Figure 4.

Ca²⁺ and Tb³⁺ binding to S100A1-TRTK12 as monitored via Tb³⁺ luminescence. Displacement of Tb³⁺ by Ca²⁺ from a Tb³⁺-S100A1-TRTK12 complex as monitored by the decrease in Tb³⁺ luminescence. The sample conditions included 50μM TRTK12, 20 mM HEPES buffer, pH 7.0, and 20 mM DTT at 25 °C in D₂O. The ^CaK_D calculated from this competition experiment (^CaK_D=8 ± 3 μM) relied on the dissociation of Tb³⁺ from the Tb³⁺-S100A1-TRTK12 complex in the same buffer (^TbK_D = 149 ± 3 nM; inset). In this titration (inset), S100A1 was titrated into a solution where [Tb³⁺] was kept constant (3 μM) and [TRTK12] was kept at a concentration (50μM) where Tb³⁺-S100A1 is fully saturated with TRTK12 peptide at all points in the titration.