Figure 2.

Structure-based design of a fluorogenic probe for folded and functional RA. (a) Schematic of a push–pull environmentally sensitive fluorophore. EWG = electron-withdrawing group. EDG = electron-donating group. (b) Structure of the retroaldol substrate S1 utilized by the de novo designed RA enzyme.²⁹ RA catalyzes a retroaldol reaction using the pK_a-perturbed lysine-210 ε-amine side chain that forms a Schiff base with S1. (c) P1 is a push–pull environmentally sensitive fluorophore featuring a reactive vinyl ketone (in red) that also serves as an EWG. The dimethyl amino group (in green) serves as an EDG. P1 covalently modifies the pK_a-perturbed lysine-210 residue of RA through 1,4-conjugate addition, rendering the RA-P1 covalent conjugate fluorescent.