PMC

(A) Firefly luciferase catalyzes the formation of an activated AMP ester of its native substrate, D-luciferin. Subsequent oxidation within the luciferase binding pocket generates an excited state oxyluciferin molecule that is responsible for light emission. (B) Synthetic alkylated aminoluciferin substrates used in this study.

Purified luciferases (10 nM) were rapidly injected at the 10-second time point into 10 μM of CycLuc1 (A), CycLuc2 (B), or 6’-Me₂NLH₂ (C) and the light emission was recorded. Burst emission behavior for all substrates and characterized mutants is detailed in , with emission wavelengths reported in .

Dose-response curves for purified luciferases (WT, F247L, F247A, F247S, and F247V) were generated with D-Luciferin, 6’-NH₂LH₂ and CycLuc1 at concentrations from 0.122-125 μM. The assays were performed in triplicate and are represented as the mean +/- SEM. Note that the emission scale for D-luciferin is 4-fold higher than 6’-NH₂LH₂ and CycLuc1. Comparison of each mutant in lysed and live CHO cells is detailed in .

(A) Mutation sites were selected based on proximity to the luciferin substrate in the crystal structure of Luciola cruciata luciferase (PDB 2D1R). (B) Selected residues were subjected to saturating mutagenesis. Mutant luciferase-expressing bacteria were screened for light emission with CycLuc1 and those that exhibited improved properties were sequenced () and the mutant protein purified for further characterization.

Dose-response curves for purified luciferases (WT, T251S, L286M, S347A, and R218K) are shown with each luciferin substrate. The assays were performed in triplicate and are represented as the mean +/- SEM. The Y-axes have been calibrated to allow comparison of mutant emission for each substrate. For comparison between substrates, note the difference in scale. Each curve was fit to the Michaelis-Menten equation by nonlinear regression (GraphPad 5.0) to determine apparent K_m and V_max values (). Comparison of the light emission of S347T and S347A and the combination of S347A with other mutations is detailed in .

CHO-K1 cells were transiently transfected with pcDNA3.1 vectors expressing WT, T251S, L286M, S347A, or R218K firefly luciferase. Dose-response curves for each luciferase with D-Luciferin, 6’-NH₂LH₂, 6’-MeNHLH₂, 6’- Me₂NLH₂, CycLuc1 and CycLuc2 were generated at concentrations from 0.122-125 μM using lysates from the transfected cells (A) or using the intact live cells (B). The assays were performed in triplicate and are represented as the mean +/- SEM. Note that the WT emission scale is larger than that of the mutants by 2-fold and 3-fold for cell lysates and live cells, respectively. Substrate-by-substrate comparison of the light emission from each mutant in cell lysates is detailed in .