Structural investigation of biomolecules is fundamental to the elucidation of their functional mechanisms. In the dogma of modern structural biology, structure determines biomolecular function. However, increasing evidence shows that also flexibility and dynamics are important for biological function and have implications for dysfunction and disease. In addition, biomolecular function is governed by the interaction between biomolecules leading to the formation of large complexes within cells. Thus, the investigation of biological structures of ever-increasing complexity requires methods on the relevant length-scale and the ability to follow complex formation within cells.
This project will employ electron paramagnetic resonance (EPR) spectroscopy that has been making increasingly important contributions to studies of structure and dynamics over the past decade. EPR can provide nanometre distances between specific points of the biomolecular assembly and monitor changes in the structure and flexibility of the biomolecule. EPR detects the quantum mechanical spin of unpaired electrons. While all matter contains electrons, these are rarely unpaired as this leads to the high reactivity associated with ‘free radicals’. However, chemists have developed tools to introduce stable radicals selectively at strategic positions that can be arbitrarily chosen and thus are able to site-selectively label the biomolecule with a spin. The exclusive and exquisite sensitivity of EPR for electron spins gives the method a very high structural contrast and makes it very appealing in complicated systems where other methods lack resolution. Nevertheless, the method has the drawbacks that the system is commonly studied in isolation (in vitro) and lacks biological context. Furthermore, the spin-labels available commonly require cooling to very low temperatures for achieving good resolution and the molecules are often needed in concentrations that exceed physiological conditions by far.
This project will combine several recent achievements made by EPR researchers in Bonn, St Andrews and elsewhere. The use of bespoke triarylmethyl (TAM)-based spin labels developed in Bonn has allowed measurements in cells, despite the reducing environment and miniscule volume available. This type of strategy has been shown also to bring experiments at ambient temperature within grasp. In parallel, St Andrews researchers have been able to perform experiments at much lower concentrations using only 200 nanograms of sample. The new experimental approaches will be combined to demonstrate distance measurements at physiological concentrations, ambient temperature and in the biological context of the interior of a cell.
Room temperature measurements depend on a sufficiently slow rotational reorientation to not average the dipolar interaction between spins. This holds for both very large complexes and membrane anchored systems. The pathogen Yersinia circumvents the host’s immune system by the use of a Type III secretion system and its YopO protein activates upon actin binding. This leads to a large scale conformational change while also immobilising the protein. YopO is an ideal target for room temperature in cell measurements but several other opportunities exist if YopO were to prove intractable (e.g. bacterial surface proteins, nucleic acid binding/remodelling enzymes, amino sugar transferases). The scholar will synthesise the TAM spin-label and produce proteins constructs that will be TAM labelled. The conditions of the experiment shall be optimised before targeting both room temperature and intracellular measurements. Observing structural transitions under native conditions will pave the way for widespread application of this approach.
The successful candidate will be supervised by Dr Bela Bode in the School of Chemistry at the University of St Andrews and by Professor Olav Schiemann in the Institute for Physical and Theoretical Chemistry at the University of Bonn.
Informal enquiries regarding this scholarship may be addressed to Dr Bela Bode – email [email protected]
What does it cover?
Duration up to 3.5 years. The student will be expected to spend approximately half of the award term at the University of St Andrews and half at the University of Bonn. The successful candidate will be expected to have completed the doctorate degree by the end of the award term. The award term excludes the continuation period and any extension periods.
The award covers full tuition fees for the period of time spent at the University of St Andrews. For this period, the student will also receive a stipend payable at the standard UK Research council rate (the 2019-2020 annual rate is £15,009). For the period spent at the University of Bonn the student will receive equivalent funding to help with living and maintenance costs.
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