PELDOR in multi-spin systems : from model systems synthesis to biological applications

Abstract

Pulsed electron-electron double resonance (PELDOR) is an emerging technique for nanometre distance measurements in nano-sized assemblies and between specific sites of molecules. Most commonly nitroxide radicals are used as probes for EPR distance measurements because they are easy to introduce in biological systems such as soluble and membrane proteins or nucleic acids. PELDOR distance measurements currently rely on data processing software which has been proven to accurately extract inter-spin distances from the dipolar coupling between two paramagnetic centres. However, when the dipolar coupling is affected by contributions from other close-by unpaired electrons inaccuracies as broadening effects and artefacts are introduced in the distance distributions derived. This challenge, commonly referred as multi-spin effects, has been affecting the extraction of accurate distance information from PELDOR measurements in chemical and biological systems with multiple spin labels. The aim of this project is to approach, identify and suppress inaccuracies introduced in PELDOR-based distance distributions by multi-spin effects. This is achieved through the synthesis of multiply labelled model systems which would allow for assessment of the impact of multi-spin effects on distance measurements of simple geometries whose behaviour can be easily predicted and modelled. In this work existing methods for suppression of multi-spin effects are tested, together with their efficiency and limitations. The results are used to devise better sets of parameters including alternative settings for extraction of accurate distances from multi-spin systems and to explore their efficiency and limitations. Additional effects influencing distance measurements by pulsed EPR are also examined; in particular the effects of orientation selection and their interplay with multi-spin effects is studied in depth. Studies on rigid symmetric and asymmetric chemical model systems together with heptameric channel membrane proteins allow for outlining of recommendations for PELDOR distance measurements settings on systems presenting similar structural features, including symmetries and inter-spin distances.