biostudies-literatureUnknown120(8)Aplin CPDutch Research Council (NWO)Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na₂SO₄) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptBiophysical journal1417-1430https://www.ebi.ac.uk/biostudies/studies/S-EPMC8105717biostudies-literatureFluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy.PMC8105717Aplin CPHeikal AAMiller RCSheets EDBoersma AJKay TMfalseFluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy.Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na₂SO₄) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the accept2021-01-01T00:00:00Z2021 Apr2025-04-04T19:41:34.504Z2025-04-04T19:41:34.504ZS-EPMC81057173358214010.1016/j.bpj.2021.01.035