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 H(2)O or in D(2)O media. Essentially, the data first show that addition of Na(+) induces appearance of peaks assigned to changes in the environment and/or orientation of alpha-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na(+) produces peaks corresponding to additional changes of alpha-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(+) co-ordination. 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.
León
Xavier
X
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.
Lemonnier
Raymonde
R
Leblanc
Gérard
G
Padrós
Esteve
E
D020816:Amino Acid Motifs; D003903:Deuterium; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008553:Melibiose / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012964:Sodium / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D029968:Escherichia coli Proteins; D008081:Liposomes; D027981:Symporters; D014867:Water; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium; D003903:Deuterium; D008239:Lysine