Abstract
Nanometre distance measurements by pulsed electron−electron double resonance (PELDOR) spectroscopy have become an increasingly important tool in structural biology. The theoretical underpinning of the experiment is well defined for systems containing two nitroxide spin-labels (spin pairs); however, recently experiments have been reported on homo-oligomeric membrane proteins consisting of up to eight spin-labelled monomers. We have explored the theory behind these systems by examining model systems based on multiple spins arranged in rotationally symmetric polygons. The results demonstrate that with a rising number of spins within the test molecule, increasingly strong distortions appear in distance distributions obtained from an analysis based on the simple spin pair approach. These distortions are significant over a range of system sizes and remain so even when random errors are introduced into the symmetry of the model. We present an alternative approach to the extraction of distances on such systems based on a minimisation that properly treats multi-spin correlations. We demonstrate the utility of this approach on a spin-labelled mutant of the heptameric Mechanosensitive Channel of Small Conductance of E. coli.
Acknowledgements
We thank Prof. Gunnar Jeschke for implementing the retention of modulation depth information during model fitting in DeerAnalysis and Prof. Olav Schiemann for helpful discussions on PELDOR of MscS. AG is grateful for a postgraduate fellowship by the EPSRC-funded doctoral training centre ‘integrated magnetic resonance’. BEB is currently supported by an EaStCHEM Hirst Academic Fellowship by the School of Chemistry, St Andrews and funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (REA 334496). JHN acknowledges BBSRC support (BB/H017917/1) and Wellcome Trust support (WT092552MA).