New design strategies for FRET-based sensor proteins

Sensor proteins based on FRET between two fluorescent proteins allow real-time imaging of mol. events in living cells. Here we report new FRET-based sensor approaches for the detection of Zn(II) and protease activity. Zn(II) sensors with a very high and tunable affinity (Kd = 30 fM -1.4 pM) have been created by connecting two fluorescently labeled metal binding domains using a series of flexible peptide linkers. Modeling the conformational distribution of the linker allows a quant. understanding of the ratiometric changes and the Zn(II) affinity. In a different sensor approach, de novo Zn(II) binding sites are introduced directly on the surface of both fluorescent proteins. This sensor displays an impressive 9-fold increase in emission ratio and allows detection of Zn(II) from 10 nM to 1 mM. Finally, we present a new concept for FRET-based protease sensors that is based on intramol. complex formation between donor and acceptor fluorescent domains. [on SciFinder (R)]