Fig 8. Ca²⁺ and Mg²⁺ titrations of CTV-TnC and CTV-Tn complex allow determination of EF hand affinities for divalent cations from F_R.

(A) Ca²⁺ titrations of CTV-TnC (1μM) were performed in the presence (solid symbols) or absence (open symbols) of 2mM Mg²⁺_free to determine the affinities of sites III and IV (high affinity), and site II (low affinity) for Ca²⁺. (B) Mg²⁺ titration of CTV-TnC (1 μM), where the binding of Mg²⁺ is shown for sites III and IV. (C) Ca²⁺ titration of CTV-TnC reconstituted in Tn complex, in the presence of 2mM Mg²⁺_free. Insets in (A) and (C) show expansion of the initial portion of the titrations for clarity. Data were fitted using nonlinear least squares regression to either a double-Hill relation (Eq 5) in (A and C), or a single Hill relation (Eq 6) in (B). All regression parameter estimates are given in Table 3. Plotted values represent mean + SE (N = 4).