Figure 2. Na⁺-coupled binding to the outward and inward facing states.

Binding data at 25 °C for Glt_Ph^out, Glt_Phⁱⁿ in and WT transporter are shown in blue (left), red (center) and black (right), respectively. (a) Asp binding isotherms derived from the ITC experiments conducted in the presence of 10 mM Na⁺. Solid black lines through the data are the fits to independent binding sites model with the following parameters for the three constructs, respectively: K_Ds, 220, 211 and 493 nM; ΔHs, −16.4, −17.8, −12.3 kcal mol⁻¹; and apparent number of binding sites, n, 0.8, 0.8 and 1.0. Insets show the Asp binding thermal powers with the corresponding scales. (b) Asp titrations derived from fluorescence-based assays in the presence of 1 mM Na⁺ at 25 C°. Solid black lines through the data are fits to the single-site quadratic equation with K_Ds of 125, 232 and 457 μM for Glt_Ph^out, Glt_Phⁱⁿ and WT, respectively. Insets show the percentage of the fluorescence change of RH421 dye as a function of time. The vertical lines mark additions of Asp aliquots. (c) Logarithmic plots of the Asp (circles) and TBA (squares) K_Ds as functions of Na⁺ activities. Data were obtained from ITC (solid symbols) and fluorescence-based (open symbols) experiments. Solid lines represent the best fits of the data to equation (1) with the parameters shown next to the graphs. The y-axis on the right shows corresponding binding free energies.