Extended Data Fig. 2: Screening, optimization, and validation of Trp-CAGE.
From: Genetically encoded bioorthogonal tryptophan decaging in living cells
A, The amber suppression efficiency of vyW by the indicated chPheRSs was tested by a GFP reporter assay (n = 2). B, The chPheRS mutants by rational design for vyW recognition. Rational mutations were labelled light brown. C, In vitro optimization of decaging conditions on GFP protein. 100 μL of GFP variant (10 μM) in PBS was treated with Tz at 37 °C for 60 min. The yields were calculated based on relative intensity of substrate and product peaks in LC-MS. D, the activity of wild-type Rluc-WT, RLuc-261-W variant and RLuc-261-vyW variant with/without Tz treatment. Error bars represent ± standard error of the mean (n = 3). Two-tailed t test was used. ****P < 0.0001. E, The decaging of RLuc-261-vyW in different incubation times. The activation signal can be observed after 1 min of Tz treatment, and the signal reached to 50% of maximum activation within 10–15 min. Error bars represent ± standard error of the mean (n = 3 biologically independent samples). Two-tailed t test was used. ****P < 0.0001. F, Summarized Km of RLuc-WT, RLuc-261-W and RLuc-261-vyW. The Km were generated by Michaelis–Menten analysis with Graphpad Prism 9.0. G, RLuc catalysed luminescence process. The peroxide intermediate is crucial for the final product generation. H, The crystal structure of coelenterazine binding to RLuc (PDB code: 7QXR). F261 is close to the heterocycle core of coelenterazine. I, the incorporated vyW may react with peroxide intermediate, thus preventing the formation of the final luminescent product. J, The Prilezhaev oxidation reaction.
