Faculty of Chemistry and Center for Molecular Materials, Bielefeld University , Universitätsstraße 25, 33615 Bielefeld, Germany.
J Am Chem Soc. 2014 Oct 29;136(43):15366-78. doi: 10.1021/ja508274d. Epub 2014 Oct 17.
Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of Xenopus laevis oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP10CP10CP10-NH2 was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into Xenopus laevis oocytes, and the Gd(III)-Gd(III) distances were determined by double electron-electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd(III) ion and the Cα of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix.
通过电子顺磁共振波谱（EPR）与定点自旋标记相结合，在纳米范围内测量距离是监测生物大分子在其自然环境中的结构和动态的非常有力的工具。然而，在细胞内的应用受到细胞内还原环境中常用的氮氧自由基自旋标记物的短寿命的阻碍。在这里，我们证明了基于 Gd（III）的自旋标记物 Gd-PyMTA 适用于细胞内的 EPR。Gd-PyMTA 被证明与细胞相容，并且在 Xenopus laevis 卵母细胞的细胞内提取物中在 18°C 下超过 24 小时保持惰性。脯氨酸丰富的肽 H-AP10CP10CP10-NH2 在两个半胱氨酸残基处被 Gd-PyMTA 定点自旋标记。所得肽 H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 以及由已知刚性间隔物组成的模型化合物 Gd-间隔物-Gd 被微注射到 Xenopus laevis 卵母细胞中，通过双电子电子共振（DEER）光谱确定 Gd(III)-Gd(III) 距离。为了分析细胞内肽构象，建立了一个旋转体库，以考虑连接 Gd(III)离子和半胱氨酸残基的 Cα之间的连接的构象灵活性。结果表明，自旋标记的肽 H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 插入细胞膜中，与寡脯氨酸从 PPII 构象转变为 PPI 螺旋相一致。
