FIGURE 9.

Microscale thermophoresis (MST) assays reveal that phosphorylation increases the Ca²⁺ affinity of the C₂C domain and reduces the Ca²⁺ affinity of the C₂F domain. (A–I) Fluorescence changes after infrared laser mediated heating of the sample indicate binding of a ligand. Data points are mean values ± SD for n = 3 technical replicates each. K_Ds were acquired by Hill fitting (solid lines) in IGOR (Wavemetrics). (A) A minor change in fluorescence for the C₂C domain or a fragment containing the C₂ABC domains did not differ from the negative controls with EDTA, suggesting no Ca²⁺ binding. (B) In contrast, the phosphomimetic (pm) C₂C domain (T449D) showed binding to Ca²⁺, but with rather low affinity. (C) Direct comparison for the wild-type and the phosphomimetic C₂C domain illustrates that phosphorylation increases the Ca²⁺ affinity. (D) The wild-type C₂F domain binds to Ca²⁺; but not when EDTA was present. (E) In comparison, the binding of Mg²⁺ to the C₂F domain is much weaker than the binding to Ca²⁺. (F) Compared to the non-phosphorylated C₂F domain, the three phosphomimetic mutations lower Ca²⁺ affinity by one order of magnitude; (G) no binding occurred in the presence of EDTA. (H) Mg²⁺ affinity is lower than Ca²⁺ affinity for the phosphomimetic C₂F domain. (I) Measuring in a high salt buffer (300 mM NaCl) slightly lowers the K_D for Ca²⁺.