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 2H2O 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 β-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 α-helices plus nonordered 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 β-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.
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1 Feb 2002|
Dave, N., Lórenz-Fonfría, V. A., Villaverde, J., Lemonnier, R., Leblanc, G., & 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. Journal of Biological Chemistry, 277, 3380-3387. https://doi.org/10.1074/jbc.M105466200