A Reactive, Rigid Gd(III) Labeling Tag for In-Cell EPR Distance Measurements in Proteins.
A Reactive, Rigid Gd(III) Labeling Tag for In-Cell EPR Distance Measurements in Proteins.
复制标题
用于蛋白质细胞内 EPR 距离测量的反应性刚性 Gd(III) 标记标签。
DOI：
10.1002/anie.201611051
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Su Xun-Cheng
中科院分区：
文献类型：
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作者：
Yang Yin;Yang Feng;Gong Yan-Jun;Chen Jia-Liang;Goldfarb Daniella;Su Xun-Cheng
蛋白质的细胞环境与大多数蛋白质结构研究的体外条件有很大不同。 因此，人们对确定细胞中蛋白质的动力学和结构越来越感兴趣。在蛋白质中通过双电子-电子共振（DEER）方法进行细胞内距离测量的一个关键因素是所使用的自旋标记的性质。在这里，我们提出了一种新设计的GdIIIspin标记，一种巯基特异性DOTA衍生物（DO 3 MA-3BrPy），其具有化学稳定性和动力学惰性，蛋白质标记效率高，刚性系链短，以及良好的光谱特性，所有这些都特别适合于在W波段频率下通过DEER方法进行的细胞内距离测量。DO 3 MA-3BrPy-GdIII在体外和HeLa细胞中对双标记的泛素D39 C/E64 C表现出高性能。 高质量的DEER数据可以在HeLa细胞中获得长达12小时的蛋白质递送后，在细胞内蛋白质浓度低至5-10 μm。
The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIIIspin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐GdIIIis demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm.