FRET-based in vivo ca²⁺ a imaging by a new calmodulin-GFP fusion molecule

Intracellular Ca²⁺ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca²⁺ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca²⁺ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca²⁺ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a twofold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca²⁺ concentration of 0.05-1 μM.