Variation of linker length in ratiometric fluorescent sensor proteins allows rational tuning of Zn(II) affinity in the picomolar to femtomolar range

Ratiometric fluorescent sensor proteins with a very high and tunable affinity for Zn(II) were created by connecting two fluorescently labeled metal binding domains, CFP-Atox1 and WD4-YFP, using a series of flexible peptide linkers. A simple random-coil model describing the conformational distribution of the linker allowed a quantitative understanding of the effect of the linker length on both the change in emission ratio and the Zn(II) affinity.