Robustness and Sensitivity of Gd(III)–Gd(III) Double Electron–Electron Resonance (DEER) Measurements: Comparative Study of High-Frequency EPR Spectrometer Designs and Spin Label Variants

In this paper, we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron–electron resonance (DEER) distance measurements in proteins for different spectrometer designs and three spin labels. To do this a protein was labeled at the same two positions with Gd(III) spin labels and measurements were performed on two home-built high-frequency (W-band, ~ 95 GHz) EPR spectrometers with different design approaches, and a commercial 150 W Q-band (34 GHz) spectrometer. The first W-band measurement approach uses a conventional, narrow band single mode cavity, while the second uses a broadband non-resonant induction mode sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use three DOTA-like Gd(III) spin labels, Gd.C12, Gd.DO3A and Gd.L¹, conjugated to the calmodulin protein. We compare measurements taken by including or excluding the Gd(III) central transition excitation. The advantages and disadvantages of the EPR spectrometers for the measurement of Gd(III)–Gd(III) DEER are discussed in terms of the robustness of the resulting distance distribution width, absolute and concentration sensitivity, sample handling, ease of use, and flexibility of measurement.