Fig. 3

Multiple Ca²⁺-binding sites in EFSAM. a–c Isothermal titration calorimetric (ITC) analyses of EFSAM-GrpE WT (a), GrpE (b), and EFSAM-GrpE D76A (c). Left panels, Heat changes measured by injecting 1 μl aliquots of 13 mM Ca²⁺ into a sample cell containing initially 130 μM protein, stated as the monomer concentration. Right panels, Integrated binding isotherm as a function of molar ratio (Ca²⁺: protein) after subtracting heats of dilution. In a, the indicated total Ca²⁺ at 40 min is the amount added to the ITC cell up to that point, corrected for the increase in volume, and free Ca²⁺ is calculated as total Ca²⁺ minus bound Ca²⁺ assuming five sites are occupied in EFSAM. d D4 sensor Ca²⁺ competition assay design. e–g D4 sensor normalized fluorescence signals in the presence of GrpE (35 μM) (e), EFSAM(D76A)-GrpE (35 μM) (f), or WT EFSAM-GrpE (35 μM) (g) are compared to the Ca²⁺-binding curve of D4 alone (black solid trace in each panel). h Method used for calculating free Ca²⁺ and Ca²⁺ bound to EFSAM (as total Ca²⁺ minus free Ca²⁺) from the competition data in g. i Plot of Ca²⁺ bound to WT EFSAM as a function of free Ca²⁺ concentration, calculated as in h. Data from two experiments plotted individually