PMID:11729178
| Citation |
Dave, N, Lórenz-Fonfría, VA, Villaverde, J, Lemonnier, R, Leblanc, G and Padrós, E (2002) Study of amide-proton exchange of Escherichia coli melibiose permease by attenuated total reflection-Fourier transform infrared spectroscopy: evidence of structure modulation by substrate binding. J. Biol. Chem. 277:3380-7 |
|---|---|
| Abstract |
The accessibility of Escherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H(+), Na(+), melibiose). Analysis of the amide II band upon (2)H(2)O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H(+)*melibiose*MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on beta-sheet accessibility, with the complex H(+)*melibiose*MelB being the most protected state against exchange, followed by Na(+)*melibiose*MelB. Although of smaller magnitude, similar changes in alpha-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly beta-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4-5 and 10-11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized. |
| Links |
PubMed Online version:10.1074/jbc.M105466200 |
| Keywords |
Amides/metabolism; Binding Sites; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen-Ion Concentration; Kinetics; Liposomes/metabolism; Protein Structure, Secondary; Proteolipids/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Spectroscopy, Fourier Transform Infrared; Symporters/chemistry; Symporters/metabolism |
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