A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies
A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies
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DOI：
10.1002/anie.201710184
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发表时间：
2018-01-26
影响因子：
16.6
通讯作者：
Mileo, Elisabetta
中科院分区：
文献类型：
--
作者：
Karthikeyan, Ganesan;Bonucci, Alessio;Mileo, Elisabetta
在细胞环境中接近蛋白质结构动力学和蛋白质-蛋白质相互作用是一个基本的挑战。由于其绝对的灵敏度和顺磁性物质的选择性，结合电子顺磁共振（EPR）的定点自旋标记（SDSL）有可能发展成为一种有效的方法来跟踪蛋白质的构象变化直接在细胞内。到目前为止，基于氮氧自由基的自旋标记物在细胞内研究中的应用一直是一个主要障碍，因为它们在细胞环境中的持久性很短。设计和合成了第一个基于马来酰亚胺基的自旋标记物（M-TETPO），该标记物具有抗还原性，并且能够有效地通过连续波和脉冲EPR探测蛋白质动力学。特别是，M-TETPO的寿命延长使得能够研究在不存在和存在其结合伴侣的情况下直接在细胞内内源性浓度的分子伴侣的结构特征。
Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido-proxyl-based spin label (M-TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M-TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.