Pulsed EPR Methods to Study Biomolecular Interactions

Orthogonal site-directed spin labelling in combination with pulsed EPR spectroscopy is a powerful approach to study biomolecular interactions on a molecular level. Following a surge in pulse EPR method development, it is now possible to access distance distributions in the nanometre range in systems of complex composition. In this article we briefly outline the necessary considerations for measurements of distance distributions in macromolecular systems labelled with two or more different types of paramagnetic centres. We illustrate the approach with two examples: an application of the Double Electron-Electron Resonance (DEER) method on a triple spin-labelled protein dimer labelled with nitroxide and Gd(III), and an optimisation study of the Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiment for the orthogonal spin pair Cu(II)-nitroxide