Nanometre distance measurements with EPR using different spin probes.

Pulse electron paramagnetic resonance (EPR) spectroscopy is an emerging technique for long range distance determination. Sparse long-range distances are very valuable for understanding structure and flexibility of complex nanostructures. 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 of up to 10 nm and beyond between two paramagnetic centres in the system studied. 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.
This project aims to explore different types of paramagnetic centres to be employed a spin labels and to firmly establish best practices for the choice of labels especially with respect to the amount of information that can be extracted from samples labelled with several spectroscopically distinct probes (orthogonal labelling). To this end chemical models bearing paramagnetic metal centres and organic radicals will be employed to benchmark the metal nitroxide distance measurements using different methods based on excitation with two frequencies (PELDOR) or relaxation based methods (RIDME). In a second step systems bearing multiple spin centres will be scrutinised. This will be extremely valuable for applications in multimeric systems in which several spin-labelled monomers assemble to the functional unit. In particular addressing specific distances within the complex structure will be a huge gain in information. These model studies will be backed up by numeric simulations and the outcome is envisioned to be transferred to a model protein multimer. This approach will add to the armoury of structural EPR techniques with the long term aim of identifying structural changes in systems not accessible with current methods.