NMR characterization of a 72 kDa transcription factor using differential isotopic labeling

NF‐κB is a major transcription factor that mediates a number of cellular signaling pathways. Crystal structure analysis gives an incomplete picture of the behavior of the protein, particularly in the free state; free monomers or dimers of NF‐κB have never been crystallized. NMR analysis gives insights into the structure and dynamics of the protein in solution, but a necessary first step is the assignment of resonances. The size of the heterodimer of the Rel homology regions of the NF‐κB monomers p65 and p50 (72 kDa) prohibits the straightforward use of triple‐resonance spectroscopy to obtain the assignments. However, the dynamic nature of the free heterodimer, in particular the independence of the DNA‐binding and dimerization domains of each monomer, allows the assignments made on differentially labeled smaller domains to be mapped successfully onto the spectrum of the larger full‐length RHR. Problematic areas such as the p65 nuclear localization sequence, which is disordered in the free protein, can be approached by residue‐specific labeling and comparison with previously‐published spectra of a short peptide with the same sequence. Overall, this NMR analysis of NF‐κB has given valuable insights into the highly dynamic nature of the free state, which is likely to play an important role in the functional cycle of NF‐κB in the cell.