- Protein-nucleic acid interactions and structures, including chromatin and proteins of the HMG domain family.
- NMR spectroscopy, fluorescence and calorimetry are my main research tools.
I am interested in how and why proteins and nucleic acids fold up and interact, all in relation to their 3D structure. Study of the molecule(s) of interest often uses a combination of molecular biophysical techniques, e.g. scattering (SAX, SANS), centrifugation (AUC), spectroscopy (NMR, fluorescence, circular dichroism, optical melting) or calorimetry (ITC and DSC) to obtain information on the how and why of their folding and interactions. Central to this work is the optimal expression and purification of recombinant proteins and nucleic acids, such as in isotopic labelling for NMR.
I graduated in 1977 from Portsmouth with a BSc (Hons) degree in Biomolecular Science. I went on to complete a PhD on chromatin in the laboratory of Prof. Colyn Crane-Robinson with a thesis entitled “Studies of Subnucleosomal Complexes containing only the Arginine-rich Histones H3 and H4” (Examiner: Dame Prof J.O. Thomas FRS, Cambridge). A range of molecular biophysical research tools (protein crosslinking, nuclease footprinting, AUC, UV melting, CD and NMR spectroscopy) was used to characterise the protein-DNA complexes. A highlight included small angle neutron scattering at the Institut Laue-Langevin, a world-leading nuclear research facility in Grenoble. More recently I have come full circle to chromatin again.
My first post-doctoral position was in the laboratory of Prof. Tom Moss (at Portsmouth) on a project to find and map in vivo replication origins in the naturally cell-cycle synchronous slime-mold Physarum polycephalum. The work continued with mapping replication origins in the chromatin loci encompassing the human alpha- and beta-globin genes.
As a part-time lecturer and research fellow, I collaborated with Tom Moss (now at the Universite de Laval, Quebec) on the regulation of ribosomal RNA transcription in the African clawed toad, Xenopus. Using point mutants we produced high resolution maps of the DNA sequences important for initiation and termination of the RNA polymerase I transcripts and for interaction with the transcription factor UBF. The confluence resulting from the recognition that UBF contains multiple DNA binding domains highly related to the two domains of the chromatin protein HMGB1, then an active project in the Crane-Robinson lab, led me into structural biology.
I was one of the first to determine the structure of an HMG box (or domain), the 2nd box of mammalian HMGB1, by multidimensional NMR at Oxford with Drs. Paul Driscoll and Dave Norman. I continued with determining structures and dynamics of the sequence-specific HMG box from mouse Sox-5 protein, free and in complex with DNA, using high-field multidimensional NMR at Portsmouth, UCL London, Frankfurt and Birmingham. The interest of the lab in the determinants of DNA sequence-specificity and of DNA bending has led to a long collaboration with Prof. Peter Privalov (Johns Hopkins University, Baltimore) in which the thermodynamics of the interaction of DNA binding protein domains with DNA has been probed by calorimetry and optical spectroscopy. It has become clear that water in the minor groove of DNA but not in the major-groove, is ice-like and mediates the entropic interaction of minor-groove DNA binding domains, such as the HMG box.
In 1999, I became a Senior Experimental Officer in the School of Biological Sciences. One of my first jobs was to commission the Biological NMR facility. I now operate our 600 MHz spectrometer having 5-channels, HCN coldprobe and z-axis gradients. I have used NMR to probe the structure, dynamics and conformational equilibria of various proteins, for example MMP-1, of stapled ER peptides (a collaboration with Pfizer UK) and of small circular peptides (e.g. Vasopressin, Urotensin).
My extensive research experience of molecular biophysical techniques has appeared in two undergraduate textbooks, including a front cover [1,2]. Additionally, I have written book chapters offering practical laboratory advice to researchers on fluorescence spectroscopy and calorimetry.
1. H. Rattle. An NMR Primer for Life Scientists, Partnership Press, Fareham, 1995, pp. 136. ISBN: 9780951643631.
2. I.N. Serdyuk, N.R. Zaccai, J. Zaccai. Methods in Molecular Biophysics: Structure, Dynamics and Function, first ed., Cambridge University Press, Cambridge, 2007, pp. 1136. ISBN: 9780521815246.