Collauto A, Frydman V, Lee M D, Abdelkader E H, Feintuch A, Swarbrick J D, Graham B, Otting G, Goldfarb D
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.
Phys Chem Chem Phys. 2016 Jul 28;18(28):19037-49. doi: 10.1039/c6cp03299k. Epub 2016 Jun 29.
Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.
基于脉冲电子顺磁共振的方法可用于测量两个自旋标记之间的电子-电子偶极耦合。在此，我们将最常用的技术——双电子-电子共振（DEER或PELDOR），与用于在W波段（94.9 GHz，≈3.5 T）测量Gd(iii)-Gd(iii)距离的无死时间5脉冲弛豫诱导偶极调制增强（RIDME）方法进行比较，使用具有小零场分裂（ZFS）的Gd(iii)标签。这类标签很重要，因为其窄中心跃迁产生了高电子顺磁共振灵敏度。研究了两个体系：（i）一个自旋间距离为2.35 nm的刚性模型化合物，以及（ii）一个同二聚体蛋白的两个突变体，二者均用基于DOTA的Gd(iii)螯合物标记，自旋间距离约为6 nm，其中一个具有窄距离分布，另一个具有宽距离分布。对模型化合物的测量表明，RIDME对弱耦合近似失效引起的复杂情况不太敏感，而这种情况会影响对具有窄中心跃迁的Gd(iii)标签间自旋距离较短的体系的DEER测量。对具有长自旋间距离的蛋白质样品的测量强调了由于S>1/2自旋体系在RIDME迹线中出现偶极相互作用频率的谐波而导致的复杂情况，以及背景扣除中不确定性的增加。在这两种情况下，均发现RIDME的灵敏度明显优于DEER。讨论了实验参数对RIDME迹线的影响。
