Figure 1. Functional characterization.

Purified MelB_St containing 100 mM NaCl or LiCl were subjected to D²G FRET and ITC measurements. (a) D²G FRET measurement. With an excitation wavelength of 290, emission spectra were collected between 415 and 575 nm. Difference spectra (ΔI) were calculated before and after addition of a given sugar (50 mM). Grey curves, melibiose; purple curves, methyl-β-D-thiogalactoside (TMG); green curves, sucrose; blue curves, glucose. (b) ITC measurement. Melibiose titration thermograms (insets) were recorded at 25 °C. Cumulative heat change (ΔQ) is plotted as a function of the molar ratio of melibiose/MelB_St, and fitted with the one-site independent binding model. (c) Melibiose-binding energetics. Enthalpy change (ΔH) and the association constant (K_a) were measured directly; dissociation constant K_d=1/K_a; free-energy change ΔG=−RT ln K_a; entropy change ΔTS=ΔH−ΔG; n, stoichiometry. Error bar, s.d., n=2.