Ratiometric detection of Zn(ll) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small mols. or metal ions in living cells are invaluable tools for understanding their physiol. function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiol. relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concn. range from 10 nM to 1 mM. Size-exclusion chromatog. and fluorescence anisotropy were used to provide a detailed picture of the conformational changes assocd. with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concn. of the fluorescent proteins and could be understood quant. by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small mols.