Q-Band Double Quantum Coherence ESR for Sensitive Nitroxide-Based Distance Measurements

Recent advances in pulsed dipolar spectroscopy have driven the sensitivity of the technique to physiologically relevant concentrations and extended its applicability to more complex biological systems. In this work, we establish double quantum coherence (DQC) ESR at Q-band as a practical technique for distance measurements in doubly nitroxide-labeled proteins. We show that 8 ns π pulses provide efficient excitation of the double quantum transition at Q-band, though more accessible 12 and 16 ns pulses also yield strong signals. Additionally, we show that the field dependence of the DQC signal is sensitive to the relative orientation of the two g-tensor axes. With careful spectrometer tuning, signal-to-noise ratios greater than 100 were possible in just 11 min of acquisition for a dipolar evolution time of 1.5 μs with 50 μM protein samples. We also show the effects of varying levels of deuteration on the sensitivity of the DQC signal, which can further extend dipolar evolution times and the measurable distance range. The DQC data is in good agreement with theoretical expectations and displays negligible contributions of intermolecular interactions on the DQC background signal at protein concentrations up to 100 μM. Overall, Q-band DQC offers distinct advantages for dual nitroxide-labeled proteins, including deep dipolar modulations, minimal background decay at micromolar concentrations, and increased sensitivity with the incorporation of deuterium. These findings position Q-band nitroxide DQC as a broadly accessible technique for pulsed ESR distance measurements.