Figure 6.

CaM binds to RCBM peptides at low Ca²⁺ concentrations. (A) Fluorescence emission spectra of the Badan-labeled control peptide (3 µM) without CaM (−CaM, left) and in the presence of 1.5 µM CaM (+CaM, middle). The free Ca²⁺ concentrations ranged from 0.007 to 10 µM. Spectra did not change with the Ca²⁺ level. Asterisks indicate the four point mutations used to perturb CaM binding. The diagram on the right displays the dependence of the wavelength for maximal emission (λ_max) on the Ca²⁺ concentration with (solid line) and without (broken line) CaM. Error bars indicate mean ± SEM. (B) The Badan-labeled RCBM peptide corresponding to ANO 1 responds to increasing Ca²⁺ in the presence of CaM with a blueshift of the fluorescence emission spectrum and an increase in fluorescence intensity (middle). The dependence of λ_max on the Ca²⁺ concentration (right) is described by a Hill function (solid line) with an EC₅₀ of 0.09 ± 0.01 µM Ca²⁺ (n = 3) and a Hill coefficient of 3.2 ± 0.6. (C) The ANO 2 peptide displays a stronger response to Ca²⁺ in the presence of CaM than the ANO 1 peptide, both with respect to the blue-shift of λ_max and to the fluorescence intensity. A fit of the Hill function yielded an EC₅₀ of 0.23 ± 0.01 µM Ca²⁺ (n = 3) and a Hill coefficient of 2.8 ± 0.2 (right).