Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer - I. Evidence for ion-induced conformational change

Further insight into the cosubstrate-induced structural change of the melib iose permease (MelB) of Escherichia coli has been sought by investigating t he binding and spectroscopic properties of the fluorescent sugar 2'-(N-5-di methylaminonaphthalene-1-sulfonyl)-aminoethyl 1 -thio-beta-D-galactopyranos ide (Dns(2)-S-Gal) and related analogs (Dns(3)-S-Gal or Dns(6)-S-Gal with a propyl or hexyl instead of an ethyl linker, respectively) interacting with MelB in membrane vesicles or in proteoliposomes. The three analogs efficie ntly inhibit melibiose transport and bind to MelB in a sodium-dependent fas hion, Their dissociation constants (K-d) are in the micromolar range in the presence of NaCl and an order of magnitude higher in its absence. In the p resence of NaCl and Dns(2)-S-Gal, sample excitation at 335 or 297 nm gives rise to a fluorescent signal at around 465 nm, whereas Dns(2)-S-Gal or Dns( 6) S-Gal emits a fluorescence light at 490 or 506 nm, respectively. Detaile d study of the Dns2-S-Gal signal elicited by a 297 nm illumination indicate s that a tryptophan-mediated fluorescence resonance energy transfer phenome non is involved in the response. All fluorescence signals below 500 nm are prevented by addition of melibiose in excess, and the kinetic constants des cribing their dependence on the probe or NaCl concentrations closely correl ate with the probe binding constants. Finally, the Dns(2)-S-Gal signal reco rded in sodium-free medium is red shifted by up to 25 nm from that recorded in the presence of NaCl, Taken together, these results suggest (i) that th e fluorescence signals below 500 nm arise from Dns-S-Gal molecules bound to MelB, (ii) the presence of a highly hydrophobic environment close to or at the sugar-binding site, the polarity of which increases on moving away fro m the sugar-binding site, and (iii) that the interaction of sodium ions wit h MelB enhances the hydrophobicity of this environment. These results are c onsistent with the induction of a cooperative change of the structure of th e sugar binding site or of its immediate vicinity by the ions.