Single-Molecule Studies of Environmental Ionic-Strength Sensors using Fluorescence Correlation Spectroscopy
Ionic strength affects many biological processes in living cells, which include protein folding, protein-protein interactions, enzymatic activity, and cellular osmosis. As a result, there is a need for ionic strength sensors that can be genetically encoded in live cells with site-specificity as a means to map out the heterogeneity of the compartmentalized ionic strength in living cells. Importantly, noninvasive analytical tools are needed to quantify the intracellular ionic strength while reducing the expression levels of those sensors to avoid interfering with native biomolecules in living cells. In this UROP project, we are investigating a newly designed, genetically encoded family of ionic strength biosensors that consist of two fluorescently proteins (cyan fluorescent protein as a donor and yellow fluorescent protein as an acceptor), linked together by a flexible linker with rationally designed amino acid sequence, which form Förster resonance energy transfer (FRET) pairs (namely, K6 and E6) in controlled salt solutions with predefined ionic strength. Using fluorescence correlation spectroscopy (FCS), we are developing a new analytical approach for quantifying the correlation between the FRET efficiency and the environmental ionic strength at the single-molecule level. Our working hypothesis is that as the environmental ionic strength increases, the positively charged amino acids in the linker region will be screened, which disrupt electrostatic interactions and therefore reducing the FRET efficiency due to the enhanced donor-acceptor distance. These single-molecule studies in controlled environments are critically important prior to in vivo studies with the inherent biocomplexity.