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Biochemistry

Melibiose permease of Escherichia coli: structural organization of cosubstrate binding sites as deduced from tryptophan fluorescence analyses.


PMID 8810910

Abstract

Binding of the coupling ion (Na+ or Li+) and sugars to the purified melibiose permease of Escherichia coli, reconstituted in proteoliposomes, produces selective and cooperative changes of the transporter tryptophan fluorescence. To assess the individual contribution of N- or C-terminal domains of the permease to these substrate-induced fluorescence variations, we replaced the two tryptophans located in its C-terminal half (W299 and W342) by a phenylalanine and compared the signal change in mutants and wild-type permease. None of the mutations significantly 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 fluorescence of W299 and W342, beta-galactosides enhance the signal of W299 and of one (or more) of the N-terminal tryptophans but quench that of W342. Moreover, addition of sugars producers 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 and that this latter is lined by the C-terminal helices IX and X. Moreover, as sugars with the beta-configuration also enhance the fluorescence of the N-terminal tryptophans, it is suggested that one (or more) helix of the N-terminal half may be also at or near the sugar binding site. This implies close proximity and/or tight functional linkage between some N-terminal helices and helices IX and X of the C-terminal domain of the transporter.

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