Milliwatt Three- and Four-Pulse Double Electron Electron Resonance for Protein Structure Determination.

Electron paramagnetic resonance (EPR) experiments for protein structure determination using double electron-electron resonance (DEER) spectroscopy rely on very high incident microwave powers (>300 W) to create the short pulse lengths needed to excite a sizable portion of the spectrum. The recently introduced self-resonant microhelix combines a high B1 conversion efficiency with an intrinsically large bandwidth (low Q-value) and a high absolute sensitivity. We report dead times as low as 14±2 ns achieved using less than 1 W of power at X-band (nominally 9.5 GHz) on a molecular ruler and a T4-lysozyme sample. These low-power experiments were performed using an active volume 120 times smaller than that of a standard pulse EPR resonator, while only a sixfold decrease in the signal-to-noise ratio was observed. Small build sizes, as realized with the microhelix, give access to volume-limited samples, while shorter dead times allow the investigation of fast relaxing spin species. With the significantly reduced dead times, the 3-pulse DEER experiment can be revisited. Here, we show experimentally that 3-pulse DEER offers superior sensitivity over the 4-pulse DEER. We assert that the microhelix paves the road for low-cost benchtop X-band pulse EPR spectrometers by eliminating the need for high-power amplifiers, accelerating the adoption of pulse EPR to a broader community.