**2.3 Expression and purification of 12 Fluc and revertants**

His10-tagged WT recombinant luciferase (WT) and mutants were expressed and purified according to the optimised protocol described in (Law *et al.*, 2006). Total protein concentrations were estimated by the method of Bradford (Bradford, 1976), using the Coomassie Blue protein assay reagent kit from Pierce according to the manufacturer's protocol, with BSA as the standard.

#### **2.4 Luciferase activity assays, kinetic analysis, pH dependence of activity, thermal inactivation and bioluminescence spectra**

Luciferase mutants were diluted from purified stock solutions into pre-chilled 0.1 M Tris/ acetate; pH 7.8, 2 mM EDTA and 10 mM MgSO4 (TEM) containing 2 mM DTT to obtain the required concentration, unless specified otherwise. Refer to caption accompanying each table or figure for method details. Bioluminescence spectra were captured using a Varian fluorometer (Palo Alto, Ca, USA). For measurements at differing pH values, TEM buffer at different pH values was used to dilute substrates and enzymes. Data were corrected for variant PMT sensitivity as previously described (Law *et al.*, 2006).
