A ligand-dependent conformational change of the Na+/galactose cotransporter of Vibrio parahaemolyticus, monitored by tryptophan fluorescence.
Purification and reconstitution of the active Vibrio parahaemolyticus Na+/galactose transporter (vSGLT) enables us to do protein chemistry studies on a representative member of this class of membrane transporters. By measuring intrinsic tryptophan (Trp) fluorescence, conformational changes on the binding of substrates could be investigated. Trp fluorescence increased by 6% on the addition of saturating levels of both Na+ and D-galactose, with a K0.5 for D-galactose of 0.6 mM. No change was seen on the addition of Na+ alone or by adding D-galactose in the presence of K+. The Trp fluorescence could be quenched by acrylamide, but not by Cs+or I?. In the presence of Na+ or K+ alone, of Na+ or K+ and D-galactose, of Na+ and L-glucose, or in the absence of ligands, the fluorescence quenches by acrylamide were similar. This indicated that the tryptophan exposure to acrylamide was unchanged in the presence or absence of ligands. No shifts in lem maximum were observed. To find the Trp responsible for the change in fluorescence, Trp 448 in transmembrane helix 11 in the putative sugar-binding pocket was mutated. It was found that W448F showed a similar change in Trp fluorescence upon the addition of D-galactose in the presence of Na+. We conclude that the Trp fluorescence properties of the purified and reconstituted Na+/galactose cotransporter are selectively changed by the transported substrates Na+ and D-galactose, but it is not the Trp (W448) in the sugar translocation pathway that is involved.
Veenstra
M
M
Department of Physiology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1751, USA. mveenstra@mednet.ucla.edu
Turk
E
E
Wright
E M
EM
D002135:Calcium-Binding Proteins; D005453:Fluorescence; D005690:Galactose / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014736:Vibrio parahaemolyticus / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011510:Proteolipids; C023550:galactose-binding protein; C020485:proteoliposomes; D014364:Tryptophan; D012964:Sodium; D005690:Galactose