Abstract
Cytosolic and organellar free Ca2+ concentrations are very dynamic; they are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations.Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jelly fish Aequorea victoria. Its cDNA can be concatenated with those encoding many other proteins, and the resulting fusion proteins are usually fluorescent and often preserve the biochemical functions and cellular localizations of the partner proteins. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation or emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET).Our new indicators consist of tandem fusions of a blue- or cyan-emitting mutant of GFP, calmodulin (CaM), the calmodulin-binding peptide Ml3, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the Ml3 domain, increasing the FRET between the flanking GFPs (Fig. 1).
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