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

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.