The transcription factor NFκB (RelA-p50) is a multidomain protein that binds DNA and its inhibitor, IκBα with apparently different conformations. We used single-molecule FRET to characterize the interdomain motions of the N-terminal DNA-binding domains in the free protein and also in various bound states. Several surprising results emerged from this study. First, the domains moved with respect to each other on several widely different timescales from hundreds of milliseconds to minutes. The free NFκB displayed stochastic motions leading to a broad distribution of states, ranging from very low-FRET states to high-FRET states. Varying the ionic strength altered the slow motions suggesting that they may be due to different weak electrostatic interactions between the domains creating a rugged energy landscape. Third, the DNA-binding domains continued to be mobile even when the protein was bound to its cognate DNA, but in this case the majority of the states were either high-FRET, a state expected from the available x-ray structures, or low-FRET, a state consistent with one of the DNA-binding domains dissociated. The fluctuations of the DNA-bound states were of lower amplitude and slightly faster frequency. Fourth, the inhibitor, IκBα freezes the domains into a low-FRET state, expected to be incapable of binding DNA. Neutralization of five acidic residues in the IκBα PEST sequence, which was previously shown to impair IκBαs ability to strip NFκB from the DNA, also impaired its ability to freeze the domains into a low-FRET state indicating that the freezing of motions of the DNA-binding domains is essential for efficient molecular stripping.
Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer