Cryogen-Free Arbitrary Waveform EPR for Structural Biology and Biophysics

This equipment upgrade will enhance a large number of molecular biosciences related projects that utilise electron paramagnetic resonance (EPR) spectroscopy and will have a major impact in the biomedical sciences and structural biology at the Universities of St Andrews and Dundee and beyond.
EPR is an ideal method for obtaining specific information at the nanoscale, as it is exquisitely sensitive to the magnetic spins of radicals and other paramagnetic centres. These are of interest as many are hotspots of biochemical activity. Recently, the number of applications utilising EPR has greatly increased. This increase was catalysed by new technology which allows researchers to selectively introduce spins into biomolecules and use them as molecular beacons. The magnetic moments of the spins interact with the magnetic moments of other paramagnets or magnetic nuclei and thus magnetically illuminate their surroundings. It has become increasingly popular to use measurements of the long-range distances between the spins to map the nanoworlds of protein conformations and interactions quite as if navigating by the relative positions and brightness of lighthouses.
Recently, developments have started to transform this science.
While spins are commonly manipulated by on/off electromagnetic irradiation, the advent of arbitrary waveform generators has opened up a whole new universe allowing access to new realms of information in experiments. This technology is often proposed to be the future of EPR and the analogous advancements have wholly transformed the field of nuclear magnetic resonance. Now that the more demanding technical requirements of EPR are fulfilled it seems a logical imperative to upgrade to this technology as the upgrade even just means a moderate additional cost compared to the initial investment.
A second development concerns the need to cool the spins to extremely low temperatures using liquid helium. Helium is an expensive, finite resource that has proven to be highly susceptible to disruption in the supply chain and poses a considerable safety hazard. Traditionally, liquid helium would be connected to the spectrometer before performing EPR experiments. Recently, cryogen-free cryostats have been developed using closed-cycle cooling that operates like an ultra-low temperature refrigerator. In these cryostats, used helium is cooled by electrically driven compression and expansion cycles to re-liquefy and thus be recycled. This greatly improves reliability and sustainability. Significantly, this also reduces risks to the researchers whilst performing experiments. Additionally, the increased stability means that the facility can operate around the clock and for a longer period of time, allowing more experiments to be performed and more scientific questions to be answered in a given time, thereby substantially improving the efficiency of the facility.
The Universities of St Andrews and Dundee have developed an extensive programme of biological applications using this methodology and now seek to implement cryogen-free arbitrary waveform EPR to enhance existing facilities for projects investigating biomedical challenges and opportunities in the bio-based economy of the future.

With the ever-growing popularity of site-directed mutagenesis and site-specific spin labelling EPR has become an established biophysical tool for tracing structural or conformational transitions and quaternary complex formation. The increasing complexity of the systems studied multiplies the demand on the sensitivity and accuracy of the EPR methods used. As a response, there is rapid development of novel labelling strategies and of more intricate experiments that demand full control of the frequency, phase and amplitude profiles of the microwave irradiation for EPR excitation. The ability to engineer arbitrary pulses is critical to new enabling experiments, designed to improve sensitivity and resolution by precisely controlling the excitation of spins. Additionally, most advanced EPR experiments require cryogenic cooling to achieve meaningful results on biological samples. Today, nearly all new EPR installations in the UK are equipped with cryogen-free (cf) cooling systems to remove the costly and inconvenient use of liquid helium. These cf systems provide better control of temperature, permitting longer experiments and reduce safety hazards related to liquefied gases.
This proposal seeks to fund arbitrary waveform generator and cryogen-free cryostat upgrades for a commercial Bruker E580 X-band pulse EPR spectrometer at the Biomedical Sciences Research Complex, St Andrews. The projects that would greatly benefit from the new technology are involving nanometre distance measurements between radical spin-labels and/or paramagnetic metal ions and the characterisation of intrinsic or engineered paramagnetic sites and of their surroundings by pulse EPR and hyperfine spectroscopy. Current systems are ranging from membrane proteins to nucleic acids, covering research topics as diverse as mechanosensation and genome maintenance.

The proposed equipment upgrade will greatly enhance an extremely broad range of molecular bioscience projects. The scope of applications will be hugely increased through bespoke experiments becoming available allowing researchers to achieve the next level of sensitivity and sustainability provided by the two upgrades. This would maintain St Andrews' and the UK's position at the forefront of molecular bioscience research expertise.
For projects investigating aspects of nucleic acid structure, dynamics and interactions with other biomolecules, results produced from the upgrades will have profound impacts on understanding and manipulating genome maintenance (with relevance to many disease conditions e.g. cancer and ageing), genome editing and biochips. Further insights in these fields will provide advancements in healthcare and wellbeing.
A second field of research where substantial advancements in understanding will be made is in the understanding of conformational changes in difficult to analyse molecules such as membrane proteins e.g. channels and transporters. A large number of these have been validated as drug-targets for conditions ranging from cardiovascular disease, neurological and immunological disorders to cancer. Many targets have also been validated for infectious diseases including bacterial and viral infections (with implications for antimicrobial resistance), parasites or fungal/yeast infections. Impacts will materialise in the pharmaceutical value chain from drug-discovery to treatment with benefits to the global community in healthcare and veterinary treatments.
Projects aligning with industrial biotechnology (e.g. biorenewable resources as exemplified by the valorisation of lignin) as well as agriculture and food security (e.g. understanding host/pathogen interactions in plants) would be feasible with the upgraded facility with implications for considerable global economic impact.
The facility will also influence the commercial Electron Paramagnetic Resonance market. The equipment will be highly specialised, consisting of state-of-the-art technology aligned with cutting-edge software. The commercial developers work closely with researchers to incorporate novel user-led developments in hardware and experimental design.
Furthermore, research staff and students from more than a dozen research groups in St Andrews and Dundee (most with a track record of BBSRC funding) and many more from across Scotland, the UK and Europe will be involved with and directly benefit from using this equipment. They will get the opportunity to explore biomolecular systems using state-of-the art techniques and world-class facilities and expertise.
The projects underpinned by the equipment have the potential to have impact in a variety of fields and will in the short to medium-term be beneficial for the UK's economy, as existing and proposed research projects will be greatly enhanced.
In the longer term, the results from research on the fundamental understanding of biomolecules will benefit the search for treatments of disease and further biomedical applications which will be transformative for health and wellbeing in the UK and around the world.
Academic impact will be leveraged by dissemination of results at conferences and seminars as well as publication in prestigious peer reviewed journals and data deposition in appropriate repositories.