Biophysical characterization of the free IkappaBalpha ankyrin repeat domain in solution.
The crystal structure of IkappaBalpha in complex with the transcription factor, nuclear factor kappa-B (NF-kappaB) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IkappaBalpha (67-317) when it is not in complex with NF-kappaB, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. Ikappa Balpha unfolds with a T(m) of 42 degrees C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IkappaBalpha are discussed.
Croy
Carrie Hughes
CH
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA.
Bergqvist
Simon
S
Huxford
Tom
T
Ghosh
Gourisankar
G
Komives
Elizabeth A
EA
D000577:Amides / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D017089:Ankyrin Repeat; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D020672:I-kappa B Proteins / Q000737:chemistry*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051996:Transcription Factor RelA
D000577:Amides; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D006859:Hydrogen; D003903:Deuterium