FIG. 4.

ITC analysis of FSUAXH1 TM2 mutants with soluble wheat arabinxylan as ligand. The ITC experiments were performed with an initial titration of 5 μl followed by 28 successive 10-μl aliquots of ligand (1.2 mg/ml arabinoxylan) into 50 μM protein sample at 25°C. The binding parameters shown in the figure were derived by subtracting the results for TM2-D148A/Y484A from those for TM2-D148A. The data should, therefore, provide estimates of the binding parameters of the CBM activity within TM2. The molar ratio of ligand indicates the concentration of binding sites on arabinoxylan. Affinity gel electrophoresis for the proteins is presented in Fig. S2 of the supplemental material.