The fluorogenic TMP-tag. a) Cartoon of the fluorogenic TMP-tag, which centers a trimeric TMP-quencher (Q) - fluorophore (F) molecule to be cleaved in a proximity-induced SN2 reaction. Specifically, we selected the Cys side chain as the nucleophile and a tosylate linker as the electrophile. When TMP binds to eDHFR, the unique Cys near binding pocket would replace the tosylate leaving group attached to the quencher, and thus the probe is switched on. b) Structure of TMP-BHQ1-Atto520 (TMP-Q-Atto520), the first fluorogenic TMP-tag.