ABSTRACT
Further insight into the cosubstrate-induced structural change of the melibiose permease (MelB) of Escherichia coli has been sought by investigating the binding and spectroscopic properties of the fluorescent sugar 2'-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl 1-thio-beta-D-galactopyranoside (Dns2-S-Gal) and related analogs (Dns3-S-Gal or Dns6-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 efficiently inhibit melibiose transport and bind to MelB in a sodium-dependent fashion. Their dissociation constants (Kd) are in the micromolar range in the presence of NaCl and an order of magnitude higher in its absence. In the presence of NaCl and Dns2-S-Gal, sample excitation at 335 or 297 nm gives rise to a fluorescent signal at around 465 nm, whereas Dns3-S-Gal or Dns6-S-Gal emits a fluorescence light at 490 or 506 nm, respectively. Detailed study of the Dns2-S-Gal signal elicited by a 297 nm illumination indicates that a tryptophan-mediated fluorescence resonance energy transfer phenomenon is involved in the response. All fluorescence signals below 500 nm are prevented by addition of melibiose in excess, and the kinetic constants describing their dependence on the probe or NaCl concentrations closely correlate with the probe binding constants. Finally, the Dns2-S-Gal signal recorded 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 the 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 from the sugar-binding site, and (iii) that the interaction of sodium ions with MelB enhances the hydrophobicity of this environment. These results are consistent with the induction of a cooperative change of the structure of the sugar-binding site or of its immediate vicinity by the ions.
