Fluorescence spectroscopy

Tryptophan fluorescence spectra (50 µg/mL) of MERS-CoV PL^pro were recorded using a Cary Eclipse Fluorescence Spectrophotometer in a 10-mm-path length cuvette. To measure tryptophan fluorescence, MERS-CoV PL^pro sample was excited at 295 nm and emission spectra were collected between 305 and 400 nm (5 nm excitation and 5 nm emission bandwidth). Temperature melting studies of MERS-CoV PL^pro were performed at 10 °C increments as well as a linear increase of 1 °C/min. MERS-CoV PL^pro incubated at different temperatures from 20 to 90 °C in Peltier-controlled Cary eclipse fluorometer. The temperature of the protein samples was monitored using an internal temperature probe. When the desired temperature was reached, the sample was allowed to equilibrate for 2 min before tryptophan fluorescence spectra were measured. The maximum fluorescence intensity (I _max) and maximum fluorescence wavelength (λ _max) were plotted with respect to temperature. In a similar experiment, MERS-CoV PL^pro was gradually heated from 20 to 80 °C at a rate of 1 °C/min during which tryptophan fluorescence was measured by exciting at 295 nm and collecting at 330 and 350 nm to obtain the temperature melting curve.