The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors
Figure 5
For the same separation distance, dynamic and static isotropic regimes have different FRET efficiency distributions and decays.
A. The FRET efficiency distributions from Gaussian populations with a mean RDA value of 5.4 nm±1% in either the dynamic random isotropic reorientational regime (κ2 = 2/3, GRAY peak) or the static random isotropic orientational regime (BLUE bimodal distribution). B. Fluorescence decays for the populations depicted in panel A. C. The dependence of 〈E〉 on RDA in these dynamic (GRAY open circles) and static (BLUE squares) regimes. The blue area between these curves depicts the region between the dynamic and static regimes into which the FRET efficiencies for samples that have rotational correlation times similar to the inverse of the energy transfer rates will fall. D and E. FRET efficiency distributions (probability densities, p(E)) used to generate the dynamic (D) or static (E) average FRET efficiency curves displayed in panel C. Note that in the static random isotropic regime, samples tend to have FRET efficiencies either near 0% or centered at F/(1+F). F. FRET efficiency distributions, p(E), for fixed separation ranging from 1 to 10 nm and a Förster separation of 5.4 nm (F-values ranging from 3.7×104 to 3.7×10−2) calculated analytically.