Structural investigations of biological systems are translated to increasingly complex systems. It emerges that more and more biological context is needed to make in vitro studies relevant to structure and function in cell or in vivo. While there is a range of extremely powerful structural techniques including electron microscopy, crystallography and nuclear magnetic resonance the addition of biological context (in the form of interacting proteins, nucleic acids, cofactors and metabolites) will most often massively reduce the structural resolution. Electron paramagnetic resonance (EPR) spectroscopy is a complementary method in the field of structural biology. The EPR technique called PELDOR (pulsed electron-electron double resonance or DEER for double electron-electron resonance) allows reliable distance measurements up to 10 nm and beyond between two paramagnetic centres. These paramagnetic centres can be native metal ions or radical cofactors, but most commonly they are deliberately introduced by site-directed mutagenesis and site-specific spin-labelling. One particular advantage of EPR is that it is only sensitive to the spin centres and can thus be used with systems of tremendous complexity without being overwhelmed by the number or complexity of signals. The PELDOR method has been shown to be highly reliable and extremely powerful especially when docking quarternary structures from substructures, evaluating structural models or tracing conformational transitions. A major challenge in these measurements is posed by the sensitivity of EPR methods. However, St Andrews has been driving the HIPER programme. The new instrument offers more than an order of magnitude increase in sensitivity relative to the most used commercial instrumentation - making PELDOR measurements at physiological concentrations possible - and this opens up a new field in biomolecular science. Together with collaborators from the Biomedical Sciences Research Complex (BSRC) St Andrews we aim to exploit this hardware to make decisive progress understanding in the structure-function relationships of nucleic acid binding proteins (2), mechanosensation (3), and bacterial surface proteins by studying protein nucleic acid interactions, structural transitions in membrane proteins and protein-protein interactions.
The St Andrews-Dundee EPR grouping has an outstanding track record in EPR applications especially for biological distance measurements. Expertise ranges from production of optimised samples (mutagenesis, protein deuteration, reconstitution of membrane proteins) to data analysis and structural modelling and interpretation. St Andrews has the UK’s largest density of state-of-the-art pulse EPR facilities dedicated to structural biology applications.
This project will exploit the combined expertise of the Bode lab, driving methodology for biological EPR spectroscopy, and the mmwave and high-field EPR group being world leaders in the development of EPR instruments. The prospective student will receive training in a wide range of skills: molecular biology and mutagenesis, protein purification, spin-labelling, hands on EPR on laboratory built instruments and data analysis.
(1) Motion, et al. (2016) J Phys Chem Lett 7(8):1411-1415.
(2) Morten, et al. (2015) Nucleic Acids Res 43(22):10907-10924.
(3) Ackermann, et al. (2017) Biophys J in press.