SPR binding data for UN2A with S100A1 at pH 7.4. (A) S100A1 demonstrates concentration dependent binding by SPR. UN2A was attached to the gold surface and varying concentrations of S100A1 was titrated across the surface at pH 7.4 and pCa 3. Data were fit to a single-state dissociation model and results in a K_D of 21.6 µM. (B) Binding affinity of S100A1 to UN2A depends on Ca²⁺ concentration. SPR-binding experiments were repeated at a range of Ca²⁺ concentrations, from pCa 10 to pCa 3. Negligible binding was observed at pCa 10 and 7 with increased binding at pCa 5, and the strongest binding observed at pCa 3. This is consistent with a model where weak binding occurs at pCa 5 due to one Ca²⁺ bound per monomer and strong binding occurs at pCa 3 when both Ca²⁺-binding sites are occupied. (C) Representative affinity fit for the binding interaction between S100A1 and UN2A at pCa 3.