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.
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.
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