title: NMR characterization of a 72 kDa transcription factor using differential isotopic labeling.
abstract: 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.
authors.affiliation: Department of Integrative Structural and Computational Biology, the Scripps Research Institute, La Jolla, California, 92037.
authors.forename: Sulakshana P
authors.initials: SP
authors.lastname: Mukherjee
authors.affiliation: Department of Integrative Structural and Computational Biology, the Scripps Research Institute, La Jolla, California, 92037.
authors.forename: Brendan
authors.initials: B
authors.lastname: Borin
authors.affiliation: Department of Integrative Structural and Computational Biology, the Scripps Research Institute, La Jolla, California, 92037.
authors.forename: Pedro O
authors.initials: PO
authors.lastname: Quintas
authors.affiliation: Department of Integrative Structural and Computational Biology, the Scripps Research Institute, La Jolla, California, 92037.
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism; D051379:Mice; D056004:Molecular Dynamics Simulation; D000072000:NF-KappaB Inhibitor alpha / Q000737:chemistry / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D000072417:Protein Domains; D055503:Protein Multimerization; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000378:metabolism
chemical_list: D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA
keywords: IκBα; NF-κB; NMR; dynamics; structure