Isothermal titration calorimetry

Zinc binding affinities were measured by isothermal titration calorimetry (ITC) on a MicroCal Auto-iTC200 instrument in UNC's Macromolecular Interactions Facility. All experiments were run at 20°C with 20 2-µl injections of zinc into 200 µl protein in sample buffer. ZE2 affinity measurements were carried out with a cell concentration of 40 µM ZE2 and a syringe concentration of 800 µM ZnSO₄; affinity measurements for Hinge2 and its mutants were performed at ∼50 µM protein and 1 mM ZnSO₄.