title: Oligomeric sensor kinase DcuS in the membrane of Escherichia coli and in proteoliposomes: chemical cross-linking and FRET spectroscopy.
abstract: DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C(4)-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by fluorescence resonance energy transfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {f(D) = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.
authors.affiliation: Institute for Microbiology and Wine Research, Johannes Gutenberg University, Becherweg 15, 55099 Mainz, Germany.
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Scheu
authors.forename: Yun-Feng
authors.initials: YF
authors.lastname: Liao
authors.forename: Julia
authors.initials: J
authors.lastname: Bauer
authors.forename: Holger
authors.initials: H
authors.lastname: Kneuper
authors.forename: Thomas
authors.initials: T
authors.lastname: Basché
authors.forename: Gottfried
authors.initials: G
authors.lastname: Unden
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Erker
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: C485182:Cyan Fluorescent Protein; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011510:Proteolipids; C020485:proteoliposomes; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; C469359:DcuS protein, E coli
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