Schematic diagram of the TIRF setup used for the smFRET measurements (only one of two possible ways the protein could be labeled with the Alexa dyes is depicted).

(A) smFRET histogram of the IκBα–NFκB complex at 37 °C demonstrating that binding NFκB to IκBα completely suppressed the low- to mid-FRET populations observed for free IκBα, even at the higher temperature. The inset illustrates the IκBα–NFκB complex (ARs 5–6 in green) and the placement of the fluorophores, which are represented by stars (only one of two possible ways the protein could be labeled with two different dyes is depicted). (B) In the absence of NFκB, the IκBα fluctuations, and hence the low-mid-FRET efficiencies, increased at 37 °C. The inset depicts an extended IκBα in the absence of NFκB at 37 °C, with ARs 5–6 in blue. (C) smFRET histogram of free IκBα in 0.1 M urea shows a marked increase in the mid-FRET population. The inset represents a schematic of an extended IκBα in the presence of 0.1 M urea, with ARs 5–6 in red. (D) smFRET histogram of N-terminally His-tagged YLTA mutant IκBα at 24 °C. The lower-FRET population is greatly suppressed compared to the wild-type, despite the higher temperature. The inset illustrates the stabilized YLTA mutant with ARs 5–6 in gray. (E) At 37 °C, the YLTA construct begins to show a lower-FRET population. (F) Cross-correlation plot averaged over the traces in each condition quantifies the degree of fluctuation. Smooth curves represent single-exponential fitting. The green and gray curves correspond to NFκB-bound and YLTA mutant IκBα; the blue curve corresponds to native IκBα at 37 °C; and the red and magenta curves correspond to IκBα in 0.1 and 0.5 M urea, respectively. The insets in A–D depict the molecules in the same color scheme.

Schematic free energy profile showing the native state and the broad, heterogeneous intrinsically disordered state. Higher temperatures or addition of small amounts of urea increase the population of the intrinsically disordered state. Below the graph are schematic drawings of possible structures in each state. The colors for ARs 5–6 follow the same scheme as Fig. 4F (magenta: IκBα in 0.5 M urea; blue: IκBα at 37 °C; gray: YLTA mutant at 24 °C; green: IκBα-NFκB complex at 37 °C).

(A) Crystal structure of IκBα (green) bound to NFκB (gray) (PDB ID code 1NFI). Amino acids 70–282 of IκBα are observed to be ordered in this structure (16). After replacing all wild-type cysteines with serines, two cysteines were introduced at positions 128 (AR 2) and 262 (AR 6) (purple spheres) for labeling with Alexa 555 and 647. (B) The IUPRED analysis of IκBα(67–287) is plotted for ARs 1–4 (black), AR5 (blue), and AR6 (magenta). The inset shows a comparison of the IUPRED results for the wild-type sequence of IκBα (magenta) vs. the Y254L/T257A sequence (black) expanded to show only AR6. The arrows indicate the Y254L and T257A mutation sites.

(A) Sample trace of an IκBα molecule showing stable high FRET. Traces similar to this one represented approximately 80% of the molecules at room temperature. (B) Sample trace of an IκBα molecule showing fluctuating FRET. Traces similar to this one represented 14–18% of the molecules at room temperature. (C) Sample trace of an IκBα molecule showing stable mid FRET. Traces similar to this one represented 2.5–7% of the molecules at room temperature. In A, B, and C, a dashed line at E = 0.5 marks the boundary for high vs low FRET and shaded blue (> 0.5 FRET efficiencies) and peach (< 0.5 FRET efficiencies) areas in the FRET efficiency traces correspond respectively to the blue and peach areas in the histograms shown in D and E. (D) smFRET histogram of N-terminally His₆-tagged IκBα in the absence of NFκB at 21 °C. The histogram revealed a major high-FRET curve centered at 0.76 and a broad shoulder centered at 0.49. (E) smFRET histogram of C-terminally His₆-tagged IκBα in the absence of NFκB at 21 °C. All histograms were fitted with up to two Gaussian peaks.

(A) Representative smFRET trace of a molecule that switched from fluctuating to a stable high-FRET state during data collection. (B) Representative smFRET trace of a molecule that switched from mid-FRET to a stable high-FRET state during data collection. A dashed line marks the boundary between high- and low-FRET regions of each trace.