Dr David Norman
We use a variety of biophysical methods to investigate the structure and function of proteins and nucleic acids. Most of our present efforts particularly focus on using pulsed Electron Paramagnetic Resonance (pEPR) in conjunction with site specific spin labeling for long distance and orientation measurements in protein complexes.
We are using Pulsed ELectron-electron DOuble Resonance (PELDOR a.k.a. DEER) to investigate the structure of nucleic acids, proteins and protein-DNA complexes. The technique uses Site Directed Spin Labeling (SDSL) to incorporate spin labels (mainly nitroxide) into the primary structure of proteins or nucleic acids. The PELDOR experiment uses the dipolar coupling between two unpaired electron spins to determine the distance between them. When applied correctly the observed dipolar coupling can be interpreted to reveal long distances giving not only an accurate distance but also an accurate distribution of the distances in molecular ensembles. We have recently developed a technique using total system deuteration, which has allowed us to measure distances of up to 102Å, and probably further. In addition to measuring extreme distances we are developing techniques for the measurement of spin label orientation and its application to structure reconstruction. Amongst the subjects being investigated at this time are several aspects of nucleosome structure including histone chaperones and remodeling complexes, protein-DNA complexes, membrane proteins and other protein chaperones. In conjunction with our colleagues in Dundee and St Andrews Universities, we run an advanced EPR centre, having facilities for running X-band, very high power Q-band and an advanced quasi-optical W-band spectrometer (HIPER).
Spin labeling and PELDOR distance measurement on fully deuterated Nap1 T302R1. A distance distribution centred at 102Å. A distance far longer is than possible without protein deuteration. A) The Nap1 dimer with spin label ensembles shown in blue and red. B) background corrected PELDOR trace showing dipolar oscillations extending past 33ms. C) Tikhonov derived distance distribution showing a modal distance of 102Å.
- Zoltner, M., Norman, D.G., Fyfe, P.K., El Mkami, H., Palmer, T., Hunter, W.N., 2013 The architecture of EssB, a Type VII secretion system integral membrane component, derived by combining crystallography and PELDOR spectroscopy. 2013 Structure 21, 595
- Zhang, W., Tyl, M., Ward, R., Sobott, F., Maman,J., Murthy, A.S., Watson, A.A. , Fedorov, O., Bowman, A. , Owen-Hughes, T. , EL Mkami, H. , Murzina, N.V., David Norman, D.G., & Laue, E.D. Structural plasticity of histones H3–H4 facilitates their allosteric exchange between RbAp48 and ASF1. 2013 Nature Structural & Molecular Biology 20, 1, 29-U43
- Urnavicius, L., S. A. McPhee, D. M. Lilley, and D. G. Norman. 2012. The Structure of Sulfoindocarbocyanine 3 Terminally Attached to dsDNA via a Long, Flexible Tether. Biophys J 102:561-568.
- Freeman, A. D. J., R. Ward, H. El Mkami, D. M. J. Lilley, and D. G. Norman. 2011. Analysis of conformational changes in the DNA junction-resolving enzyme T7 endonuclease I on binding a four-way junction, using EPR. Biochemistry
- Bowman, A., R. Ward, N. Wiechens, V. Singh, H. El-Mkami, D. G. Norman, and T. Owen-Hughes. 2011. The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation. Molecular Cell 41:398-408.
- Ward, R., A. Bowman, E. Sozudogru, H. El-Mkami, T. Owen-Hughes, and D. G. Norman. 2010. EPR distance measurements in deuterated proteins. Journal of Magnetic Resonance 207:164-167.
- Ellis, I. R., S. J. Jones, D. Staunton, I. Vakonakis, D. G. Norman, J. R. Potts, C. M. Milner, N. A. G. Meenan, S. Raibaud, and G. Ohea. 2010. Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor). Experimental cell research 316:2465-2476.
- Bowman, A., R. Ward, H. El-Mkami, T. Owen-Hughes, and D. G. Norman. 2010. Probing the (H3-H4) 2 histone tetramer structure using pulsed EPR spectroscopy combined with site-directed spin labelling. Nucleic acids research 38:695-707.
- Ward, R., M. Zoltner, L. Beer, H. El Mkami, I. Henderson, T. Palmer, and D. Norman. 2009. The orientation of a tandem POTRA domain pair, of the beta-barrel assembly protein BamA, determined by PELDOR spectroscopy. Structure 17:1187-1194.
- Ward, R., A. Bowman, H. El-Mkami, T. Owen-Hughes, and D. G. Norman. 2009. Long distance PELDOR measurements on the histone core particle. Journal of the American Chemical Society 131:1348-1349.
- Lleres, D., J. James, S. Swift, D. G. Norman, and A. I. Lamond. 2009. Quantitative analysis of chromatin compaction in living cells using FLIM‚ FRET. The Journal of Cell Biology 187:481-496.
- Lipfert, J., J. Ouellet, D. G. Norman, S. Doniach, and D. M. J. Lilley. 2008. The complete VS ribozyme in solution studied by small-angle X-ray scattering. Structure 16:1357-1367.
- Iqbal, A., L. Wang, K. C. Thompson, D. M. J. Lilley, and D. G. Norman. 2008. The Structure of Cyanine 5 Terminally Attached to Double-Stranded DNA: Implications for FRET Studies‚. Biochemistry 47:7857-7862.
- Iqbal, A., S. Arslan, B. Okumus, T. J. Wilson, G. Giraud, D. G. Norman, T. Ha, and D. M. J. Lilley. 2008. Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids. Proceedings of the National Academy of Sciences 105:11176.
- Ward, R., D. J. Keeble, H. El Mkami, and D. G. Norman. 2007. Distance determination in heterogeneous DNA model systems by pulsed EPR. ChemBioChem 8:1957-1964.