MELIBIOSE PERMEASE OF ESCHERICHIA-COLI - STRUCTURAL ORGANIZATION OF COSUBSTRATE BINDING-SITES AS DEDUCED FROM TRYPTOPHAN FLUORESCENCE ANALYSES

Binding of the coupling ion (Na+ or Li+) and sugars to the purified me libiose permease of Escherichia coli, reconstituted in proteoliposomes , produces selective and cooperative changes of the transporter trypto phan fluorescence. To assess the individual contribution of N- or C-te rminal domains of the permease to these substrate-induced fluorescence variations, we replaced tile two tryptophans located in its C-termina l half (W299 and W342) by a phenylalanine and compared the signal chan ge in mutants and wild-type permease. None of the mutations significan tly impairs transport activity. Persistence of the ion-induced signal quenching in a permease carrying only the six other tryptophans of the N-terminal domain is consistent with a previous suggestion that this domain accommodates the ion-binding site. On the other hand, the sugar -induced fluorescence increase varies from mutant to mutant in a sugar -specific fashion. While alpha-galactosides increase essentially the f luorescence of W299 and W342, beta-galactosides enhance the signal of W299 and of one (or more) of the N-terminal tryptophans but quench tha t of W342. Moreover, addition of sugars produces a 10 nm blue shift of both W299 and W342 emission spectra, suggesting reduced accessibility of these residues to solvent following substrate binding, These data suggest that W299 and W342 are at or close to the sugar binding site a nd that this latter is lined by the C-terminal helices IX and X. Moreo ver, as sugars with the beta-configuration also enhance the fluorescen ce of the N-terminal tryptophans, it is suggested that one (or more) h elix of the N-terminal half may be also at or near the sugar binding s ite. This implies close proximity and/or tight functional linkage betw een some N-terminal helices and helices IX and X of the C-terminal dom ain of the transporter.