Substrate-induced conformational changes of melibiose permease from Escherichia coli studied by infrared difference spectroscopy.
Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.
León
Xavier
X
Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Lórenz-Fonfría
Víctor A
VA
Lemonnier
Raymonde
R
Leblanc
Gérard
G
Padrós
Esteve
E
D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008094:Lithium / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D011522:Protons; D012680:Sensitivity and Specificity; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011522:Protons; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D008094:Lithium; D012964:Sodium