Substrate effects on EIIA^Glc binding to MelB_St by ITC. EIIA^Glc-binding experiments were conducted at 25°C with the sample cell containing MelB_St. The thermogram was plotted as the corrected heat rate (µJ/s; left axis) versus time (bottom axis) for the titrant to MelB_St (black) or buffer (gray) under an identical scale. (a) EIIA^Glc injected into MelB_St in UDM at 50 µM. (b) EIIA^Glc injected MelB_St in nanodiscs at 20 µM. EIIA^Glc binding was measured in four different conditions. From right to left, MelB_St in the main buffer (the apo MelB_St), MelB_St preequilibrated with NaCl at 100 mM (MelB_St/Na⁺ binary complex), MelB_St preequilibrated with melibiose at 50 mM (MelB_St/melibiose binary complex), and MelB_St preequilibrated with 50 mM melibiose and 100 mM NaCl (MelB_St/melibiose/Na⁺ ternary complex). The normalized heat change (kJ/mol; filled blue symbol) was plotted against the EIIA^Glc/MelB_St molar ratio using the top/right axes. The K_d value was obtained by fitting the data using the one-site independent-binding model with a fixed binding stoichiometry N number of 1 (for most data, the curve fitting can yield the N number near 1). For EIIA^Glc binding to the ternary complex, a smaller heat change prevents accurate curve fitting, so there is no K_d result presented. The number of tests, mean, and standard error are reported under each panel.