Dr David Norman
Nucleic Acid Structure, School of Life Sciences
+44 (0)1382 384798
Medical Sciences Institute
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 labelling for long-distance and orientation measurements in protein complexes.
See figure 1 below
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 remodelling 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 labelling 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.
Magnetic Resonance, FRET, EPR, Spin-labeling and PELDOR