Fig. 1. Spectroscopic properties of CaM-EGFP prove that the fusion protein preserves the CaM characteristics with respect to calcium and peptide binding. (A) Absorption spectra of CaM-EGFP: Ca²⁺-free and with saturating Ca²⁺; inset: CaM-EGFP absorbance (489 nm) with Ca²⁺ titration. Different Ca²⁺ saturation levels do not affect the EGFP characteristic absorption spectra (with maximum at 489 nm); at 280 nm there is the same change in the single Tyr absorbance as for wtCaM. (B) Ca²⁺ titration of CaM-EGFP: 5,5'-Br₂BAPTA absorbance at 263 nm; CaM-EGFP competes with Br₂BAPTA showing no change of the average Ca²⁺ affinity of the fusion protein compared with wtCaM. (C) WFF peptide fluorescence titration of Ca²⁺- saturated CaM-EGFP (_ex=290 nm, _em=330 nm) indicates a 1:1 stoichiometry and dissociation constant similar to that of wtCaM.