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  • Title: 8-Anilino-1-naphthalenesulfonate is a fluorescent probe of conformational changes in the D-galactose-H+ symport protein of Escherichia coli.
    Author: Walmsley AR, Martin GE, Henderson PJ.
    Journal: J Biol Chem; 1994 Jun 24; 269(25):17009-19. PubMed ID: 8006005.
    Abstract:
    The binding of sugars and antibiotics to the overexpressed D-galactose-H+ symport protein (GalP) can be monitored from changes in the fluorescence of 8-anilino-1-naphthalenesulfonate (ANS) equilibrated with inside-out vesicles. Transported sugars, such as D-glucose and D-galactose, cause an enhancement in the ANS fluorescence of up to 13%. Nontransported sugars that have little, if any, affinity for GalP, such as L-galactose and L-glucose, have no effect upon the ANS fluorescence. Cytochalasin B and forskolin, which are potent inhibitors of the transporter, produce little change in the fluorescence, but are capable of reversing the D-galactose/D-glucose enhancement in fluorescence. Sugars that bind to GalP but are not transported, such as methyl-beta-D-glucose, produce only a slight quench in the ANS fluorescence, but again reverse the enhancement in fluorescence induced by transported sugars. A simple interpretation is that the increase in ANS fluorescence is attributable to the sugar-induced reorientation of the transporter from an inward- to an outward-facing conformation. Nontransported sugars and antibiotics, which are thought to bind at the inner membrane face of the transporter, are able to reverse the fluorescence enhancement by binding to the inward-facing conformation. The postulated reorientation process was sufficiently slow to follow its progress by stopped-flow fluorometry. The Kd for the binding of D-galactose to the inward-facing site was 7.22 (+/- 1.49) mM, and the rate constants for outward and inward reorientation of the transporter were 4.06 (+/- 0.16) s-1 and 1.36 (+/- 0.18) s-1, respectively. The overall Kd values for a range of sugars and antibiotics have been determined, and the involvement of each sugar hydroxyl group in the recognition and translocation processes has been assessed.
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