Cloning, Purification and NMR Studies on β-catenin C-terminal Domain
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β-catenin is a key signaling protein which regulates cell signaling and gene transcription. Abnormal activation of β-catenin is linked to many cancers, particularly with colorectal cancers. Although many genetic and biological studies on Wnt/β-catenin have been reported and structures of the complex between β-catenin and its diverse binding partners have been published, many of them have focused on armadillo repeat domain of β-catenin. Both N- and C-terminal domains have been suggested to regulate interactions of β-catenin with other molecules, but still little is known about the C-terminal unstructured domain. To investigate the structure of this domain, construct of C-terminus was designed and structural studies were performed using size exclusion chromatography (SEC), circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy. We observed that not only the purified full-length construct but the purified C-terminal construct also dimerizes in solution by SEC, suggesting that this domain involves in dimerization of β-catenin. CD and fluorescence data indicate its flexibility and structural formation in the presence of membrane environments.
Abstract
β-catenin is a key signaling protein which regulates cell signaling and gene transcription. Abnormal activation of β-catenin is linked to many cancers, particularly with colorectal cancers. Although many genetic and biological studies on Wnt/β-catenin have been reported and structures of the complex between β-catenin and its diverse binding partners have been published, many of them have focused on armadillo repeat domain of β-catenin. Both N- and C-terminal domains have been suggested to regulate interactions of β-catenin with other molecules, but still little is known about the C-terminal unstructured domain. To investigate the structure of this domain, construct of C-terminus was designed and structural studies were performed using size exclusion chromatography (SEC), circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy. We observed that not only the purified full-length construct but the purified C-terminal construct also dimerizes in solution by SEC, suggesting that this domain involves in dimerization of β-catenin. CD and fluorescence data indicate its flexibility and structural formation in the presence of membrane environments.
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