FIG 6. The effects of Ca²⁺ binding to the C-terminal EF hand pair in CaM on its affinities for reporters containing mutant neuromodulin IQ domains altered to match the PEP19 sequence at positions -1, 3 and 6.

Data for binding of N_xCCaM to the following reporter constructs are presented: B_IQK⁻¹A (■), B_IQA³Q (●), B_IQG⁶K (▲), and B_IQTr (◆), which contains all three amino acid changes. The complete amino acid sequences of the IQ domains in these reporters are listed in Fig. 1. At the ~250 µM free Ca²⁺ concentration used for these experiments the native EF hand pair in N_xCCaM is Ca²⁺ saturated. Apparent K_d values derived from fits of a hyperbolic binding equation to these (solid lines) and similar data are listed in Table 1. Additional experimental details are given in “Experimental Procedures” or in the caption to Fig. 2. Fitted curves for binding of Ca²⁺-saturated N_xCCaM to B_IQ (-.-) and B_IQPEP (…) are presented for comparative purposes (see Fig. 2).