Advanced biomolecular applications of electron paramagnetic resonance spectroscopy

Abstract

The understanding of the structures, interactions, and functions of biological systems as well as their impact on health and disease is of fundamental importance. Electron paramagnetic resonance (EPR) spectroscopy has become a powerful tool for the investigation of structures and interactions of various systems. If not intrinsically present in the biological system paramagnetic centres can be introduced into the system of interest via site directed spin labelling (SDSL) to enable the use of EPR methods. Pulsed electron-electron double resonance (PELDOR) as well as relaxation-induced dipolar relaxation enhancement (RIDME) measurements exploit the dipole-dipole coupling between paramagnetic centres. Both methods have been used to investigate the distances between two spin labels, giving valuable insight into the structure of the biological systems investigated as well as sparse long-distance restrains used for structure refinement. PELDOR has in addition been used to determine the number of interacting spins, concluding to the number of interacting molecules. In combination with computational methods for structure prediction this opens a wide range of methods for the investigation and understanding of biological structures and structural transitions. The self-multimerisation of a single stranded DNA binding protein could be shown by PELDOR measurements and the flexibility of the system was validated by computational structure prediction. In another project good insight into the tertiary structure of the M3 protein could be gained using a combined approach of computational structure prediction, PELDOR, and RIDME measurements. ¹⁹F electron-nuclear double resonance (ENDOR) measurements enable the investigation of distances between a nitroxide spin label and a ¹⁹F nucleus. The investigation of a fluoride sensing riboswitch by nuclear magnetic resonance (NMR) spectroscopy and PELDOR showing a fluoride-free, a magnesium-stabilised and a fluoride-bound form is shown and an approach for the determination of the fluoride position in the solution structure based on ¹⁹F ENDOR measurements is introduced.