Department of Chemical and Biological Physics, Weizmann Institute of Science, 234 Herzl St., Rehovot, 7610001, Israel.
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Angew Chem Int Ed Engl. 2023 May 8;62(20):e202218780. doi: 10.1002/anie.202218780. Epub 2023 Apr 12.
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 Gd - F 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 Gd - F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin-labeled with rigid Gd tags. The proteins were delivered into human cells via electroporation. The solution and in-cell derived Gd - F 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 Gd and F regions in the cell.
蛋白质结构和动力学的研究通常在稀缓冲溶液中进行，这些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附着的自旋标记之间的距离分布来跟踪细胞中蛋白质的构象。然而，该技术无法测量低于 1.8nm 的距离。在这里，我们表明 Gd-F Mims 电子-核双共振（ENDOR）测量可以覆盖该短程的一部分。对氟化 GB1 和泛素（Ub）进行低温溶液和细胞内 ENDOR 测量，并结合室温溶液和细胞内 Gd-F PRE（顺磁弛豫增强）NMR 测量，这些蛋白通过电穿孔递送至人细胞。溶液和细胞内衍生的 Gd-F 距离基本相同，位于 1-1.5nm 范围内，表明 GB1 和 Ub 在细胞内的 Gd 和 F 区域均保留了其整体结构。
