FRET Analysis of Ionic-Strength Sensors using Time-Resolved Fluorescence

Abstract

Ionic strength influences many aspects of the cell such as cell volume, catalytic activities of enzymes, protein activities, and protein-protein interactions. A family of genetically encoded novel ionic strength sensors has been developed to quantify the ionic strength in vivo. These ionic strength sensors consist of a single polypeptide chain that is made up of mCerulean (a cyan fluorescent protein) connected to mCitrine (a yellow fluorescent protein) via a basic α-helix (enriched in either lysine or arginine residues), a short flexible hinge, and an acidic α-helix (enriched in either aspartate or glutamate residues). These sensors are capable of undergoing fluorescence resonance energy transfer (FRET). The two α-helices are electrostatically attracted to each other, and they become less so as the ionic strength of the environment surrounding the sensor increases due to electrostatic screening. The increase in the intramolecular distance between donor, mCerulean, and acceptor, mCitrine, causes a decrease in the energy transferred. E6G2, which has uncharged α-helices and thus insensitive to ionic strength changes, is used as a control. To quantify the effects of ionic strength as a function of potassium chloride concentration, fluorescence lifetime is used to assess energy transfer between mCerulean and mCitrine. In addition to determining the sensitivity of these protein sensors, these experiments serve as a critical control for the single molecule approach of using fluorescence correlation spectroscopic measurements of molecular brightness as a measure of energy transfer efficiency. The combination of fluorescence lifetime and fluorescence correlation spectroscopy approaches will be useful tools for our long-term goal, which is to dynamically map ionic strength changes in living cells.