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Local conformational changes in the Vibrio Na+/galactose cotransporter
Marloes Veenstra1, Seren Lanza, Bruce A Hirayama
1Department of Physiology, The David Geffen School of Medicine at UCLA, University of California, Los Angeles, California 90095-1751, USA.
Biochemistry
|March 24, 2004
Summary
Researchers used fluorescence to pinpoint conformational changes in a bacterial sodium/galactose cotransporter. Ligand binding, particularly sodium, influences these changes near transmembrane helices 10 and 11, revealing an ordered transport mechanism.
Area of Science:
- Membrane Biology
- Biochemistry
- Structural Biology
Background:
- Cotransporter function relies on ligand-induced conformational changes.
- Understanding these changes is key to elucidating transport mechanisms.
Purpose of the Study:
- To localize conformational changes in a bacterial sodium/galactose cotransporter.
- To investigate the order of ligand binding during cotransport.
Main Methods:
- Cysteine-scanning mutagenesis of a Vibrio parahaemolyticus sodium/glucose cotransporter.
- Fluorescence labeling with ThioGlo3 and pyrene maleimide.
- Transport assays in bacteria and proteoliposomes.
Main Results:
- Mutant transporters retained activity; Cysteine 423's fluorescence was ligand-sensitive.
- D-galactose reduced labeling rate of Cys423; Na(+) and D-galactose quenched its fluorescence.
- Ligand concentrations affected apparent affinities for both Na(+) and D-galactose.
Conclusions:
- Conformational changes occur at the extracellular domain between transmembrane helices 10 and 11.
- Results suggest an ordered binding mechanism with Na(+) binding first.
- This study provides insights into the molecular mechanism of Na(+)/galactose cotransport.