Figure 2.
Measurements of substrate binding to MelBSt by ITC and temperature dependence. Heat changes from substrate binding to MelBSt were collected with ITC calorimeters (TA Instruments) at 15°C, 20°C, 25°C, 30°C, or 35°C, respectively. For each measurement, an aliquot of 80 µM MelBSt in the ligand-free main buffer (20 mM Tris-HCl, pH 7.5, 50 mM ChoCl, 0.035% UDM, and 10% glycerol) without addition (apo MelBSt) or with addition of 100 mM NaCl or 50 mM melibiose (the binary complex) was placed in the sample cell. Melibiose or NaCl solution prepared in matching buffers was placed in the syringe and incrementally injected with 2-µl aliquots into the sample cell at a 300-s interval as described in Materials and methods. (a) Melibiose binding to Apo MelBSt. Melibiose at 80 mM was titrated to the main buffer with MelBSt. (b) Na+ binding to apo MelBSt. NaCl at 5 mM was titrated in MelBSt in the main buffer. (c) Melibiose binding to MelBSt/Na+ binary complex. Melibiose at 10 mM was titrated in MelBSt in the main buffer with addition of 100 mM NaCl. (d) Na+ binding to MelBSt/Mel binary complex. NaCl solution at 2 mM was titrated to MelBSt in the main buffer with addition of 50 mM melibiose. For the determination of the temperature dependence, each measurement was performed under identical testing protocols, including identical buffer compositions and fixed concentrations for ligands (titrant) and protein (titrand), as well as the ITC measurement settings. The thermogram was plotted as the baseline-corrected heat rate (µJ/s; left axis) versus time (bottom axis) for the titrant to MelBSt (black) or buffer (gray) under an identical scale. The heat change Q (µJ; filled blue symbol) was plotted against the ligand/MelBSt molar ratio based on the top/right axes. The binding isotherm was obtained by fitting the data using the one-site independent-binding model included in the NanoAnalyze software (version 3.6.0). The binding stoichiometry N number was fixed to 1 in all cases. The determination of thermodynamic parameters was determined as described in Materials and methods, and the number of tests is presented in Table 2.

Measurements of substrate binding to MelBSt by ITC and temperature dependence . Heat changes from substrate binding to MelBSt were collected with ITC calorimeters (TA Instruments) at 15°C, 20°C, 25°C, 30°C, or 35°C, respectively. For each measurement, an aliquot of 80 µM MelBSt in the ligand-free main buffer (20 mM Tris-HCl, pH 7.5, 50 mM ChoCl, 0.035% UDM, and 10% glycerol) without addition (apo MelBSt) or with addition of 100 mM NaCl or 50 mM melibiose (the binary complex) was placed in the sample cell. Melibiose or NaCl solution prepared in matching buffers was placed in the syringe and incrementally injected with 2-µl aliquots into the sample cell at a 300-s interval as described in Materials and methods. (a) Melibiose binding to Apo MelBSt. Melibiose at 80 mM was titrated to the main buffer with MelBSt. (b) Na+ binding to apo MelBSt. NaCl at 5 mM was titrated in MelBSt in the main buffer. (c) Melibiose binding to MelBSt/Na+ binary complex. Melibiose at 10 mM was titrated in MelBSt in the main buffer with addition of 100 mM NaCl. (d) Na+ binding to MelBSt/Mel binary complex. NaCl solution at 2 mM was titrated to MelBSt in the main buffer with addition of 50 mM melibiose. For the determination of the temperature dependence, each measurement was performed under identical testing protocols, including identical buffer compositions and fixed concentrations for ligands (titrant) and protein (titrand), as well as the ITC measurement settings. The thermogram was plotted as the baseline-corrected heat rate (µJ/s; left axis) versus time (bottom axis) for the titrant to MelBSt (black) or buffer (gray) under an identical scale. The heat change Q (µJ; filled blue symbol) was plotted against the ligand/MelBSt molar ratio based on the top/right axes. The binding isotherm was obtained by fitting the data using the one-site independent-binding model included in the NanoAnalyze software (version 3.6.0). The binding stoichiometry N number was fixed to 1 in all cases. The determination of thermodynamic parameters was determined as described in Materials and methods, and the number of tests is presented in Table 2.

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