Significantly Improved Sensitivity of Q-Band PELDOR/DEER Experiments Relative to X-Band Is Observed in Measuring the Intercoil Distance of a Leucine Zipper Motif Peptide (GCN4-LZ)
Significantly Improved Sensitivity of Q-Band PELDOR/DEER Experiments Relative to X-Band Is Observed in Measuring the Intercoil Distance of a Leucine Zipper Motif Peptide (GCN4-LZ)
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DOI：
10.1021/bi900781u
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发表时间：
2009-06-30
期刊：
影响因子：
2.9
通讯作者：
Lorigan, Gary A.
中科院分区：
文献类型：
--
作者：
Ghimire, Harishchandra;McCarrick, Robert M.;Lorigan, Gary A.
脉冲电子双共振 (PELDOR)/双电子-电子共振 (DEER) 光谱是一种非常强大的结构生物学工具，可测量两个不成对电子自旋（定点硝基氧自旋标记）之间的偶极耦合。这些测量通常使用四脉冲 DEER 序列在 X 波段 (9.4 GHz) 微波激励下进行，通常需要对生物样品进行长达 12 小时的信号平均（取决于自旋标记浓度）。在这项工作中，我们首次提出，与 X 波段相比，通过在 Q 波段（34 GHz）收集 DEER 光谱，获得了 DEER 灵敏度的大幅提高。 Q 波段的灵敏度大幅提升（13 倍）意味着数据收集时间减少了 169 倍，显着改善了频谱和更高质量的距离数据，并显着提高了样本吞吐量。因此，Q 波段 DEER 光谱的可用性将对使用定点自旋标记 EPR 技术的结构生物学研究产生重大影响。
Pulsed electron double resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin-labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, we present for the first time a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and higher-quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.