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Phys Chem Chem Phys. 2014 Apr 14;16(14):6480-5. doi: 10.1039/c3cp54427c. Epub 2014 Mar 6.

Predicted disorder-to-order transition mutations in IκBα disrupt function.

Author information

1
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA. ekomives@ucsd.edu.

Abstract

IκBα inhibits the transcription factor, NFκB, by forming a very tightly bound complex in which the ankyrin repeat domain (ARD) of IκBα interacts primarily with the dimerization domain of NFκB. The first four ankyrin repeats (ARs) of the IκBα ARD are well-folded, but the AR5-6 region is intrinsically disordered according to amide H/D exchange and protein folding/unfolding experiments. We previously showed that mutations towards the consensus sequence for stable ankyrin repeats resulted in a "prefolded" mutant. To investigate whether the consensus mutations were solely able to order the AR5-6 region, we used a predictor of protein disordered regions PONDR VL-XT to select mutations that would alter the intrinsic disorder towards a more ordered structure (D → O mutants). The algorithm predicted two mutations, E282W and P261F, neither of which correspond to the consensus sequence for ankyrin repeats. Amide exchange and CD were used to assess ordering. Although only the E282W was predicted to be more ordered by CD and amide exchange, stopped-flow fluorescence studies showed that both of the D → O mutants were less efficient at dissociating NFκB from DNA.

PMID:
24605363
PMCID:
PMC4040282
DOI:
10.1039/c3cp54427c
[Indexed for MEDLINE]
Free PMC Article

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