The DEER (double electron-electron resonance, also called PELDOR) experiment, which probes the dipolar interaction between two spins and thus reveals distance information, is an important tool for structural studies. In recent years, shaped pump pulses have become a valuable addition to the DEER experiment. Shaped pulses offer an increased excitation bandwidth and the possibility to precisely adjust pulse parameters, which is beneficial especially for demanding biological samples. We have noticed that on our home built W-band spectrometer, the dead-time free 4-pulse DEER sequence with chirped pump pulses suffers from distortions at the end of the DEER trace. Although minor, these are crucial for Gd(III)-Gd(III) DEER where the modulation depth is on the order of a few percent. Here we present a modified DEER sequence—referred to as reversed DEER (rDEER)—that circumvents the coherence pathway which gives rise to the distortion. We compare the rDEER (with two chirped pump pulses) performance values to regular 4-pulse DEER with one monochromatic as well as two chirped pulses and investigate the source of the distortion. We demonstrate the applicability and effectivity of rDEER on three systems, ubiquitin labeled with Gd(III)-DOTA-maleimide (DOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) or with Gd(III)-DO3A (DO3A, 1,4,7,10-Tetraazacyclododecane-1,4,7-triyl) triacetic acid) and the multidrug transporter MdfA, labeled with a Gd(III)-C2 tag, and report an increase in the signal-to-noise ratio in the range of 3 to 7 when comparing the rDEER with two chirped pump pulses to standard 4-pulse DEER.
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