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Changes in Secondary Structures and Acidic Side Chains of Melibiose Permease upon Cosubstrates Binding

Xavier León ^*, Raymonde Lemonnier , Gérard Leblanc  and Esteve Padrós ^*

^* Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Barcelona, Spain; and Service de Biophysique des Fonctions Membranaires, Département de Biologie Joliot Curie, Commissariat à l'Energie Atomique Saclay, LRC-CEA16V, Villefranche sur mer, France

Correspondence: Address reprint requests to Esteve Padrós, Unitat de Biofísica, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel.: 34-93-5811870; E-mail: esteve.padros{at}uab.es.

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 -helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na⁺ produces peaks corresponding to additional changes of -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.

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