Increasing nitroxide lifetime in cells to enable in-cell protein structure and dynamics measurements by electron spin resonance spectroscopy
Increasing nitroxide lifetime in cells to enable in-cell protein structure and dynamics measurements by electron spin resonance spectroscopy
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DOI：
10.1016/j.jmr.2018.12.005
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发表时间：
2019-02-01
影响因子：
2.2
通讯作者：
Saxena, Sunil
中科院分区：
文献类型：
--
作者：
Singewald, Kevin;Lawless, Matthew J.;Saxena, Sunil
越来越多的证据表明，许多蛋白质的稳定性、结构、动力学和功能在细胞中与在体外不同。确定蛋白质的结构和动力学在原生细胞环境可能导致更好地了解蛋白质的行为。电子自旋共振（ESR）作为一种可以报道细胞内蛋白质结构和动力学的技术已经出现。基于氮氧化物的自旋标记能够报告蛋白质动力学，结构和骨干灵活性，但由于细胞中发生的氮氧化物还原而受到限制。为了克服这一限制，我们使用了氧化剂铁氰化钾(K3Fe（CN)(6)）和抗劈裂自旋标记3-malemidoPROXYL （5-MSL）。此外，我们假设注射浓度是影响氮氧化物还原动力学的重要参数。通过增加双5-MSL标记蛋白的注射浓度，我们发现非洲爪蟾卵母细胞的氮氧化物寿命增加。我们的工作证明了使用氮氧化物自旋标签进行双电子-电子共振（DEER）实验的细胞内3小时和细胞质内5小时的前所未有的孵育时间。这允许更有意义的测量较大的蛋白质系统，可能需要更长的孵育时间来平衡在细胞环境中。通过将我们的方法与更多屏蔽氮氧化物和q波段相结合，甚至可以延长孵化时间。(C) 2018爱思唯尔公司版权所有。
There is increasing evidence that the stability, structure, dynamics, and function of many proteins differ in cells versus in vitro. The determination of protein structure and dynamics within the native cellular environment may lead to better understanding of protein behavior. Electron spin resonance (ESR) has emerged as a technique that can report on protein structure and dynamics within cells. Nitroxide based spin labels are capable of reporting on protein dynamics, structure, and backbone flexibility but are limited due to nitroxide reduction occurring in cells. In order to overcome this limitation, we used the oxidizing agent potassium ferricyanide (K3Fe(CN)(6)) as well as the cleavage resistant spin label 3-malemidoPROXYL (5-MSL). Furthermore, we hypothesized that injection concentration is an important parameter regarding nitroxide reduction kinetics. By increasing the injection concentration of doubly 5-MSL labeled protein into Xenopus laevis oocytes, we found an increased nitroxide lifetime. Our work demonstrates unprecedented incubation times of 3-h in-cell and 5-h in-cytosol for double electron-electron resonance (DEER) experiments using nitroxide spin labels. This allows for more meaningful measurements of larger protein systems which may require longer incubation times for equilibration in the cellular milieu. Even longer incubation times are possible by combining our approach with more shielded nitroxides and Q-band. (C) 2018 Elsevier Inc. All rights reserved.