Figure 3.

Bioorthogonal labeling of the TM4 sites of Rho. A) The heterologous expression of Rho with single azF substitution at the indicated positions. HEK293F cells were transfected with three plasmids encoding the suppressor tRNA, the amino-acyl tRNA synthetase, and Rho with an amber mutation. The cell lysates were analyzed by Western blot (100 μg total protein per lane). The receptor was probed with 1D4 mAb specific to the C-terminus of Rho, followed by goat-anti-mouse 800 CW (LI-COR). The full-length receptor was detected only when the expression medium was supplemented with azF, indicating specific incorporation of azF into Rho. B) Fluorescent labeling of azF-Rho by Alexa488-DIBO. The cells expressing azF-Rho were regenerated with 11-cis-retinal, solubilized in 1% DM, immunoprecipitated by 1D4 mAb-sepharose resin, and reacted with Alexa488-DIBO (a final concentration of 50 μM in the resin/buffer mixture, 25 °C, for 18 hrs). The purified receptor was analyzed by in-gel fluorescence (50 ng purified receptor per lane) and UV–Vis spectroscopy (Supplementary Figure S1). The labeling stoichiomeries of the azF-Rho variants calculated as dye-to-protein ratios are indicated below.