Measuring Ionic Strength Changes using Fluorescence Lifetime and Time-Resolved Anisotropy

Abstract

Intracellular ionic strength affects many factors such as cell volume, catalytic activities, and molecular interactions. A family of hetero-FRET sensors has been designed to detect changes in ionic strength in vivo and in vitro. These proteins consist of a donor (mCerulean) and an acceptor (mCitrine) that are connected via a single flexible polypeptide hinge with a basic helix and an acidic helix. The basic helices are enriched with either arginine or lysine, and the acidic helices are enriched with either glutamate or aspartate. The FRET sensors (RE, RD, and KE) are designed to have maximal energy transfer at low ionic strength and to decrease monotonically as electrostatic screening of the charged helices occurs with increasing ionic strength. In this study, we investigate the effects that different linker structures have on the energy transfer efficiencies as measured using fluorescence lifetime and time-resolved anisotropy. We find that RE consistently has the greatest energy transfer across all salt concentrations and RD has the lowest, with KE having intermediate sensitivity. This family of proteins has been characterized as a function of potassium chloride and other ions in the Hofmeister series. Our solution studies indicate that these proteins are useful sensors of biologically relevant ionic strengths. Importantly they have potential for dynamically mapping ionic strength changes in response to biological stimuli.