Fluorescence fluctuation spectroscopy of mEGFP-Linker-mScarlet-I environmental crowding sensor at the single-molecule level

Living cells are complex, crowded, and dynamic with heterogeneous ionic strength, which influences biological processes that are essential to cellular function and survival. Recently, we have investigated a family of newly developed donor-linker-acceptor constructs for environmental sensing of macromolecular crowding and ionic strength using integrated, ultrafast time-resolved fluorescence spectroscopy methodologies. In this contribution, we highlight a novel single-molecule approach to investigate the sensitivity of these sensors to environmental variables using fluorescence fluctuation analysis and molecular brightness spectroscopy. These single-molecule studies complement the traditional, ensemble methods for protein-protein interactions. In addition, our findings represent a stop forward towards the development of a systematic, rational design strategy for environmental sensors.