title: Local conformational changes in the Vibrio Na+/galactose cotransporter.
abstract: Na(+) and sugar transport by cotransporters (symporters) is thought to occur as a series of ordered ligand-induced conformational changes. To localize these conformational changes in a bacterial Na(+)/galactose cotransporter, we have employed a combination of cysteine-scanning and fluorescence techniques. Single or pairs of cysteine residues were introduced into the external face of a cysteine-less Vibrio parahaemolyticus sodium/glucose cotransporter for expression in Escherichia coli, and each transporter was purified using affinity chromatography. All the mutant proteins retained transport activity in bacteria and proteoliposomes. Each mutant was exposed to two different fluorescence reagents, ThioGlo3 or pyrene maleimide, that are essentially nonfluorescent until they react with a thiol. Fluorescence was recorded as a function of time and ligand concentrations. The reagents specifically labeled six of the seven cysteine mutants, but only in Cysteine 423 was the fluorescence affected by ligands. The rate of labeling of Cys423 by ThioGlo3 or pyrene maleimide was reduced by D-galactose in Na(+) buffer. Furthermore, the fluorescence of Thioglo3-labeled Cys423 was quenched by D-galactose, but only in the presence of Na(+). This quench was not accompanied by a Stokes shift and was not produced by nontransported sugars, e.g., L-glucose. Reducing the sodium concentration from 200 to 10 mM decreased the apparent affinity for d-galactose without altering the maximum quench with saturating D-galactose. Reducing the galactose concentration from 20 to 0.5 mM reduced both the apparent affinity for Na(+) and the maximum quench at saturating Na(+). These results suggest an ordered reaction scheme with Na(+) binding first. The fluorescence results with ThioGlo3-labeled Cys423 indicate that conformational changes underlying Na(+)/galactose cotransport occur at or near the extracellular domain between transmembrane helices 10 and 11.
authors.affiliation: Department of Physiology, The David Geffen School of Medicine at UCLA, University of California, Los Angeles, California 90095-1751, USA.
authors.forename: Marloes
authors.initials: M
authors.lastname: Veenstra
authors.forename: Seren
authors.initials: S
authors.lastname: Lanza
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Hirayama
authors.forename: Eric
authors.initials: E
authors.lastname: Turk
authors.forename: Ernest M
authors.initials: EM
authors.lastname: Wright
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003545:Cysteine / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005690:Galactose / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry; D051247:Sodium-Glucose Transport Proteins; D051269:Sodium-Glucose Transporter 1; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014736:Vibrio parahaemolyticus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D008081:Liposomes; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D051247:Sodium-Glucose Transport Proteins; D051269:Sodium-Glucose Transporter 1; C099811:sodium glucose symporter, Vibrio parahaemolyticus; D014364:Tryptophan; D012964:Sodium; D003545:Cysteine; D005690:Galactose