Fig. 5. Implementation of LG-approach in firefly luciferase.
a Locations of the final 22 LG pairs selected using the procedure outlined in Fig. 2. b Pluc variants were synthesized using CFPS and activity of D-luciferin oxidation was assessed. Reaction rates (RLU) are shown in response to increasing Ni(II) concentrations (0–100 µM) as a heat map. Inhibited variants are highlighted in red. c Steady-state kinetic assays were performed at room temperature with CFPS-produced enzyme variants Pluc202/532, Pluc108/508, and PlucWT in either the absence (black) or presence of Ni(II) (green). Maximum rate (RLU) is plotted versus substrate concentration (µM) and data was fit to a Michaelis-Menten equation that accounts for the observed inhibition using OriginPro. Each data point represents the average of three replicates and error bars represent standard deviations. Source data are provided as a Source Data file.