Substrate-Induced Conformational Changes of Melibiose Permease from Escherichia coli Studied by Infrared Difference Spectroscopy
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- 作者：Xavier Leó ; n ; Ví ; ctor A. Ló ; renz-Fonfrí ; a ; Raymonde Lemonnier ; Gé ; rard Leblanc ; and Esteve Padró ; s
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 8, 2005
- 年：2005
- 卷：44
- 期：9
- 页码：3506 - 3514
- 全文大小：143K
- 年卷期：v.44,no.9(March 8, 2005)
- ISSN：1520-4995
文摘
Fourier transform infrared difference spectroscopy has been used to obtain information aboutsubstrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstitutedinto liposomes. Binding of the cosubstrate Na+ gives rise to several peaks in the amide I and II regionsof the difference spectrum Na+·MelB minus H+·MelB, that denote the presence of conformational changesin all types of secondary structures (
-helices, 
-sheets, loops). In addition, peaks around 1400 and at1740-1720 cm-1 are indicative of changes in protonation/deprotonation or in environment of carboxylicgroups. Binding of the cosubstrate Li+ produces a difference spectrum that is also indicative ofconformational changes, but that is at variance as compared to that induced by Na+ binding. To analyzethe following transport steps, the melibiose permease with either H+, Na+, or Li+ bound was incubatedwith melibiose. The difference spectra obtained by subtracting the spectrum cation·MelB from the respectivecomplex cation·melibiose·MelB were roughly similar among them, but different from those induced bycation binding, and more intense. Therefore, major conformational changes that are induced during melibiosebinding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm-1, are similarfor the three cotransporting cations. Changes in the protonation state and/or in the environment of givencarboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.
-helices, -sheets, loops). In addition, peaks around 1400 and at1740-1720 cm-1 are indicative of changes in protonation/deprotonation or in environment of carboxylicgroups. Binding of the cosubstrate Li+ produces a difference spectrum that is also indicative ofconformational changes, but that is at variance as compared to that induced by Na+ binding. To analyzethe following transport steps, the melibiose permease with either H+, Na+, or Li+ bound was incubatedwith melibiose. The difference spectra obtained by subtracting the spectrum cation·MelB from the respectivecomplex cation·melibiose·MelB were roughly similar among them, but different from those induced bycation binding, and more intense. Therefore, major conformational changes that are induced during melibiosebinding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm-1, are similarfor the three cotransporting cations. Changes in the protonation state and/or in the environment of givencarboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.