investigated by combining crystallographic and EPR spectroscopic methods. The protein forms a dimer that straddles the cytoplasmic membrane. A helical fold is observed for the C-terminal segment, which is positioned on the exterior of the membrane. This segment contributes most to dimer formation. The N-terminal segment displays a structure related to the pseudokinase fold and may contribute to function by recognizing substrates or secretion system partners. The remaining part of EssB may serve as an anchor point for the secretion apparatus, which is embedded in the cytoplasmic membrane with the C-terminal domain protruding out to interact with partner proteins or components of peptidoglycan.
  Structure 03/2013; · 6.35 Impact Factor
• Article: Structural plasticity of histones H3-H4 facilitates their allosteric exchange between RbAp48 and ASF1.

  Wei Zhang, Marek Tyl, Richard Ward, Frank Sobott, Joseph Maman, Andal S Murthy, Aleksandra A Watson, Oleg Fedorov, Andrew Bowman, Tom Owen-Hughes, Hassane El Mkami, Natalia V Murzina, David G Norman, Ernest D Laue
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  ABSTRACT: The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of the histone H3-H4 complex with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3-H4 between these two histone chaperones has a central role in the assembly of new nucleosomes, and we show here that the H3-H4 complex has an unexpected structural plasticity, which is important for this exchange.
  Nature Structural &#38 Molecular Biology 11/2012; · 12.71 Impact Factor
• Article: The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether.

  Linas Urnavicius, Scott A McPhee, David M J Lilley, David G Norman
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  ABSTRACT: Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ∼30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5'-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information.
  Biophysical Journal 02/2012; 102(3):561-8. · 3.65 Impact Factor
• Article: Analysis of conformational changes in the DNA junction-resolving enzyme T7 endonuclease I on binding a four-way junction using EPR.

  Alasdair D J Freeman, Richard Ward, Hassane El Mkami, David M J Lilley, David G Norman
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  ABSTRACT: The four-way (Holliday) DNA junction is the central intermediate in homologous recombination. It is ultimately resolved into two nicked-duplex species by the action of a junction-resolving enzyme. These enzymes are highly selective for the structure of branched DNA, yet as a class these proteins impose significant distortion on their target junctions. Bacteriophage T7 endonuclease I selectively binds and cleaves DNA four-way junctions. The protein is an extremely stable dimer, comprising two globular domains joined by a β-strand bridge with each active site including amino acids from both polypeptides. The crystal structure of endonuclease I has been solved both as free protein and in complex with a DNA junction, showing that the protein, as well as the junction, becomes distorted on binding. We have therefore used site-specific spin-labeling in conjunction with EPR distance measurements to analyze induced fit in the binding of endonuclease I to a DNA four-way junction. The results support the change in protein structure as it binds to the junction. In addition, we have examined the structure of wild type and catalytically inactive mutants alone and in complex with DNA. We demonstrate the presence of hitherto undefined metastable conformational states within endonuclease I, showing how these states can be influenced by DNA-junction binding or mutations within the active sites. In addition, we demonstrate a previously unobserved instability in the N-terminal α1-helix upon active site mutation. These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme.
  Biochemistry 11/2011; 50(46):9963-72. · 3.42 Impact Factor
• Source

  Available from: dundee.ac.uk

  Article: EPR distance measurements in deuterated proteins.

  Richard Ward, Andrew Bowman, Erman Sozudogru, Hassane El-Mkami, Tom Owen-Hughes, David G Norman
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  ABSTRACT: One of the major problems facing distance determination by pulsed EPR, on spin-labeled proteins, has been the short relaxation time T(m). Solvent deuteration has previously been used to slow relaxation and so extend the range of distance measurement and sensitivity. We demonstrate here that deuteration of the underlying protein, as well as the solvent, extends the T(m) to a considerable degree. Longer T(m) gives greatly enhanced sensitivity, much extended distance measurement, more reliable distance distribution calculation and better baseline correction.
  Journal of Magnetic Resonance 11/2010; 207(1):164-7. · 2.14 Impact Factor