Figure 1.

FRET Efficiency and Z Parameter Histograms of the Labeled Wild-Type D34 and Variants under Native Conditions

(A) Schematic of the D34 structure.

(B) Locations of the Alexa dye pairs in wild-type D34 (ANK3-5) and the other variants are shown schematically to the left of the histograms. FRET efficiency histograms (left histograms) were obtained by single (blue) laser excitation of the double-labeled samples. FRET efficiency, E, was calculated as E = I_A / (γI_D + I_A), where I_D and I_A are the donor (blue channel) and acceptor (red channel) fluorescence intensities, respectively, and γ is the instrument factor determined previously as 0.54. FRET Z parameter histograms (middle histograms) of the same data set are presented in the form Z = ln(I_A / I_D). TCCD Z parameter histograms (right histograms) were obtained by simultaneous two-laser (blue and red) excitation of the double-labeled samples. The data were acquired at 25 pM protein concentration in 50 mM Tris-HCl buffer (pH 8), 150 mM NaCl, and 0.001% Tween 20 at 25°C. The orange and green lines on the FRET efficiency histogram and FRET Z parameter histogram of ANK9-12 are the Gaussian fits of the data at low FRET and high FRET, respectively.