Isothermal Titration Calorimetry of Galectin

Titrations were performed under standard conditions, as applied previously, to ensure comparability (Kutzner et al. 2019 (link); Ludwig et al. 2019a (link)). Removal of cognate sugar from protein preparations was guaranteed by dialysis over the course of 72 h with five buffer exchanges (2 L each). Briefly, 2 µL aliquots of ligand-containing solution (from a total of 36.4 µL) were injected per step into the galectin-containing solution in 20 mM phosphate buffer at pH 7.2 with 10 mM NaCl and 10 mM β-mercaptoethanol, starting at a volume of 200 µL. Injections were performed every 180 s at 25 °C and 500 rpm in a PEAQ-ITC calorimeter (Malvern, Westborough, MA, USA). Protein concentrations were based on absorbance applying a sequence-based extinction coefficient calculated with ExPASy ProtParam software. Data were routinely processed by MicroCal PEAQ-ITC analysis software with the one-set-of-sites/sequential models and a fitted offset parameter to account for potential background signal.