BioEmPiRe; Accessing uncharted but essential landscapes to biological machineries by pulse EPR

To understand biological processes at a molecular detail one needs to capture and visualize proteins in action. Modern structural biology methods such as X-ray crystallography and cryo-electron microscopy (CryoEM) could provide static high-resolution snapshots of protein structures, but they are unable to capture proteins in motion to reveal dynamic function. The new state-of-the-art electron paramagnetic resonance (EPR) spectrometer in Leeds will be capable of extracting accurate distances between pairs of unpaired electrons engineered on protein sites and thus acting as a molecular nanoscale-ruler. The accurate measurement of such distances over a protein's functional cycle will thereby enable the elucidation of fundamental biological processes. Pulsed EPR is a powerful method in modern biomolecular research and has seen tremendous technical advances over the last 10 years with sensitivity increasing by more than an order of magnitude. As a network of protein structural molecular biologists in Leeds (including several leading EPR specialists), consider PELDOR as a key approach in the future of biosciences. We have a very large base of users in Leeds, nationally (Imperial, King's, Glasgow, St Andrews) and abroad (EU, Australia and India) and an unmatched variety of fundamental biological systems with representative proteins across all kingdoms of life. These proteins are involved in a wide range of disease-related biological mechanisms from cancer and neurodegeneration to antimicrobial resistance and metabolism. Novel information for fundamental biological machineries in molecular detail and currently inaccessible by other methods, would be first revealed by the new EPR spectrometer. Our investigators, collaborators and industrial partners come from a wide range of national and international institutions. We have an extensive track record in the field of EPR and biological and medical sciences and anticipate this installation will substantially increase the UK's capability and reputation in biological EPR worldwide. Our business case will ensure sustainability for the Leeds-based centre and will serve the North East and other Universities as demonstrated by our list of groups and investigators actively supporting BioEmPiRe. The position of an EPR staff scientist will be secured for an initial period of two years through a contribution by the University of Leeds. In addition, intended location will be fully refurbished and a chiller will be purchased to enable the optimal installation and operation of the spectrometer. The instrument will be part of the UK academic and industrial networks further ensuring sustainability. These upgrades will allow the UK to remain internationally competitive and to continue developing and applying the EPR methodology to important problems across the biosciences.

This proposal will establish a new biological electron paramagnetic resonance (EPR) centre of excellence in Leeds to address fundamental biological questions by enabling pulsed electron-electron double resonance (PELDOR, or DEER) distance measurements on challenging proteins. We aim to install and operate a new high power (300W) Bruker Qband pulsed EPR spectrometer with Arbitrary Waveform Generator (AWG) and cryogen-free cooling system, the first of its kind in the North East, integrated within the Astbury Centre bio-structure facilities. This instrument will be dedicated to elucidating bio-molecular mechanisms and enable distance measurements within membrane proteins and complexes. The existing sensitivity would be further enhanced through pulse shaping via the AWG, significantly decreasing measurement time. Dynamic states are most crucial for understanding complex biological machineries in molecular detail and these essentially require an ensemble method such as PELDOR, which does not rely on software- or user- biased single particle selection, such as cryoEM or FRET. PELDOR further enables low yield proteins (high sensitivity) to be studied, and distinct and low populated protein conformational states to be resolved in space (subÅ resolution) and time (us flash mixing and freezing methods) and in cell (unnatural spin labelled amino acids). In addition, PELDOR offers no disruption of protein function (label of the size of an amino acid), sample condition flexibility and allows for measurements within membranes and cellular environment. Within BioEmPiRe we will use PELDOR to first reveal the entire conformational ensemble of proteins of fundamental importance, such as GPCRs, ion channels (including mechanosensitive Piezo and TRPs), ABC transporters, kinases, chaperones, lectins and enzymes involved in folding, cancer, antimicrobial resistance and natural product biosynthesis, systems which will be brought along by our world-class investigators.