Macromolecular crowding effects on the excited-state dynamics of novel FRET probes using time-resolved fluorescence

Abstract

Living cells are crowded with large molecules (proteins and nucleic acids) and organelles. These macromolecules are known to have many effects on cellular processes, yet there is still a need to develop a technique to quantify crowding concentrations in live cell studies. Recently, several fluorescence-based probes have been engineered to potentially quantify crowding within living cells. These dynamic probes contain a pair of fluorophores (mCerulean3 and mCitrine, the donor and acceptor, respectively) that are capable of fluorescence resonance energy transfer (FRET). This series of probes are connected by a linker of variable length and rigidity that allow them to undergo varying degrees of conformational changes upon increases in macromolecular crowding. Due to the newness of these FRET probes, there is a need to characterize the excited-state dynamics of these new protein-based sensors using fluorescence techniques that are compatible with non-invasive and imaging modes. Here we used steady-state spectroscopy and time-resolved fluorescence to accomplish this characterization. We also used cleaved versions of these probes to control for complexities resulting from changes in refractive indices. These measurements allow us to develop a kinetic model for the depopulation of the donor’s excited-state, and to estimate the FRET efficiencies of these probes in both heterogeneous and homogenous environments. We find that these probes undergo conformational changes in heterogeneous environments and favor a more compact structure, thereby increasing energy transfer rates. Conversely, in homogenously viscous environments, our probes do not favor conformational changes when compared to pure buffer solutions. These results serve as the next advancement in developing the full potential of these probes for future studies in live cells.