Mocanu Elena M, Ben-Ishay Yasmin, Topping Lydia, Fisher S Ronan, Hunter Robert I, Su Xun-Cheng, Butler Stephen J, Smith Graham M, Goldfarb Daniella, Lovett Janet E
SUPA School of Physics and Astronomy and BSRC, University of St Andrews, North Haugh, St Andrews, KY16 9SS UK.
Department of Chemical and Biological Physics, Weizmann Institute of Science, 7610001 Rehovot, Israel.
Appl Magn Reson. 2025;56(5):591-611. doi: 10.1007/s00723-024-01741-0. Epub 2025 Jan 3.
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.L, 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.
The online version contains supplementary material available at 10.1007/s00723-024-01741-0.
在本文中，我们探讨了针对不同光谱仪设计和三种自旋标记的蛋白质中钆（III）-钆（III）双电子-电子共振（DEER）距离测量的稳健性和灵敏度。为此，用钆（III）自旋标记在同两个位置对一种蛋白质进行标记，并在两台采用不同设计方法的自制高频（W波段，约95吉赫兹）电子顺磁共振光谱仪以及一台商用150瓦Q波段（34吉赫兹）光谱仪上进行测量。第一种W波段测量方法使用传统的窄带单模腔，而第二种方法使用宽带非共振感应模式样品架。这两种系统都配备了先进的任意波形发生器（AWG），可在激发带宽方面提供灵活性。我们使用三种与钙调蛋白结合的类DOTA钆（III）自旋标记，即Gd.C12、Gd.DO3A和Gd.L。我们比较了包含或排除钆（III）中心跃迁激发时所进行的测量。从所得距离分布宽度的稳健性、绝对和浓度灵敏度、样品处理、易用性以及测量灵活性等方面讨论了电子顺磁共振光谱仪在测量钆（III）-钆（III）DEER方面的优缺点。
在线版本包含可在10.1007/s00723-024-01741-0获取的补充材料。
