Figure 5.

Determination of the FRET efficiencies and kinetic parameters using the maximum likelihood method with the eight-state model, which includes blinking states of the two acceptors. (a) Relaxation rate k (= k_F + k_U). (b) Fraction of the folded state p_F. Errors in (a) and (b) are standard deviations determined from the curvature at the maximum of the likelihood function. (c) Fractions of the acceptor count rates for the folded (open circle) and unfolded (filled square) states determined from the three-color segments. (d) Comparison of the measured two-color FRET efficiencies (eq 33) determined from DA1 segments ($E_{1\text{F}}^{2\text{c}}$, purple circle; $E_{1\text{U}}^{2\text{c}}$, purple square) and DA2 segments ($E_{2\text{F}}^{2\text{c}}$, magenta circle; $E_{2\text{U}}^{2\text{c}}$, magenta square), measured three-color FRET efficiencies (eq 34) ($E_{1\text{F}}^{3\text{c}}$, blue circle; $E_{1\text{U}}^{3\text{c}}$, blue square; $E_{2\text{F}}^{3\text{c}}$, green circle; $E_{2\text{U}}^{3\text{c}}$, green square), and calculated three-color FRET efficiencies for the unfolded state using the Gaussian chain model ($E_{1\text{U}}^{\text{G}}$ and $E_{2\text{U}}^{\text{G}}$, orange filled diamond). FRET efficiencies were corrected for background, donor leak, direct acceptor excitation, and γ-factors.