The citrate carrier CitS probed by single-molecule fluorescence spectroscopy.
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 C(5) maleimide (AF(546), AF(568)). 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-AF(546), 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-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS.
Kästner
Christopher N
CN
Eidgenössische Technische Hochschule Zürich, Institut für Mikrobiologie, Institut für Physikalische Chemie, Switzerland.
Prummer
Michael
M
Sick
Beate
B
Renn
Alois
A
Wild
Urs P
UP
Dimroth
Peter
P
D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019343:Citric Acid / Q000737:chemistry*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D027981:Symporters
D001426:Bacterial Proteins; D002352:Carrier Proteins; C490118:CitS protein, E coli; C086246:CitS protein, bacteria; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D027981:Symporters; D019343:Citric Acid; D003545:Cysteine