Fig. 2: Changes in luminescence of NanoLuc-based biosensors upon titration with their cognate ligands.
From: Engineering and exploiting synthetic allostery of NanoLuc luciferase
a Titration of 200 μl solution of 1 nM Rapamycin biosensor supplemented with 0.25 μl furimazine stock solution in buffer containing 20 mM Tris-HCl pH 7.2, 100 mM NaCl with increasing concentrations of rapamycin. The data was fitted to a Kd value of 0.4 nM. b As in a but using a chimeric protein where fusion of Calcineurin A and Calcineurin B replaces FRB domain and using tacrolimus (FK506) as a titrant. The data was fitted to a Kd value of 0.4 nM. c As in a but using cpNanoLuc fused to anti α-amylase VHH domains for ligand recognition and α-amylase as a titrant. The data was fitted to a Kd value of 0.5 nM. d A schematic representation of a two component Nanoluc based biosensor with calmodulin inserted into the loop connecting the last β-strand rendering the molecule inactive. Ligand-induced scaffolding of this chimeric unit with the calmodulin-binding peptide induces the conformation change of calmodulin and activation of the enzyme. e Activity of a two-component rapamycin biosensor shown in d. Here, a solution of 10 nM CaM-NanoLuc-FKBP and 30 nM FRB-CaM-BP in reaction buffer described in a was titrated with increasing concentrations of rapamycin. The data was fitted to a Kd value of 9.6 nM. f As in e but using 10 nM CaM-NanoLuc-FKBP and 30 nM Calcineurin A and Calcineurin B fusion with calmodulin binding peptide and tacrolimus as titrant. The data was fitted to a Kd value of 6 nM. Source data are provided as a Source Data file.
