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The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation.

Biochimica et biophysica acta (2013-03-19)
Yibin Lin, Oliver Fuerst, Meritxell Granell, Gérard Leblanc, Víctor Lórenz-Fonfría, Esteve Padrós
ABSTRACT

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H(+), Na(+) or Li(+) to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na(+) in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Melibiose, ≥98% (HPLC)
Millipore
D-(+)-Melibiose, suitable for microbiology, ≥99.0%

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