8-ANILINO-1-NAPHTHALENESULFONATE IS A FLUORESCENT-PROBE OF CONFORMATIONAL-CHANGES IN THE D-GALACTOSE-H-COLI( SYMPORT PROTEIN OF ESCHERICHIA)

The binding of sugars and antibiotics to the overexpressed D-galactose -H+ symport protein (GalP) can be monitored from changes in the fluore scence of 8-anilinol-naphthalenesulfonate (ANS) equilibrated with insi de-out vesicles. Transported sugars, such as D-glucose and D-galactose , cause an enhancement in the ANS fluorescence of up to 13%. Nontransp orted sugars that have little, if any, affinity for GalP, such as L-ga lactose and L-glucose, have no effect upon the ANS fluorescence. Cytoc halasin B and forskolin, which are potent inhibitors of the transporte r, produce little change in the fluorescence, but are capable of rever sing the D-galactose/D-glucose enhancement in fluorescence. Sugars tha t bind to GalP but are not transported, such as methyl-beta-D-glucose, produce only a slight quench in the ANS fluorescence, but again rever se the enhancement in fluorescence induced by transported sugars. A si mple interpretation is that the increase in ANS fluorescence is attrib utable to the sugar-induced reorientation of the transporter from an i nward- to an outward-facing conformation. Nontransported sugars and an tibiotics, which are thought to bind at the inner membrane face of the transporter, are able to reverse the fluorescence enhancement by bind ing to the inward-facing conformation. The postulated reorientation pr ocess was sufficiently slow to follow its progress by stopped-flow flu orometry. The K-d for the binding of D-galactose to the inward-facing site was 7.22 (+/- 1.49) mM, and the rate constants for outward and in ward reorientation of the transporter were 4.06 (+/- 0.16) s(-1) and 1 .36 (+/- 0.18) s(-1), respectively. The overall K-d values for a range of sugars and antibiotics have been determined, and the involvement o f each sugar hydroxyl group in the recognition and translocation proce sses has been assessed.