Fluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy

Cody P. Aplin, Robert C. Miller, Taryn M. Kay, Ahmed A. Heikal, Arnold J. Boersma, Erin D. Sheets

Research output: Contribution to journalArticlepeer-review

Abstract

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 acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies.

Original languageEnglish (US)
Pages (from-to)1417-1430
Number of pages14
JournalBiophysical journal
Volume120
Issue number8
DOIs
StatePublished - Apr 20 2021
Externally publishedYes

Bibliographical note

Funding Information:
We thank Prof. Paul Siders (UMD Chemistry and Biochemistry) for a useful discussion and thoughtful comments. We also thank Ryan Leighton, Rowan Simonet, and Chioma Nwachuku for their technical help during the early stages of this project. E.D.S. and A.A.H. acknowledge the financial support provided by the University of Minnesota Grant-in-Aid, a Chancellor's Small Grant, the Department of Chemistry and Biochemistry, the Swenson College of Science and Engineering, University of Minnesota Duluth. A.J.B. acknowledges the financial support of the Netherlands Organisation for Scientific Research Vidi grant (723.015.002). C.P.A. and R.C.M. were supported by teaching fellowships from the Department of Chemistry and Biochemistry. T.M.K. acknowledges the support of Mylan Radulovich Graduate Fellowship as well as the teaching fellowship from the Department of Physics and Astronomy, University of Minnesota Duluth. The authors also acknowledge the Minnesota Supercomputing Institute at the University of Minnesota for providing resources that contributed to the research results reported within this work.

Funding Information:
E.D.S. and A.A.H. acknowledge the financial support provided by the University of Minnesota Grant-in-Aid , a Chancellor’s Small Grant , the Department of Chemistry and Biochemistry, the Swenson College of Science and Engineering, University of Minnesota Duluth . A.J.B. acknowledges the financial support of the Netherlands Organisation for Scientific Research Vidi grant ( 723.015.002 ). C.P.A. and R.C.M. were supported by teaching fellowships from the Department of Chemistry and Biochemistry. T.M.K. acknowledges the support of Mylan Radulovich Graduate Fellowship as well as the teaching fellowship from the Department of Physics and Astronomy, University of Minnesota Duluth. The authors also acknowledge the Minnesota Supercomputing Institute at the University of Minnesota for providing resources that contributed to the research results reported within this work.

Publisher Copyright:
© 2021 Biophysical Society

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