Fig. 3.

High-affinity Ca²⁺-binding sites on WT and mutant CaMs. (A–C) Stoichiometry of Ca²⁺ binding to WT and mutant CaMs measured by electrospray mass spectrometry at 10 μM CaM and 200 μM Ca²⁺. (A) WT CaM. (B) CaM-C^WT. (C) CaM-N^WT. (D) Ca²⁺ binding to WT and mutant CaMs measured in a competition assay with the fluorescent Ca²⁺-binding dye, Fluo4FF. Ca²⁺ was titrated into a solution of Fluo4FF (10 μM) alone (●), Fluo4FF plus 10 μM CaM-C^WT (▿),10 μM CaM-N^WT (▴), or 10 μM WT CaM (■). The data were fit with a model assuming four high-affinity Ca²⁺-binding sites for WT CaM, two for CaM-C^WT and CaM-N^WT, and a single Ca²⁺-binding site for Fluo4FF (Table 2).