Fig. 4: Characterization of NanoLuc mutants.

a Heatmap showing the relative luciferase activities of NanoLuc mutants with CTZ (left column) and FMZ (right column) luciferins. NanoLuc wild-type = 100%. b Structures of FMA-bound asymmetric dimer of NanoLuc wild-type (i) and luciferin-free symmetric dimer of NanoLuc-Y94A mutant (ii), and their superposition (iii). The tyrosine-tyrosine gate Y81 and Y94 (A94) residues and FMA-luciferin are shown as space-filling spheres. c Close-up view of the CEI-bound luciferin-binding surface site of NanoLuc luciferase. Three residues, namely D9, H57 and K89, whose mutations increase bioluminescence with CTZ are shown as cyan space-filling spheres. The other protein residues are shown as cyan sticks, and the CEI-luciferin is shown as green sticks. d Heatmap showing the relative luciferase activities of NanoLuc mutants with CTZ (left column) and FMZ (right column) luciferins; NanoLuc wild-type = 100%. e Relative fold increases ± standard errors (s.e.) of kinetic parameters (K_m, k_cat and k_cat/K_m) observed in the double NanoLuc-D9R/K89R and triple NanoLuc-D9R/H57A/K89R mutants; NanoLuc wild-type = 1. Absolute values of kinetic parameters (K_m, k_cat and k_cat/K_m) are summarized in Table 1. f, g Long-term live-cell bioluminescence imaging of cultured ARPE-19 cells expressing either original NanoLuc or engineered NanoLuc^CTZ and powered by EnduRen (f) or Nano-Glo Endurazine (g) substrate. The luciferase activities upon addition of the corresponding luciferin were measured by LuminoCell device⁵³, integration time was 5 min. Each data point represents the mean enzymatic activity of three replicates. The standard error is represented as a shaded area around the mean curve.