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The citrate carrier CitS probed by single-molecule fluorescence spectroscopy
Christopher N Kästner1, Michael Prummer, Beate Sick
1Eidgenössische Technische Hochschule Zürich, Institut für Mikrobiologie, Institut für Physikalische Chemie, Switzerland.
Biophysical Journal
|March 1, 2003
Summary
The Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae undergoes conformational changes upon citrate binding, as shown by single-molecule fluorescence spectroscopy. This study also provides evidence for CitS existing as a homodimer.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- The Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is crucial for citrate transport.
- Loop X-XI in CitS is a conserved region containing a potential citrate binding site.
Purpose of the Study:
- To investigate conformational changes in CitS using single-molecule fluorescence spectroscopy.
- To explore the substrate binding mechanism and oligomeric state of CitS.
Main Methods:
- Site-directed mutagenesis to create single-cysteine mutants (CitS-sC398, CitS-sC414).
- Selective labeling of cysteine residues with thiol-reactive fluorophores (AlexaFluor 546/568).
- Single-molecule fluorescence spectroscopy to monitor fluorescence quenching and dual-color detection for dimerization studies.
Main Results:
- Fluorophore labeling was tolerated only at C398, yielding a functional CitS-sC398-AF(546) conjugate.
- Citrate addition caused complete fluorescence quenching, indicating a citrate-induced conformational change in the labeled domain.
- Dual-color single-molecule studies provided evidence for the homodimeric association of CitS.
Conclusions:
- Citrate binding induces a significant conformational change in the CitS protein, likely reflecting its transport mechanism.
- CitS functions as a homodimer, a structural feature important for its transport activity.