Carr-Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses
Carr-Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses
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DOI：
10.1021/acs.jpclett.5b01933
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发表时间：
2015-11-05
影响因子：
5.7
通讯作者：
Prisner, Thomas F.
中科院分区：
文献类型：
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作者：
Spindler, Philipp E.;Waclawska, Izabela;Prisner, Thomas F.
脉冲电子顺磁共振（EPR）光谱允许测定的距离，在1.5-8 nm的范围内，两个自旋标记连接到含有质子的大分子之间。不幸的是，对于疏水性的脂质结合或洗涤剂溶解，因为电子自旋的相干时间大大减少。在这里，膜蛋白，最大可达距离要低得多，我们引入了一个脉冲序列，基于观察者自旋的Carr-Purcell解耦方案，其中每个π脉冲伴随着施加到第二个自旋的成形sech/tanh反转脉冲，以克服偶极相互作用，从而实现更长的观察时间窗口。这增加了可以在膜蛋白复合物中确定的距离的上限和准确度。我们验证了双氮氧模型化合物的方法，并将此技术应用于三聚甜菜碱转运BetP。可以可靠地确定长达6 nm的原异构体间距离，这是现有方法无法实现的。
Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized because of a strongly reduced coherence time of the electron spins. Here membrane proteins, the maximum distance accessible is much lower, we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each pi-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.