Browsing by Subject "Fluorescence anisotropy"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Investigating Novel Hetero-Fret Biosensors for Environmental Ionic Strength Using Time-Resolved Fluorescence And Anisotropy
    (2020-08) Aplin, Cody
    One aspect of the biocomplexity of eukaryotic cells is the compartmentalized ionic strength, which impacts a myriad of biological functions. In this thesis, we investigate a class of environmental ionic strength sensors (mCerulean3–linker–mCitrine) that can be genetically encoded in site-specific compartments in living cells. In these sensors, mCerulean3–mCitrine acts as a Förster resonance energy transfer (FRET) pair that is tethered by two oppositely charged α-helices in the linker region. We hypothesize that as the ionic strength is increased, the attractive force between these charged alpha helices will be screened by the dissolved ions, resulting in an increase in the donor–acceptor distance (i.e., reduced FRET efficiency). To test this hypothesis, we investigated the rotational dynamics of these sensors using a newly developed time-dependent polarization anisotropy approach of the acceptor (mCitrine) emission, following pulsed excitation of the mCerulean3 (donor) in cleaved and intact mCerulean3–linker–mCitrine sensors (KE, RD, and RE) in different Hofmeister salt solutions (namely, KCl, NaCl, NaI, and Na2SO4). Our results show that the rotational dynamics of the intact and cleaved sensors are distinct under the excitation-detection conditions. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases, with slight sensitivity to the Hofmeister salt type. In contrast, FRET efficiency of E6G2 with electrostatically neutral amino acids in the linker region exhibits salt-independent rotational and FRET dynamics. Our time-resolved anisotropy data were also used to test existing theorical models concerning the steady-state anisotropy of these hetero-FRET sensors, while revealing the ionic strength effects on the angle between the dipole moments of the donor and acceptor in these sensors. Using our complementary, traditional time-resolved fluorescence method, we optimized our time-resolved anisotropy approach for FRET analysis and developed a theoretical model using Debye ionic screening of the two charged alpha helices in the linker region. These results help establishing time-resolved anisotropy of donor-acceptor pairs as a quantitative means for FRET analysis, which complement other traditional methods such as time-resolved fluorescence.