Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: the Copper(II)-Trityl Case

Recent sensitivity enhancements in pulse dipolar EPR spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science, as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin labelling using copper(II) ions and trityl yields a more than 3-fold sensitivity increase compared to the established copper(II)-nitroxide labelling strategy. Application of the recently developed variable-time RIDME method yields a further approximately 2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, more than two times lower than previously reported. We expect that experiments at single digit nanomolar concentrations are imminent, which has the potential to transform biological PDS applications.