Publication type: Article in scientific journal
Type of review: Peer review (publication)
Title: The citrate carrier CitS probed by single-molecule fluorescence spectroscopy
Authors: Kastner, C.
Prummer, M.
Sick, Beate
Renn, A.
Wild, U.P.
Dimroth, P.
DOI: 10.1016/S0006-3495(03)74974-1
Published in: Biophysical Journal
Volume(Issue): 84
Issue: 3
Pages: 1651
Pages to: 1659
Issue Date: 2003
Publisher / Ed. Institution: Cell Press
ISSN: 1542-0086
Language: English
Subjects: Optics; Membrane protein; Single molecule; Biology
Subject (DDC): 572: Biochemistry
Abstract: A prominent region of the Naþ-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitSsC398, CitS-sC414) were selectively labelled with the thiol-reactive fluorophores AlexaFluor 546/568 C5 maleimide (AF546, AF568). While both single-cysteine mutants were catalytically active citrate carriers, labelling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF546, complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labelled CitSsC398-AF546/568 and dual-color detection provided strong evidence for a homodimeric association of CitS.
Further description: Content available via Elsevier Open Archive
Fulltext version: Published version
License (according to publishing contract): Licence according to publishing contract
Departement: School of Engineering
Organisational Unit: Institute of Data Analysis and Process Design (IDP)
Appears in collections:Publikationen School of Engineering

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