Conformational equilibrium analysis of mCerulean3–linker–mCitrine constructs using time-resolved fluorescence measurements in controlled environments

Macromolecular crowding and ionic strength in living cells influence a myriad of biochemical processes essential to cell function and survival. For example, macromolecular crowding is known to affect diffusion, biochemical reaction kinetics, protein folding, and protein-protein interactions. In addition, enzymatic activities, protein folding, and cellular osmosis are also sensitive to environmental ionic strength. Recently, genetically encoded mCerulean3-linker-mCitrine constructs have been developed and characterized using time-resolved fluorescence measurements as a function of the amino acid sequence of the linker region as well as the environmental crowding and ionic strength. Here, we investigate the thermodynamic equilibrium of structural conformations of mCerulean3-linker-mCitrine constructs in response to the environmental macromolecular crowding and ionic strength. We have developed a theoretical framework for thermodynamic equilibrium of the structural conformations of these environmental sensors. In addition, we tested these theoretical models for thermodynamic analysis of these donor-linker-acceptor sensors using time-resolved fluorescence measurements as a function of the amino acid sequence of the linker region. Employing ultrafast time-resolved fluorescence measurements for gaining thermodynamic energetics would be helpful for Förster resonance energy transfer (FRET) studies of protein-protein interactions in both living cells and controlled environments.