Differential perturbation of intersubunit and interdomain communications by glycine 141 mutation in Escherichia coli CRP

Biochemistry
Xiaodong Cheng, J C Lee

Abstract

Upon binding of cAMP, concomitant changes in CRP structure across the subunit and domain interfaces are observed. In order to identify the structural elements involved in the coupling of interfacial interactions, structural perturbation was introduced at residue 141 by site-directed mutagenesis. Thermodynamic parameters defining protein stability, cAMP binding, and subunit assembly of the mutant were determined. Conformational changes probed by proteolytic digestion and fluorescence signal reported by the fluorescein-labeled C178 lead to a dissection of the contribution of the intersubunit and interdomain interactions, respectively, in the cAMP-modulated DNA binding of CRP. In the absence of cAMP, mutant G141Q is sensitive to protease attack at the subunit interface, an established property of wild type CRP observed only in the presence of cAMP. Although the G141Q mutant assumes a subunit alignment similar to that of the activated CRP, this mutant absolutely requires cyclic nucleotide for specific DNA interaction. Monitoring the fluorescence probe attached to the C-terminal DNA binding domain of the G141Q mutant showed that the DNA binding domain responds quantitatively to the binding of cyclic nucleotide to the N-terminal doma...Continue Reading

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Citations

Oct 7, 2009·Proceedings of the National Academy of Sciences of the United States of America·Hitesh SharmaThomas A Steitz
Mar 11, 2008·International Journal of Biological Macromolecules·Xianrong ShenShaoning Yu
Aug 29, 2006·Archives of Biochemistry and Biophysics·Yusuf Tutar, James G Harman
Sep 27, 2003·The Journal of Biological Chemistry·Bi-Hung PengGerald A Campbell
Jul 23, 2011·Molecular Biology Reports·Yusuf Tutar

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