Effect of flavonoids on the structural changes of MTB-MurI

For determining the secondary structural alterations of MTB-MurI in the presence of flavonoid compounds, CD measurements were recorded for Far- UV CD spectra in a Jasco J-810 spectropolarimeter equipped with a Peltier-type temperature controller. Purified MTB-MurI protein (0.5–0.6 mg/ml) was incubated overnight with different concentrations of either quercetin or naringenin. Secondary structure estimation from the Far-UV CD spectra was calculated using Yang’s method⁴⁴. Additionally, for determining changes in tertiary structure in terms of aromatic amino acid, fluorescence spectra of the purified MTB-MurI protein (0.1 mg/ml) in the absence and presence of either naringenin or quercetin was measured. Perkin Elmer LS 55 Spectro-fluorimeter with both excitation and emission slits set at 10 nm in a 3 mm quartz cuvette was used. For tyrosine fluorescence measurements, MTB-MurI protein was excited at 268 nm, while the emission spectra were recorded from 290 to 450 nm. All the measurements were recorded in triplicate with subtraction of necessary blanks.