Replacement EPR spectrometer to the multiuser Biomedical EPR Facility at the University of Essex

We propose to replace the current Electron Paramagnetic Resonance (EPR) spectrometer, at the Biomedical EPR Facility at the University of Essex, with the latest state of the art X-band spectrometer with capabilities for future upgrades.

EPR spectroscopy allows detection of molecules in the states, called paramagnetic, that are formed in all oxidation/reduction reactions. These paramagnetic states, of which the best known are free radicals, can be transient (with either short or long life time) or stable. The processes of paramagnetic states formation within enzymes and proteins, as well as their disappearance, are of great interest since the sequential events of one type of paramagnetic centre transforming into another can be followed in time, thus providing a picture of the molecular mechanism revealing how an enzyme works. Such information is important for understanding a wide range of biological processes.

The current EMX EPR spectrometer (Bruker) is 23 years old and has developed a serious intermittent fault. Throughout these years, the Biomedical EPR Facility has contributed significantly to the research focused on proteins and enzymes conducted by a range of research groups in the UK. The Facility has developed highly specialised expertise in protein-based free radicals and metallo-proteins and enzymes. Our in-house built apparatus for freezing samples for EPR spectroscopy of rapid reactions between enzyme and substrate (funded by BBSRC, BB/E02355X/1), allows valuable kinetic data pertaining to the appearance and transformation of paramagnetic species over a reaction time course to be obtained. Such data sets can be used in computer generated kinetic models to verify hypotheses about the enzyme mechanism.

To maintain the high quality output of the Facility documented in publications, grant awards and staff training, we require an EPR spectrometer of a similar class (E500 Bruker) but with substantial advantages compared to the current model, namely: a) up-to-date electronics and software package; b) the system can be upgraded in the future to operate at a second working frequency and a pulsed regime.

The consortium of applicants consists of 11 academics from two Schools of the University of Essex and 8 external collaborators, all with excellent international reputations in their fields. The scope of research by these scientists is wide but all require EPR spectroscopy as a method for their research.

The proposal features 10 projects that require and will benefit greatly from the capabilities of the requested instrument:

1. Exploring the biotechnological potential of Streptomyces lividans enzymes
2. Electron transfer through and between proteins
3. Kinetics of the phase transition in thermoresponsive smart materials on temperature jump
4. Integrated time-resolved EPR and time-resolved crystal structures of metalloproteins
5. Deep neural network analysis of the EPR spectra of enzymes and proteins
6. Dynamics of membrane proteins sequestered in polymer based lipid particles
7. New biochemistry of bacterioferritin
8. De novo haem peroxidases
9. Iron deficiency in diet and cardiac muscle tissue respiration
10. New transporter-like membrane protein from Streptomyces

The existing Bruker EMX spectrometer at the Biomedical EPR Facility at Essex has been the key instrument in producing high quality publications and obtaining grant awards with a clear upward trajectory of funds and research outputs over the period of the Facility existence. The replacement spectrometer would ensure that our world leading position in EPR spectroscopy of biomolecules will be maintained.

We propose to replace the 23 years old Bruker continuous wave (CW) X-band EPR spectrometer (EMX) at the Biomedical EPR Facility of the University of Essex with a state of the art spectrometer of the same class (E500, Bruker), upgradable to two frequencies (X- and Q-band) CW class and pulsed functionality in the future.

The Facility has played a key role in producing many high impact publications and grant awards. A unique part of the Facility, which often attracts collaborations, is the isopentane-free Rapid Freeze-Quench (RFQ) apparatus for making samples with a freezing time from 45 ms, funded by a BBSRC grant (BB/E02355X/1). In recent times the EMX spectrometer has developed a serious intermittent fault, but the Facility has continued to stay active until the time of this application, with 340 l of helium used, 7 papers published and 3 papers under review in 2019. The concern is, however, that many collaborative works, including the currently active BBSRC programme (BB/R003203/1) together with University of East Anglia (BB/R002363/1), may grind to a halt if the intermittent fault becomes permanent.

The requested EPR spectrometer will be housed, next to the RFQ apparatus, in a lab area planned for refurbishment to a Biosafety Level 2 (BSL-2). This will create an unprecedented combination of possibilities, available on a single certified site, to perform fast kinetic EPR studies of the biological samples (such as tissues and fluids) associated with human diseases.

The consortium of applicants comprises 11 academics form two Schools of the University of Essex and 8 from other UK universities (Bristol, Manchester, Birmingham, Southampton, Dundee, East Anglia, Coventry and King's College London). The co-applicant are proposing 10 research projects in collaboration with the PI, in which the new spectrometer will be used.

The requested Electron Paramagnetic Resonance (EPR) spectrometer will enable a range of biomedical research themes, focusing on structure, function and mechanisms of enzymes and other biomolecules, to be addressed. Together with our unique rapid freeze-quench apparatus, this will create unprecedented instrumental configuration for the time resolved detection of different paramagnetic species, their evolution and interconversion, and will provide a new tool for uncovering the mechanisms of bio-processes as they unfold in time.

There are several groups of potential beneficiaries of the proposed research, including the following.

* The healthcare sector. The proposed study of the link between heart failure and iron in the diet (Project 9), as well as the study of new chemistry exhibited by ferritins (Project 7), will provide important information relevant to several pathological conditions linked to impaired iron metabolism. The study of the thermoresponsive smart materials (Project 3) will benefit both the healthcare sector, particularly NHS, and the industries seeking for new drug delivery vehicles, such as Oakwood Labs.

* The biotechnology sector dealing with large scale production of enzymes to be used in the areas of biofuel production, protection of environment and drug design and delivery will benefit from the fundamental knowledge that will be acquired in the proposed projects studying Streptomyces metalloenzymes (Project 1), thermoresponsive materials (Project 3) and de-novo haem peroxidases (Project 8). This sector will also benefit from employment of biochemists and spectroscopists trained at Essex by highly experienced staff on state of the art equipment in state of the art laboratory.

* Health and environment related policy makers, such as Regional Directorates of Health and Social Care, and commercial stakeholders, such as insurance companies who sell health coverage plans and pharmaceutical firms who develop and market medications, will benefit from anticipated advances:
o in understanding of biochemistry of iron metabolism from the perspective of healthy ageing and treatment of neurological disorders that have a huge social and economic impact in the UK and internationally;
o in improving performance of enzyme systems, as well as developing new ones, in biotechnological processes of huge social and economic significance, such as drug production, biofuel production and protecting the environment.

* Scientific equipment manufacturers, such as Bruker, Jeol, Rigaku, Jasco, SEE and many other, will benefit from further development of our rapid freeze-quench apparatus. Recently demonstrated feasibility of making rapid freeze-quenched samples in anaerobic conditions (Project 7) expands the area of application of such apparatus beyond EPR spectroscopy and encompasses NMR, resonance Raman, CD/MCD and Mössbauer spectroscopies. This technological development is expected to expand the demand for such spectrometers on the market as the new methodology of making sample for them is emerging.

* General public including school and college students will benefit from the public lectures we commit to deliver as well as school and college talks and summer placements for A-level students at the laboratories of the consortium members.