Abstract
Recently, there have been remarkable improvements in pulsed ESR sensitivity, paving the way for broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Nevertheless, submicromolar distance measurements with the commonly used nitroxide spin label take multiple days. Therefore, there remains a need for rapid and reliable methods of measuring distances between spins at nanomolar concentrations. In this work, we demonstrate the power of double quantum coherence (DQC) experiments at Q-band frequencies. With the help of short and intense pulses, we showcase DQC signals on nitroxide-labeled proteins with modulation depths close to 100%. We show that the deep dipolar modulations aid in the resolution of bimodal distance distributions. Finally, we establish that distance measurements with protein concentrations as low as 25 nM are feasible. This limit is approximately 4-fold lower than previously possible. We anticipate that nanomolar concentration measurements will lead to further advancements in the use of ESR, especially in cellular contexts.