Structural biology tackles ever more complex systems with high-resolution structures produced by EM, crystallography, NMR and predictions (e.g., AlphaFold2). However, understanding of biological function requires identification of conformational transitions, complex formation, ligand binding and dynamics. Pulse dipolar EPR spectroscopy (PDS) can provide this information without requiring ordered samples. It can accurately measure nanometre distances distributions with shapes and widths informing on conformational states and flexibilities. PDS also yields multimerisation degrees and binding constants in soluble and membrane proteins, nucleic acids, and their complexes in vitro and in cells. The technique requires two or more paramagnetic centres that are most often deliberately introduced by site-directed mutagenesis and site-specific spin-labelling. One advantage of PDS is the exclusive sensitivity to these spin centres allowing systems of tremendous complexity without being overwhelmed by the number of overlapping or unresolved signals.
Recent advances using double-histidine (dHis) motif CuII-based spin-labels have also facilitated extraction of more precise distances and accurate binding constants.1-3 Here, we aim to establish this approach to study the conformational landscape of surface proteins of pathogens, such as the coiled-coil dimeric M3 protein of Streptococcus pyogenes, responsible for initiating rheumatic heart disease through collagen binding in the human host, and, more ambitiously, the trimeric SARS-CoV-2 S-protein. The S-protein is highly cysteine-rich, making conventional thiol-based spin-labelling impractical, however the absence of endogenous histidine residues in dHis configurations, suggests exogeneous dHis CuII-labelling as an alternative strategy. The role of cleavage at the S2’ site is proposed to initiate large-scale conformational changes that are implicated in fusion competence and cell entry. Ultimately observing the conformational change as a distance change in PDS might become the foundation of an assay to probe binding inhibitors and S-protein mutants.
A major challenge in these measurements is posed by the sensitivity of EPR methods. However, the HIPER spectrometer developed in St Andrews offers more than an order of magnitude increase in sensitivity relative to the most used commercial instrumentation - making PDS measurements at physiological concentrations possible - and this opens up a new field in biomolecular science.
This project will exploit the combined expertise of the Bode lab, driving methodology for biological PDS applications, and the Smith 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 and structural modelling.
Infrastructure: The EPR infrastructure available in St Andrews is truly world-leading (a Bruker EMX cw-X-Band, a Bruker E580 pulse X-Band, a Bruker E580 pulse X/Q-band hybrid and a purpose-built high power pulse W-band all spectrometers having variable temperature accessories 4-300 K, and all pulse spectrometers capable of high power ELDOR excitation). The BSRC provides outstanding facilities for performing biochemical and molecular biology work and analysis (mass spectrometry, protein crystallography, protein NMR). The School of Chemistry hosts excellent synthesis and characterisation facilities (solution and solid-state NMR, ATR-IR, HPLC, UV-Vis ...).
Informal enquiries can be made to Dr Bela Bode (firstname.lastname@example.org).
Founded in the early 15th century, St Andrews is Scotland’s first university and the third oldest in the English speaking world. The University of St Andrews is a diverse and international community of over 9000, comprising students and staff of over 100 nationalities. It has 7,500 students, 6,000 of them undergraduates, and employs approximately 2,460 staff. The University is one of Europe’s most research intensive seats of learning. It is the top rated University in Scotland for teaching quality and student satisfaction and among the top rated in the UK for research.
HOW TO APPLY
Application instructions can be found on the EASTBIO website- http://www.eastscotbiodtp.ac.uk/how-apply-0
1) Download and complete the Equality, Diversity and Inclusion survey.
2) Download and complete the EASTBIO Application Form.
3) Submit an application to St Andrews University through the Online Application Portal- https://www.st-andrews.ac.uk/study/apply/postgraduate/research/
Your online application must include the following documents:
- Completed EASTBIO application form
- 2 References (to be completed on the EASTBIO Reference Form, also found on the EASTBIO website)
- Academic Qualifications
- English Language Qualification (if applicable)
Unfortunately due to workload constraints, we cannot consider incomplete applications. Please make sure your application is complete by the 16th December 2021.