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. 2021 Oct 6;49(19):11211–11223. doi: 10.1093/nar/gkab874

Figure 3.

Figure 3.

smFRET revealed a continuum of long-lived conformations for DNA-bound NF-κB. (A) A biotinylated hairpin DNA with a κB site was used for immobilization through biotin–NeutrAvidin interactions. (B) FRET histogram showing two major populations at low and high FRET efficiencies contributed by stable traces. (C) FRET histogram constructed by counting each visit to a long-lived state showing a continuous distribution with two dominating populations. (D) Distance distribution of between labeling positions by AWSEM simulations showing two dominating populations in DNA-bound NF-κB. (E) Representative stable traces showing long-lived states with low, mid, and high FRET efficiencies for DNA-bound NF-κB. (F) A representative trace showing transitions between long-lived states from high to mid to low FRET efficiencies. (G) smFRET titration experiments with immobilized NF-κB and varying DNA concentrations. A series of NF-κB:DNA molar ratios from 10:1 to 1:100 showed similar FRET histograms with a broad band from free NF-κB and two slightly higher peaks at low and high FRET efficiencies from DNA-bound NF-κB.