Intra-monomer EPR distances in multimeric systems

Electron paramagnetic resonance (EPR) spectroscopy is an emerging technique for applications in the field of structural biology. Specifically, using an EPR method called PELDOR (pulsed electron-electron double resonance) or DEER (double electron-electron resonance), it is possible to reliably measure distances in the nanometre range between two paramagnetic centres in the system of choice. These paramagnetic centres can be native metal ions or radical cofactors, but most commonly they are deliberately introduced by a technique called site-directed spin-labelling.
In recent years, the PELDOR technique has been increasingly applied to complex biological systems, consisting of (homo-)oligomers, i.e. several copies of the same constituent. This leads to the presence of multiple spin centres even though only one spin-label per constituent is attached. These multi-spin systems are by far more challenging than the established two-spin systems. The theory behind the methods used to extract the distance information from the experimental data is limited to two spin systems. Thus, there is a high potential to confound the results in the more complicated cases. However, several approaches (modifying experiment or analysis) to relieve these limitations have been suggested recently.
With the research we propose we aim at obtaining structural information which is complementing the established methods for EPR on multimeric complexes. Here, we want to distinguish the distance within a single constituent from all those possible in-between the constituents of a complex. In other words, while the established approach is based on measuring the distance in-between monomers forming the multimers and bearing one spin-label each, we want to target distances within one doubly-labelled monomer of the multimer.
Keeping all other aspects of the sample preparation the same, this double labelling will double the number of spin-labels incorporated. This will severely increase the issues caused by multi-spin effects and additionally increase complexity by a higher number of different inter-spin distances. Furthermore, in the standard approach it will be impossible to distinguish the intra-monomer distance of interest from the other distances present. Thus, we propose a proof-of-principle study addressing how much preference has to be given to the intra-monomer distance to be reliably extracted. In other words, how much does the doubly-labelled monomer have to be "diluted" with un-labelled monomer? This will be addressed in a holistic approach integrating numerical simulations, synthetic model systems and new approaches for data acquisition and processing to demonstrate applicability on biological samples. In addition, we will transfer the gained knowledge to more complex model systems mimicking different dimerisation equilibria in biological systems. In the final stage of this project, the extracted principles are to be applied to a suitable multimeric protein to demonstrate the value of the new approach for structural biology.
This study will significantly advance the current knowledge and methodology in the field of EPR, especially with respect to PELDOR on proteins. Furthermore, our approach will add to the armoury of structural techniques and may allow tackling structural challenges in specific systems which are not accessible with the methods available to date.

The proposed research will in the short- to medium-term be beneficial for the UK's economy, with a range of opportunities for commercialisation and exploitation. In the longer term, by applying the methodologies developed in the course of this project to biomedical questions in the fields of structural biology (academic) and drug design (commercial), the research will improve the UK's health and wellbeing. Furthermore, the research staff involved in this project (PDRA) will benefit from this research by enhancing both professional project-related as well as transferable skills.
This research project can be considered as a benchmarking study for a specific fast-growing technique in EPR spectroscopy with a wide range of inter-disciplinary research applications. The study has potential for significant impact on the commercial EPR market. The equipment used is highly specialised, consisting of cutting-edge pieces of technology and software. The respective commercial developers are working closely together with researchers to incorporate novel developments in hardware and experimental design. Respective connections with commercial companies are already in place and will be exploited to the good use and incorporation of any methodology-related results. Thus, in addition to academic knowledge transfer via publications and conferences the developments from this project potentially become available through commercial distribution channels.
Even though the project itself is fundamental research, the design of the study is application-driven with the vision that its results are straight-forwardly incorporated into ongoing biomedical research within the field of structural biology and beyond. Once established, the methodology will be open for everyone to use and implement in their line of research, and collaborations within the UK and beyond will be sought by the investigator, with local collaborations currently being established. All results will be openly available through open access publication.
The outcomes of this project will enhance the armoury of structural biology methods by complementing established technologies. This will not only be of great interest in academia, but also in the commercial areas involving structural analysis of biomolecules (e.g. drug design and targeting). Thus, in the medium- to long-term the research can be expected to have a significant impact on the health and wellbeing of people both within the UK and around the world.
Furthermore, both research staff and students involved will benefit from this research. They will get the opportunity to explore specifically designed molecular systems using state-of-the art techniques and to acquire skills and expertise highly relevant for future employment within academia and beyond. In particular, the PDRA will expand her professional expertise in a wide range of techniques and will further strengthen her project management skills and organisational efficiency.