Changes in secondary structures and acidic side chains of melibiose permease upon cosubstrates binding

Infrared difference spectroscopy analysis of the purified melibiose permease of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na+, melibiose) in either H2O or in D2O media. Essentially, the data first show that addition of Na+ induces appearance of peaks assigned to changes in the environment and/or orientation of α-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na+ produces peaks corresponding to additional changes of α-helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+) cm-1/1576 (-) cm-1) appearing in the Na+-induced difference spectrum is assigned to the antisymmetric stretching of COO- groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na + coordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (-) cm-1/1567 (+) cm -1), appear in the melibiose-induced difference spectra, and may again be assigned to COO- groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding. © 2006 by the Biophysical Society.