Applications of Model Systems to Investigate Multi-spin Effects in EPR spectroscopy.

In short my project uses biological macromolecules as biologically valid model systems for investigation of multi-spin effects such as combination frequencies and distance distribution peak broadening in EPR spectroscopy. This is a problem in the field which has only recently been addressed in the archetypal PELDOR experiment, but largely remains unexplored in other methodological setups; including many single frequency techniques as RIDME, DQC and SIFTER. I have so far attempted to reproduce findings of a recent publication applying a new spin label; a Cu(II) chelate which selectively interacts with a double histidine motif in proteins. After further characterisation of this system, it is my intention to apply the Cu(II) chelate spin label to a larger, more complex protein and subsequently introduce nitroxide radicals (another type of spin label) to observe how they interact. This is the cornerstone of the RIDME methodology, utilising a fast relaxing species such as a metal centre, accompanying a slow relaxing species such as nitroxide and monitoring their interaction. As mentioned multi-spin effects are still somewhat poorly characterised in RIDME, and so we will likely incrementally increase the Cu(II) chelate stoichiometry relative to the nitroxide and attempt to observe any multi-spin effects or distortions.
I am also working on long distance measurements using PELDOR in combination with deuteration; on the order of 160 angstroms. This work will be conducted on TRIM-25, a member of the TRIM protein superfamily; a group of fibrous proteins that are elongated and lend themselves to long inter-spin separation. This work will be coupled with new developments in the field concerning the use of chirp pulses and so called arbitrary waveform generators (AWGs). These facilitate pulse shaping, moving away from 'hard' rectangular pulses in the time domain, and sinc pulses in the frequency domain, towards sech/tanh pulses, having a much broader excitation profile. This allows for recovery of transverse magnetization during the PELDOR experiment and as such means one can measure a longer time window because the parameter T2 is suitably elongated. The longer time window is imperative to measure such long distances accurately; and pending the results of the Cu(II) chelate study, it is possible we will attempt measurement using this label, as opposed to the more common and better characterised nitroxide labels.