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 conformational properties of purified melibiose permease after reconstitution in liposomes. Melibiose permease emission fluorescence is selectively enhanced by sugars, which serve as substrates for the symport reaction, alpha-galactosides producing larger variations (13-17%) than beta-galactosides (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 fluorescence signal (2%). Evidence suggesting that sugars and cations compete for their respective binding sites is also given. Both the sugar-induced fluorescence variation and the NaCl(or LiCl)-dependent potentiation effect exhibit saturation kinetics. In each ionic condition, the half-maximal fluorescence change is found at a sugar concentration corresponding to the sugar-binding constant. Also, half-maximal potentiation of the fluorescence change by sodium or lithium occurs at a concentration comparable to the activation constant of sugar binding by each ion. The sugar- and ion-dependent fluorescence variations still take place after selective inactivation of the permease substrate translocation capacity by N-ethylmaleimide. Taken together, the data suggest that the changes in permease fluorescence reflect conformational changes occurring upon the formation of ternary sugar/cation/permease complexes.
Mus-Veteau
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Laboratoire J. Maetz, Departement de Biologie Cellulaire et Moleculaire du Commissariat à l'Energie Atomique, Villefranche sur mer, France.
Pourcher
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Leblanc
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D001692:Biological Transport / Q000187:drug effects; D002412:Cations; D004357:Drug Synergism; D004926:Escherichia coli / Q000201:enzymology*; D005697:Galactosides / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry; D018021:Lithium Chloride / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D027981:Symporters; D014364:Tryptophan / Q000737:chemistry*
D002412:Cations; D005697:Galactosides; D008081:Liposomes; D026901:Membrane Transport Proteins; D027981:Symporters; D012965:Sodium Chloride; D014364:Tryptophan; C021548:melibiose permease; D018021:Lithium Chloride