Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein

Biochemistry
R C Stewart, Ricaele VanBruggen

Abstract

In the chemotaxis signal transduction pathway of Escherichia coli, the response regulator protein CheY is phosphorylated by the receptor-coupled protein kinase CheA. Previous studies of CheY phosphorylation and CheY interactions with other proteins in the chemotaxis pathway have exploited the fluorescence properties of Trp(58), located immediately adjacent to the phosphorylation site of CheY (Asp(57)). Such studies can be complicated by the intrinsic fluorescence and absorbance properties of CheA and other proteins of interest. To circumvent these difficulties, we generated a derivative of CheY carrying a covalently attached fluorescent label that serves as a sensitive reporter of phosphorylation and binding events and that absorbs and emits light at wavelengths well removed from potential interference by other proteins. This labeled version of CheY has the (dimethylamino)naphthalene fluorophore from Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] attached to the thiol group of a cysteine introduced at position 17 of CheY by site-directed mutagenesis. Under phosphorylating conditions (or in the presence of beryllofluoride), the fluorescence emission of Badan-labeled CheY(M17C) exhibited an approximately 10 nm blue shift and ...Continue Reading

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Citations

Feb 26, 2009·The Journal of Biological Chemistry·Andreas BuschTino Krell
Sep 10, 2010·Annual Review of Microbiology·Tino KrellJuan Luis Ramos
Mar 10, 2010·Current Opinion in Microbiology·Robert B Bourret
Mar 14, 2012·Wiley Interdisciplinary Reviews. Systems Biology and Medicine·Marcus J TindallJudith P Armitage
Mar 13, 2015·Biophysical Journal·Anne-Florence Bitbol, Ned S Wingreen
Jan 24, 2015·Organic & Biomolecular Chemistry·Laura S KocsisEmmanuel A Theodorakis
Jun 23, 2009·Progress in Biophysics and Molecular Biology·M J TindallJ P Armitage
Oct 16, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Malgorzata MagochGrzegorz Dubin

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