Figure 4.

(a) Western blot image of detergent-extracted MelB_St. MTGs (MTG-A11/A12/A13/A14 and MTG-E11/E12/E13/E14), DDM and OGNG were used at 1.5 wt% for MelB_St extraction conducted at 0 °C for 90 min. Detergent-extracted samples were further treated at a higher temperature (45, 55, or 65 °C ) for another 90 min. The detergent -extracted samples at 0 °C or thermally treated samples were subjected to ultracentrifugation and then analyzed by Western blot (top panel). The amounts of soluble MelB_St are represented as relative percentages (%) of total MelB_St in the histogram (bottom panel). Error bars, SEM, n = 2. (b) Galactoside binding of MelB in MTG-E13. MelB function was assessed using FRET reversal elicited by a ligand-substrate exchange in the active site. Dansyl-2-galacotside (D²G) and excess melibiose were added to a solution containing DDM/MTG-E13-extracted MelB (MelB_St and MelB_Ec) at 1-min and 2-min time points, respectively. The resulting responses in fluorescence intensity of the samples were then monitored over the course of the additions. Control data was obtained from addition of water instead of melibiose. Melibiose addition is indicated in black (DDM)/green (MTG-E13), with water addition indicated in gold.