Integrated Laser-Induced Fluorescence Spectroscopy of Donor-Linker-Acceptor Constructs for Bioenvironmental Sensing

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.