Gd III ‐ 19 F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance
Gd III ‐ 19 F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance
复制标题
通过电子-核双共振测量细胞中蛋白质的 Gd III – 19 F 距离
DOI：
10.1002/anie.202218780
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Goldfarb, Daniella
中科院分区：
文献类型：
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作者：
Seal, Manas;Zhu, Wenkai;Dalaloyan, Arina;Feintuch, Akiva;Bogdanov, Alexey;Frydman, Veronica;Su, Xun‐Cheng;Gronenborn, Angela M.;Goldfarb, Daniella
蛋白质结构和动力学的研究通常是在稀释的缓冲溶液中进行的，这种条件与细胞中拥挤的环境有很大的不同。双电子-电子共振（DEER）技术可以通过提供两个自旋标签之间的距离分布来跟踪细胞中蛋白质的构象。然而，这种技术无法处理1.8纳米以下的距离。在这里，我们表明GdIII‐19fmims电子-核双共振（ENDOR）测量可以覆盖这一短范围的一部分。低温溶液和细胞内ENDOR测量，以及室温溶液和细胞内GdIII - 19F PRE（顺磁弛豫增强）NMR测量，对氟化GB1和泛素（Ub）进行，用刚性GdIIItags自旋标记。这些蛋白质通过电穿孔进入人体细胞。溶液和细胞内衍生的GdIII - 19F距离基本相同，位于1-1.5 nm范围内，这表明GB1和Ub在细胞的GdIII - 19F区域保留了它们的整体结构。
Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII‐19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIIItags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIIIand19F regions in the cell.