FIG 2. Comparison of the CaM-binding properties of reporters containing the neuromodulin and PEP19 IQ domains.

Binding was evaluated based on the fractional changes in the fluorescence emission of the B_IQ (■, □) or B_IQPEP (●,○) reporter constructs. Binding experiments were performed in the presence of ~250 µM free Ca²⁺ (filled symbols) or 3 mM BAPTA (empty symbols). Apparent K_d values derived from fits of these (solid lines) and similar data to a hyperbolic binding equation are listed in Table 1. Fluorescent reporter concentrations were 25–100 nM. The standard experimental buffer contained 25 mM Tris (pH 7.5), 100 mM KCl, 100 µg/mL BSA, 25 µM TPEN, and other components as specified. The fitted F_min value for binding of Ca²⁺-free CaM to B_IQPEP is 0.55±0.07. This is not different from the value of 0.56±0.07 derived for the Ca²⁺-free CaM complex with B_IQ.