Site-Specific Tagging of Channelrhodopsins with Genetically-Encoded Azido Groups

Channelrhodopsins (ChRs) are light-gated cation channels widely used in optogenetics because they can trigger depolarization of membrane potential upon illumination. In order to investigate the mechanism of channel opening, we used amber stop codon suppression to introduce the unnatural amino acid (uaa) p-azido-phenylalanine (azF) into expressed ChRs with high efficiency. Based on the recent crystal structure of a ChR-hybrid, amino acid residues in vicinity of regions that might be involved in the channel gating process were chosen as targets for replacement with azF. AzF-containing mutants were purified from mammalian cells in satisfactory yields with expression levels of up to ∼35% compared with wild-type receptor, which matches earlier experience with CCR5 and bovine rhodopsin. We also developed a simple procedure to reconstitute ChR azF mutants into POPC-bilayer-membranes for future spectroscopy studies. The site-specific azF tag provides a useful FT-IR (Fourier Transform Infrared) spectroscopy probe because of its small size and its unique vibrational signature, which is well separated from intrinsic protein backbone signals. FT-IR difference spectroscopy in combination with uaa-mutagenesis can be used to track changes in the electrostatic environment of the azido probe and reveal local structural movements without impairing significantly the native protein architecture. In addition to direct interrogation of azF tags, exploiting the chemical property of the azido group as bio-orthogonal coupling site could allow specific functionalization of ChR. ChR azF mutants were reacted with fluorophore adducts using strain-promoted azide-alkyne cycloaddition chemistry. Coupling efficiency at multiple sites was determined by in-gel fluorescence scanning and UV-Vis spectroscopy. The current work describes for the first time the successful introduction of uaas into ChR variants, demonstrating a robust and powerful technology to investigate function and mechanism of this important class of photoreceptors.