Department of Chemical and Biological Physics , Weizmann Institute of Science , Rehovot 76100 , Israel.
State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) , Nankai University , Tianjin 300071 , China.
J Phys Chem B. 2019 Feb 7;123(5):1050-1059. doi: 10.1021/acs.jpcb.8b11442. Epub 2019 Jan 25.
Double electron-electron resonance (DEER) measures distances between spin labels attached at well-defined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work, we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd(III) and delivered into human HeLa cells by electroporation (EP) and hypotonic swelling (HS). Gd(III)-Gd(III) DEER measurements were carried out on cells frozen after different incubation times, following delivery to test the stability of the spin label inside the cell. For both delivery methods, it was possible to derive distance distributions up to 12 h after delivery, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, only one mutant reported a significant change in the distance distribution with time and only for HS delivery. On the basis of in vitro exchange experiments with Mn(II) and comparison with the same mutant labeled with BrPSPy-DO3MA-Gd(III) and considering the presence of Mn(II) in the cell, we hypothesized that the change occurred as a consequence of partial Gd(III)/Mn(II) exchange with endogenous Mn(II). These experiments also showed that the relative Gd(III)/Mn(II) binding affinity depends on the labeling site in the protein, which accounts for the lack of change with the other mutants delivered under HS conditions. We conclude that 4PS-PyMTA-Gd(III) is a good spin label for in-cell DEER for delivery by EP, but caution should be taken when HS is used.
双电子-电子共振（DEER）测量附着在蛋白质中明确定位点的自旋标记之间的距离，因此有可能报告细胞中蛋白质的构象状态。在这项工作中，我们评估了小而刚性的 4PS-PyMTA-Gd(III)自旋标记用于细胞内距离测量的适用性。三个泛素双突变体用 4PS-PyMTA-Gd(III)标记，通过电穿孔（EP）和低渗肿胀（HS）递送到人 HeLa 细胞中。在递送后不同的孵育时间后将细胞冷冻，进行 Gd(III)-Gd(III) DEER 测量，以测试自旋标记在细胞内的稳定性。对于这两种递送方法，都可以在递送后 12 小时内得出距离分布，尽管我们观察到随着时间的推移，递送的蛋白质数量减少。令人惊讶的是，只有一种突变体报告说距离分布随时间发生了显著变化，而且仅在 HS 递送时如此。基于与 Mn(II)的体外交换实验，并与用 BrPSPy-DO3MA-Gd(III)标记的相同突变体进行比较，并考虑到细胞中存在 Mn(II)，我们假设这种变化是由于与内源性 Mn(II)的部分 Gd(III)/Mn(II)交换所致。这些实验还表明，相对的 Gd(III)/Mn(II)结合亲和力取决于蛋白质中的标记位点，这解释了在 HS 条件下递送其他突变体时没有变化的原因。我们得出结论，4PS-PyMTA-Gd(III)是用于 EP 递送的细胞内 DEER 的良好自旋标记，但在使用 HS 时应谨慎。
