Integrated Fluorescence Spectroscopy for FRET Analysis of Novel Ionic Strength Sensors in the Presence of a Hofmeister Series of Salts

Abstract

Living eukaryotic cells are complex, crowded, and dynamic organisms that continually respond to environmental and intracellular stimuli. In addition, these cells have heterogeneous ionic strength with compartmentalized variation of both intracellular concentrations and types of ions. The underlying mechanisms associated with ionic strength variations that trigger different biological functions and response to environmental cues remain largely unknown. Therefore, there is a need to develop a quantitative method for mapping the compartmentalized ionic strength and their temporal fluctuations within living cells. In this work, we investigate a class of novel ionic- strength sensors that consists of tethered mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) via a linker of varied amino acids. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing fluorescence resonance energy transfer (FRET) based on both the linker amino acids and the environmental ionic strength. The energy transfer efficiency and the donor-acceptor distance of these sensors can be quantified noninvasively using integrated fluorescence methods in response to intracellular ionic strength in living eukaryotic cells. We employed time-resolved fluorescence methods to monitor the excited-state dynamics of the donor in the presence and absence of the acceptor as a function of the environmental ionic strength using potassium chloride (KCl, 0–500 mM). Towards mapping out the response to of these sensors towards biologically relevant salts, we carried out time- resolved fluorescence for FRET analysis of these sensors as a function of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). We also used these results towards technique development for FRET analysis based on time-resolved fluorescence polarization anisotropy. Our results show that the energy transfer efficiency of these sensors is sensitive to both the linker amino acid sequence and the environmental ionic strength. These studies in a controlled environment complement previous steady-state spectroscopy analysis of these sensors in a cuvette with the advantage of the compatibility of our approach with fluorescence lifetime imaging microscopy on living cells.