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Biophysical Journal 84:1651-1659 (2003)
© 2003 The Biophysical Society

The Citrate Carrier CitS Probed by Single-Molecule Fluorescence Spectroscopy

Christopher N. Kästner*, Michael Prummer{dagger}, Beate Sick{dagger}, Alois Renn{dagger}, Urs P. Wild{dagger} and Peter Dimroth*

Eidgenössische Technische Hochschule Zürich, * Institut für Mikrobiologie, {dagger} Institut für Physikalische Chemie, CH-8092 Zürich, Switzerland

Correspondence: Address reprint requests to Peter Dimroth, E-mail: dimroth{at}micro.biol.ethz.ch.

A prominent region of the Na+-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 (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C5 maleimide (AF546, AF568). While both single-cysteine mutants were catalytically active citrate carriers, labeling 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-labeled CitS-sC398-AF546/568 and dual-color detection provided strong evidence for a homodimeric association of CitS.




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