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


ABSTRACT: NF-?B 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-?B 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-?B 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-?B 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-?B in the cell.

SUBMITTER: Mukherjee SP 

PROVIDER: S-EPMC4815403 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Mukherjee Sulakshana P SP   Borin Brendan B   Quintas Pedro O PO   Dyson H Jane HJ  

Protein science : a publication of the Protein Society 20151221 3


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  ...[more]

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