Mandato Alysia, Hasanbasri Zikri, Saxena Sunil
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, United States.
J Phys Chem Lett. 2023 Oct 12;14(40):8909-8915. doi: 10.1021/acs.jpclett.3c02372. Epub 2023 Sep 28.
Recently, there have been remarkable improvements in pulsed ESR sensitivity, paving the way for broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Nevertheless, submicromolar distance measurements with the commonly used nitroxide spin label take multiple days. Therefore, there remains a need for rapid and reliable methods of measuring distances between spins at nanomolar concentrations. In this work, we demonstrate the power of double quantum coherence (DQC) experiments at Q-band frequencies. With the help of short and intense pulses, we showcase DQC signals on nitroxide-labeled proteins with modulation depths close to 100%. We show that the deep dipolar modulations aid in the resolution of bimodal distance distributions. Finally, we establish that distance measurements with protein concentrations as low as 25 nM are feasible. This limit is approximately 4-fold lower than previously possible. We anticipate that nanomolar concentration measurements will lead to further advancements in the use of ESR, especially in cellular contexts.
最近，脉冲电子自旋共振（ESR）灵敏度有了显著提高，为ESR在测量生物距离限制方面更广泛的应用铺平了道路，例如在生理浓度和更复杂的系统中。然而，使用常用的氮氧化物自旋标记进行亚微摩尔距离测量需要数天时间。因此，仍然需要快速可靠的方法来测量纳摩尔浓度下自旋之间的距离。在这项工作中，我们展示了Q波段频率下双量子相干（DQC）实验的能力。借助短而强的脉冲，我们展示了在氮氧化物标记的蛋白质上具有接近100%调制深度的DQC信号。我们表明，深度偶极调制有助于解决双峰距离分布问题。最后，我们确定蛋白质浓度低至25 nM时的距离测量是可行的。这个极限比以前可能达到的极限低约4倍。我们预计，纳摩尔浓度测量将推动ESR的进一步发展，特别是在细胞环境中。
