Living cells are complex, crowded, and dynamic and continually respond to environmental and intracellular stimuli. They also have heterogeneous ionic strength with compartmentalized variations in both intracellular concentrations and types of ions. These challenges would benefit from the development of quantitative, noninvasive approaches for mapping the heterogeneous ionic strength fluctuations in living cells. Here, we investigated a class of recently developed ionic strength sensors that consists of mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) tethered via a linker made of two charged α-helices and a flexible loop. The two helices are designed to bear opposite charges, which is hypothesized to increase the ionic screening and therefore a larger intermolecular distance. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing Förster resonance energy transfer (FRET) that is dependent on both the linker amino acids and the environmental ionic strength. Using time-resolved fluorescence of the donor (mCerulean3), we determined the sensitivity of the energy transfer efficiencies and the donor-acceptor distances of these sensors at variable concentrations of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). As controls, similar measurements were carried out on the FRET-incapable, enzymatically cleaved counterparts of these sensors as well as a construct designed with two electrostatically neutral α-helices (E6G2). Our results show that the energy transfer efficiencies of these sensors are sensitive to both the linker amino acid sequence and the environmental ionic strength, whereas the sensitivity of these sensors to the identity of the dissolved ions of the Hofmeister series of salts seems limited. We also developed a theoretical framework to explain the observed trends as a function of the ionic strength in terms of the Debye screening of the electrostatic interaction between the two charged α-helices in the linker region. These controlled solution studies represent an important step toward the development of rationally designed FRET-based environmental sensors while offering different models for calculating the energy transfer efficiency using time-resolved fluorescence that is compatible with future in vivo studies.
Bibliographical noteFunding Information:
We would like to thank Anh Cong, Elsie A. Johnson, Alexander Naughton, Jessica Marshik, Margaret Gurumani, and Emma Kauffman for their technical help and useful discussions during the course 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 Organization for Scientific Research Vidi grant. 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 (MSI) at the University of Minnesota for providing resources that contributed to the research results reported within this paper.
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