Non-covalent spin labelling of TRPC5 ion channels enables EPR studies of protein-ligand interactions

Robin S., Bon, Aidan V., Johnson, Sebastian Alin, Porav, Kasia L. R., Hammond, Anokhi, Shah, Joshua L., Wort, Yue, Ma, Hassane, El Mkami, Christopher M., Pask, Stephen P., Muench, Andrew J., Wilson, Christos, Pliotas
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Abstract

Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for the study of proteins in solution and under native conditions. Paramagnetic spin centres are usually introduced by site-directed spin labelling (SDSL) of engineered cysteine residues. However, for many (membrane) protein classes, cysteine engineering is not possible without affecting their structural and functional integrity. Here, we report the development of xanthine-based aminoxyl spin probes that allow non-covalent spin labelling of human TRPC5 ion channels. The compounds retained high potency as TRPC5 modulators and allowed assessment of ligand interaction and inter-ligand distances by continuous wave EPR (CW EPR) and double electron-electron resonance (DEER) spectroscopy. The results from EPR experiments were supported by high-resolution cryo-electron microscopy (cryoEM) structures of the TRPC5:spin probe complexes. This work shows that non-covalent, ligand-based spin labelling can be used for EPR studies of large, cysteine-rich membrane proteins and their complexes.
对 TRPC5 离子通道进行非共价自旋标记可实现蛋白质-配体相互作用的 EPR 研究
电子顺磁共振(EPR)光谱是研究溶液中和原生条件下蛋白质的一种强大技术。顺磁自旋中心通常是通过对工程半胱氨酸残基进行定点自旋标记(SDSL)而引入的。然而,对于许多(膜)蛋白质类别来说,半胱氨酸工程不可能不影响其结构和功能的完整性。在此,我们报告了基于黄嘌呤的氨基己基自旋探针的开发情况,这种探针可以对人类 TRPC5 离子通道进行非共价自旋标记。这些化合物作为 TRPC5 调制剂保持了很高的效力,并可通过连续波 EPR(CW EPR)和双电子电子共振(DEER)光谱评估配体相互作用和配体间距离。TRPC5:自旋探针复合物的高分辨率冷冻电镜(cryoEM)结构为 EPR 实验结果提供了支持。这项工作表明,基于配体的非共价自旋标记可用于富含半胱氨酸的大型膜蛋白及其复合物的 EPR 研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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