Figure 5.

Elimination of conformational heterogeneity of NF-κB by IκBα but not by the 5xPEST IκBα. (A) The His-tag on IκBα was used to immobilize the NF-κB through the interaction with anti-His antibody biotin conjugate. (B) FRET histogram of NF-κB bound to wildtype (WT) IκBα showing only a stable low FRET state was observed. (C) The stripping impaired 5xPEST IκBα mutant contains five mutations in the PEST sequence to neutralize negatively charged residues. (D) FRET histogram for NF-κB bound to the 5xPEST IκBα showing a broad distribution and a larger proportion of fluctuating traces. (E) A representative trace of NF-κB bound to WT IκBα showing the stable low-FRET state. (F) Representative traces of NF-κB bound to 5xPEST IκBα showing heterogeneous long-lived states with a range of FRET efficiencies and domain fluctuations.