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. 2011 Oct 5;101(7):1710–1719. doi: 10.1016/j.bpj.2011.08.025

Figure 2.

Figure 2

MFD smFRET analysis of the Nup153FG segments γ and ε. (a) Burst-integrated fluorescence-lifetime analysis of segment ε (upper row) and segment γ (lower row) under native conditions (PBS). The two-dimensional plots are color-coded for frequency of occurrence. (Top and right histograms) Maximum projections lifetime (τ) and EFRET. (Dashed circles) Result from two-dimensional Gaussian fits of both the 0-peak and the FRET-peak population. (Dashed lines) Centers of the fits to their position in the one-dimensional representation of EFRET and τ data. (b) Relationship between donor fluorescence anisotropy (r) and corresponding τ. (Solid line) Expected trend according to the Perrin equation. (c) Burst-integrated fluorescence-lifetime histograms analogous to (a) for segment ε and γ under unfolding conditions (4 M GdmCl). (d) EFRET from all experiments plotted as a function of GdmCl concentration for all mutants α to ζ. FRET efficiencies (from lifetime) in native conditions range from EFRET = 0.34 (segment γ) to 0.59 (segment ε) and decrease during denaturation to a final value of EFRET = 0.14 (segment γ) to 0.40 (segment ε) in 4 M GdmCl. (Solid lines) Result of monoexponential fits.