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.
The heterogeneous cellular environment influences a myriad of biological processes. For example, macromolecular crowding affects biochemical reactions, protein-protein interactions, and protein folding. Additionally, the structure-function relationship of biomolecules and enzymatic activities are sensitive to the surrounding ionic strength. In this contribution, we highlight our recent studies on a family of donor–linker– acceptor constructs, which were designed for mapping the macromolecular crowding and ionic strength in living cells. Integrated ultrafast laser spectroscopy methods have been employed to quantify the Förster resonance energy transfer (FRET) and the donor-acceptor distance as a measure of the sensitivity of these constructs to environmental changes. The donor-acceptor FRET pairs are intrinsically fluorescent cyan and yellow proteins, respectively, that can be genetically encoded in living cells. The sensitivity of these constructs to environmental biomimetic crowding and ionic strength was investigated as a function of the sequence and charge of the linker regions, as well as the identity of the donor protein. Integrating noninvasive, quantitative laser-induced fluorescence methods with FRET, as a molecular ruler, provides a powerful tool for cellular studies towards mapping out macromolecular crowding and ionic strength in living cells. Our results are key for the development of rational design strategies for engineering enhanced noninvasive biosensors with better environmental sensitivities. The same sensors were used as a model system for developing new experimental approaches for protein-protein interaction and FRET studies. Importantly, these diagnostic molecular and analytical tools set the stage for understanding the correlation between these environmental factors and cellular functions.
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