Fig. 3.
Var64 binds both (2′S)- and (2′R)-GR24 enantiomers (A and B), Tm curves Var64 protein preincubated with (−)-(2′S)-GR24 or (+)-(2′R)-GR24. A first derivative of the change in fluorescence due to gradual protein melting was plotted against Tm. These curves were used to calculate inflection points of fluorescence versus Tm curves, as well as Kd values. Each line represents the average protein melt curve for three replicate samples. (C) Quantification of hydrolysis of GR24 enantiomers by liquid chromatography–mass spectrometry (LC-MS). The rate of hydrolysis was monitored in triplicate by production of the ABC-ring–hydrolyzed product by LC-MS.