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Details of Grant 

EPSRC Reference: EP/M024660/1
Title: Intra-monomer EPR distances in multimeric systems
Principal Investigator: Bode, Dr BE
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Researcher Co-Investigators:
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Department: Chemistry
Organisation: University of St Andrews
Scheme: First Grant - Revised 2009
Starts: 14 August 2015 Ends: 13 August 2016 Value (£): 98,529
EPSRC Research Topic Classifications:
Analytical Science Chemical Structure
EPSRC Industrial Sector Classifications:
No relevance to Underpinning Sectors
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Panel History:
Panel DatePanel NameOutcome
14 May 2015 EPSRC Physical Sciences Chemistry - May 2015 Announced
Summary on Grant Application Form
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.

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