Volume 18, Issue 12, Pages 1649-1657 (December 2011) Identification of Mutant Firefly Luciferases that Efficiently Utilize Aminoluciferins  Katryn R. Harwood, David M. Mofford, Gadarla R. Reddy, Stephen C. Miller  Chemistry & Biology  Volume 18, Issue 12, Pages 1649-1657 (December 2011) DOI: 10.1016/j.chembiol.2011.09.019 Copyright © 2011 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2011 18, 1649-1657DOI: (10. 1016/j. chembiol. 2011 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Luciferase Mechanism and Substrates (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. Chemistry & Biology 2011 18, 1649-1657DOI: (10.1016/j.chembiol.2011.09.019) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Characterization of Phenylalanine 247 Mutants Dose-response curves for purified luciferases (WT, F247L, F247A, F247S, and F247V) were generated with D-luciferin, 6′-NH2LH2, and CycLuc1 at concentrations of 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 four-fold higher than that of 6′-NH2LH2 and CycLuc1. Comparison of each mutant in lysed and live CHO cells is detailed in Figure S1. Chemistry & Biology 2011 18, 1649-1657DOI: (10.1016/j.chembiol.2011.09.019) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Creation of Mutant Luciferases (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; those that exhibited improved properties were sequenced (Table S1), and the mutant protein was purified for further characterization. Chemistry & Biology 2011 18, 1649-1657DOI: (10.1016/j.chembiol.2011.09.019) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Characterization of Luciferase Mutants 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 Km and Vmax values (Table S2). Comparison of the light emission of S347T and S347A and the combination of S347A with other mutations is detailed in Figure S2. Chemistry & Biology 2011 18, 1649-1657DOI: (10.1016/j.chembiol.2011.09.019) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 Burst Kinetic Profiles with WT and R218K Luciferases (A–C) Purified luciferases (10 nM) were rapidly injected at the 10 s 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 Figure S3, with emission wavelengths reported in Table S3. Chemistry & Biology 2011 18, 1649-1657DOI: (10.1016/j.chembiol.2011.09.019) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 Dose-Response Profiles from Luciferases Expressed in CHO-K1 cell lysates and in live cells (A and B) 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 of 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 two-fold and three-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 Figure S4. Chemistry & Biology 2011 18, 1649-1657DOI: (10.1016/j.chembiol.2011.09.019) Copyright © 2011 Elsevier Ltd Terms and Conditions