Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range.

Genetically encoded Förster resonance energy transfer (FRET) sensors enable the visualization of ions, molecules, and processes in live cells. However, despite their widespread use, the molecular states that determine sensor performance are usually poorly understood, which limits efforts to improve them. We used dynamic light scattering (DLS) and time-resolved fluorescence anisotropy to uncover the sensing mechanism of ZifCV1.173, a Zn ²⁺ FRET sensor. We found that the dynamic range (DR) of ZifCV1.173 was dominated by the high FRET efficiency of the Zn ²⁺ -free state, in which the donor and acceptor fluorescent proteins were closely associated. Mutating the donor-acceptor interface revealed that the DR of ZifCV1.173 could be increased or decreased by promoting or disrupting the donor-acceptor interaction, respectively. Adapting the same mutations to a related sensor showed the same pattern of DR tuning, supporting our sensing mechanism and suggesting that DLS and time-resolved fluorescence anisotropy might be generally useful in the biophysical characterization of other FRET sensors.