MELIBIOSE PERMEASE OF ESCHERICHIA-COLI - SUBSTRATE-INDUCED CONFORMATIONAL-CHANGES MONITORED BY TRYPTOPHAN FLUORESCENCE SPECTROSCOPY

Tryptophan fluorescence spectroscopy has been used to investigate the effects of sugars and coupling cations (H+, Na+, or Li+) on the confor mational properties of purified melibiose permease after reconstitutio n in liposomes. Melibiose permease emission fluorescence is selectivel y enhanced by sugars, which serve as substrates for the symport reacti on, a-galactosides producing larger variations (13-17%) than beta-gala ctosides (7%). Moreover, the sugar-dependent fluorescence increase is specifically potentiated by NaCl and LiCl (5-7 times), which are well- established activators of sugar binding and transport by the permease. The potentiation effect is greater in the presence of LiCl than NaCl. On their own, sodium and lithium ions produce quenching of the fluore scence signal (2%). Evidence suggesting that sugars and cations compet e for their respective binding sites is also given. Both the sugar-ind uced fluorescence variation and the NaCl(or LiCl)-dependent potentiati on effect exhibit saturation kinetics. In each ionic condition, the ha lf-maximal fluorescence change is found at a sugar concentration corre sponding to the sugar-binding constant. Also, half-maximal potentiatio n of the fluorescence change by sodium or lithium occurs at a concentr ation comparable to the activation constant of sugar binding by each i on. The sugar- and ion-dependent fluorescence variations still take pl ace after selective inactivation of the permease substrate translocati on capacity by N-ethylmaleimide. Taken together, the data suggest that the changes in permease fluorescence reflect conformational changes o ccurring upon the formation of ternary sugar/cation/permease complexes .