Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: The Cu <sup>II</sup> -Trityl Case
Katrin Ackermann, Caspar A. Heubach, Olav Schiemann, Bela E. Bode
Abstract
High Resolution Image Download MS PowerPoint Slide Recent sensitivity enhancements in pulse dipolar electron paramagnetic resonance 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-labeling using Cu II ions and trityl yields a >3-fold increase in sensitivity compared to that of the established Cu II -nitroxide labeling strategy. Application of the recently developed variable-time relaxation-induced dipolar modulation enhancement (RIDME) method yields a further ∼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, >2 times lower than the previously reported concentration. We expect that experiments at single-digit nanomolar concentrations are imminent, which have the potential to transform biological PDS applications.