Enabling Shaped Pulse Capability for Superior Biological Structural Determination Using EPR Spectroscopy.

Biophysical techniques are a powerful way to explore the nature of biological processes and the biological molecules involved. The electron paramagnetic resonance (EPR) spectrometer is capable of extracting exquisitely detailed information about the local atomic environment surrounding atoms within these biological molecules that contain unpaired electrons - we refer to these as radicals or paramagnetic centres. Biological molecules such as proteins may contain natural paramagnetic centres, such as copper or iron, which are often fundamental to how the protein works, or an experimenter may incorporate paramagnetic metals or attach spin labels to specific parts of proteins as a molecular "spy".

These molecular spies can be used to measure local dynamics or measure long-range nanometre scale distances in the region to about 10 nm between pairs of paramagnetic centres. This is achieved by measuring the magnetic interaction between these pairs using pulsed EPR, which is analogous to measuring the force between two bar magnets (and is therefore highly dependent on their separation distance). Both distances and distance distributions can be found. Examples of these experiments are double electron electron resonance (DEER) and relaxation-induced dipolar modulation enhancement (RIDME) and collectively the techniques are referred to as pulsed dipolar spectroscopy (PDS).

This ability has proved very useful to the study of the structure and interactions of a wide variety of biomacromolecules and has now become a standard tool in biomolecular research. It is also a field which has seen tremendous technical advances over the last 10 years with sensitivity increasing by more than an order of magnitude, which has been transformative. Advances have come from higher frequency and higher power spectrometers, and our previous "state-of-the-art" commercial work-horse "Q-band" spectrometer operates almost continuously. However recent advances in fast digital electronics mean that further significant increases in both sensitivity and sample throughput have become possible. A large part of this comes from the ability to shape the phase, frequency and amplitude of microwave pulses in complex sequences. For many experiments this reduces measurement time 10-fold, dramatically increasing usage and capability for a multi-user, multi-project facility focussed on biological applications.

We, and nearly all leading EPR experts, see this technology as the future of the EPR technique for the biosciences. We have a large base of potential users and a wide variety of biological systems that would become interrogatable for the first time. Our investigators, collaborators and partners come from a wide range of national and international institutions. We have an extensive track record in the field and believe this upgrade will substantially increase the UK's capability and reputation in biological EPR.

This proposal will ensure sustainability for our centre based at St Andrews/Dundee. The position of our technical and applications manager will be secured for a further three years, and our fifteen-year-old spectrometer will be given a new lease of life. These improvements will allow us to remain internationally competitive and to continue developing and applying the EPR technique to relevant problems across the biosciences.

The onset of arbitrary waveform generators (AWGs) operating on the nanosecond timescale and therefore able to manipulate electron spins in electron paramagnetic resonance (EPR) experiments is set to revolutionise the EPR field. Just as the introduction of AWGs, and therefore pulse shaping, transformed NMR from the 1980s onwards. The EPR signal is often very broad and relaxes quickly which leads to issues with detection sensitivity. The ability to increase the sensitivity of the experiment from the tens of micromolar regime to hundreds of nanomolar will have profound consequences on the biological systems that EPR will be able to investigate. Such low concentrations are useful for many systems which cannot be reliably over expressed or synthesized, or for naturally occurring levels inside cells or to avoid aggregation effects - particularly important for membrane proteins. EPR experiments are well developed for investigating structure in the biosciences, for example pulsed dipolar spectroscopic experiments measure nanometre scale distances and distance distributions - a range complementary to other biophysical techniques - in proteins, protein complexes and nucleic acids in (usually frozen) solutions not limited to simple buffers. Crucially, the investigators on this proposal are experts in their application. Therefore, the AWG implementation will greatly increase the range and power of possible experiments impacting both on the future of St Andrews as a major EPR centre and on the ground-breaking bioscience research proposed.

This application will underpin a wide range of research across the biosciences within a number of institutions. The investigators have a good track record of both individual and significant collaborative work. The proposed upgrades will maintain this group at the cutting edge of what is a fast-developing field and allow continuing, major contributions in structural biology, which is applicable to such diverse areas as drug discovery for infectious diseases, antimicrobial resistance, cell signalling, cancer, virology, ageing and biocatalyst research. The investigation of biology, by EPR, at an atomic and nano scale is vital underpinning technology that supports and, in many cases, initiates research that has wide application, both academic and industrial. The EPR equipment will enable the users will also be able to continue contributing to graduate and post graduate education in magnetic resonance at a local, national and international level.