Absorbance and fluorescence spectroscopy measurements

All absorption spectra were acquired in a Cary 50 Bio UV–Visible spectrophotometer. Each purified protein or metabolite was diluted to 20 μM in a buffer containing 150 mM NaCl and 50 mM Tris–HCl pH 7.4. Samples were placed in a Fisher brand quartz cuvette, and the absorbance was collected from 600 to 200 nm on the slowest scan speed.

Fluorescence emission spectra were collected on a Tecan Infinite M1000 PRO spectrophotometer. For native fluorescence, each protein was diluted to 20 μM and each metabolite was diluted to 2 μM in a buffer of 150 mM NaCl and 50 mM Tris–HCl pH 7.4. Samples were excited at 450 nm with a 5 nm bandwidth. Emission data was collected from 500 to 600 nm in 1 nm steps with a 5 nm bandwidth. All samples were measured in triplicate and plotted with Datagraph as the mean value ± standard deviation. For denatured fluorescence, each protein and metabolite were diluted as above (20 μM and 2 μM, respectively) plus 7 M guanidine hydrochloride (GuHCl). Measurements, parameters, and plotting were identical for the both the denatured and native fluorescence samples.