Macromolecular crowding is prevalent in all living cells due to the presence of large biomolecules and organelles. Cellular crowding is heterogeneous and is known to influence biomolecular transport, biochemical reactions, and protein folding. Emerging evidence suggests that some cell pathologies may be correlated with compartmentalized crowding. As a result, there is a need for robust biosensors that are sensitive to crowding as well as quantitative, noninvasive fluorescence methods that are compatible with living cells studies. Here, we have developed a model that describes the rotational dynamics of hetero-Förster resonance energy transfer (FRET) biosensors as a means to determine the energy-transfer efficiency and donor-acceptor distance. The model was tested on wavelength-dependent time-resolved fluorescence anisotropy of hetero-FRET probes (mCerulean3-linker-mCitrine) with variable linkers in both crowded (Ficoll-70) and viscous (glycerol) solutions at room temperature. Our results indicate that the energy-transfer efficiencies of these FRET probes increase as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor compact structures with enhanced energy-transfer efficiencies and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the extended conformation of these FRET probes is more favorable in viscous, homogeneous environments with a reduced energy-transfer efficiency compared to those in pure buffer, which we attribute to reduced structural fluctuations of the mCerulean3-mCitrine FRET pair in the glycerol-enriched buffer. Our results represent an important step toward the application of quantitative and noninvasive time-resolved fluorescence anisotropy of hetero-FRET probes to investigate compartmentalized macromolecular crowding and protein-protein interactions in living cells as well as in controlled environments.
Bibliographical noteFunding Information:
Melissa Maurer-Jones, Robert Miller, Cody Aplin, and Anh Cong (all from the University of Minnesota Duluth) is deeply appreciated. E.D.S. and A.A.H. acknowledge the financial support provided by the University of Minnesota Grant-in-Aid. Additionally, the financial support was provided by a Chancellor’s Small Grant, the Department of Chemistry and Biochemistry, and the Swenson College of Science and Engineering, the University of Minnesota Duluth. A.J.B. acknowledges the financial support from the Netherlands Organization for Scientific Research Vidi grant. J.S. and H.J.L. were supported by teaching fellowships from the University of Minnesota at Duluth Department of Chemistry and Biochemistry. R.L. was supported by the Swenson Family Foundation, the summer undergraduate research program (SURP), and the undergraduate research opportunity program (UROP), the University of Minnesota Duluth. The authors further acknowledge the support from the Minnesota Supercomputing Institute (MSI) at the University of Minnesota.
© 2018 American Chemical Society.