High-sensitivity Gd³⁺-Gd³⁺ EPR distance measurements that eliminate artefacts seen at short distances

Gadolinium complexes are attracting increasing attention as spin labels for EPR dipolar distance measurements in biomolecules and particularly for in-cell measurements. It has been shown that flip-flop transitions within the central transition of the high spin Gd³⁺ ion can introduce artefacts in dipolar distance measurements, particularly when measuring distances less than 3–4 nm. Previous work has shown some reduction of these artefacts through increasing the frequency separation between the two frequencies required for the Double Electron-Electron Resonance (DEER) experiment. Here we use a high power (1 kW), wideband, non-resonant, system operating at 94 GHz to evaluate DEER measurement protocols using two rigid Gd(III)-rulers, consisting of two [Gd^III(PyMTA)] complexes, with separations of 2.1 nm and 6.0 nm, respectively. We show that by avoiding the |−1/2⟩ → |1/2⟩ central transition completely, and placing both the pump and the observer pulses on either side of the central transition, we can now observe apparently artefact-free spectra and narrow distance distributions, even for a Gd-Gd distance of 2.1 nm. Importantly we still maintain excellent signal-to-noise ratio and relatively high modulation depths. These results have implications for in-cell EPR measurements at naturally occurring biomolecule concentrations.