Magnetic Resonance, FRET, EPR, Spin-labeling and PELDOR

Research

We are interested in using a variety of biophysical methods to investigate the structure and function of proteins and nucleic acids. Of particular interest is the use of EPR and particularly the PELDOR experiment.

With the advent of pulsed techniques, Electron Paramagnetic Resonance (EPR) spectroscopy has become a powerful tool in biological structure investigation. 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 nitroxide spin labels 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 within the context of the tertiary or quaternary structure of the biomolecule. When applied correctly the observed dipolar coupling can be interpreted to reveal distances of up to about 80Å, giving not only an accurate distance but also an accurate distribution of the distances in molecular ensembles. We are currently investigating the structure and dynamics of the nucleosome, core tetramer and octamer. An associated effort is also aimed at determining the structures and interactions of a number of histone chaperone proteins. Other targets include The BamA, a protein chaperone system from Gram negative Bacteria, and cytoplasmic integrin interactions.

Other techniques we use include:-

• Magnetic resonance (EPR and NMR)
• Fluorescence (FRET)
• Computational techniques (homology modeling and drug design)
• Diffraction techniques (SAXS and crystallography)

Teaching

Publications

1. Andrew Bowman, Richard Ward, Hassane El-Mkami, Tom Owen-Hughes, and David G. Norman "Probing the (H3-H4)2 histone tetramer structure using pulsed EPR spectroscopy combined with site-directed spin labelling" Nucl. Acids Res. 2010 38: 695-707
2.  R.Ward, M. Zoltner, L.Beer, H.El Mkami, I. R. Henderson, T. Palmer, D.G.Norman “The Orientation of a Tandem POTRA Domain Pair, of the Beta-Barrel Assembly Protein BamA, Determined by PELDOR Spectroscopy” Structure 2009, 17(9), 1187-1194
3.  Ward R., Bowman A., El-Mkami H., Owen-Hughes T., Norman D.G. “ Long Distance PELDOR Measurements on the Histone Core Particle” JACS 2009 131 (4), pp 1348–1349
4.  Iqbal A., Wang L., Thompson K.C., Lilley D.M.J., and Norman D.G. “The Structure of Cyanine 5 Terminally Attached to Double-Stranded DNA: Implications for FRET studies” Biochemistry 2008 47(30), 7857-7862
5.  Lipfert J., Ouellet J., Norman D.G., Doniach S., and Lilley D.M.J. “The structure of the complete VS ribozyme in solution by small-angle X-ray scattering” Structure 2008 16(9), 1357-1367
6.  A. Iqbal, S. Arslan, B. Okumus, T. J. Wilson, G. Giraud, D. G. Norman, T. Ha and D. M. J. Lilley, “Orientation dependence in fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids” PNAS 2008 105(32), 11176-11181
7.  Ward R., Keeble J.D., El-Mkami H., Norman D.G. “Distance Determination in Heterogeneous DNA Model Systems by Pulsed EPR” ChemBioChem 2007, 8, 1957-1964