Microcal vp itc isothermal titration calorimeter

ITC titrations were performed with a MicroCal™ VP-iTC isothermal Titration Calorimeter (Malvern) at 20 °C. Samples of STP10 wild type and mutants were prepared in an identical manner as described above. For Size-exclusion chromatography a buffer with 20 mM NaCitrate, 250 mM NaCl, 10% glycerol and 0.03% DDM adjusted to pH 5.5 was used. For the high pH data, STP10 C77A mutant was purified in an identical buffer using 20 mM MOPS and adjusted to pH 7.5. Fractions containing the purified protein were pooled and directly used for ITC experiments. Sample concentration was avoided to minimize any mismatch derived from empty detergent micelles. D-glucose was dissolved in the size-exclusion chromatography buffer and both protein and ligand were degassed prior to use. The sample cell was loaded with ~1800 µl of STP10 WT (50–80 µM) or STP10 C77A (20–40 µM) and titrated with a 5–10 fold higher concentration of D-glucose. A total of 36 injections of 8 µl aliquots were titrated into the protein sample. Each injection had a duration of 7 s and spaced with a 250 s interval. The stirring speed was set to 312 r.p.m. Data was corrected for nonspecific heat and analyzed using MicroCal Origin 7.0 software using a one-site binding model. The experiments were performed in triplicate and showed similar results.