Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.bioenergetics | membrane protein crystal structure | MTS reagents | sugar binding | affinity labeling T he Major Facilitator Superfamily (MFS) is one of the largest and most diverse families of membrane transporters with members from Archae to Homo sapiens that utilize uniport, symport, or antiport mechanisms to transport a broad range of substrates across membranes (1). Most members of the family have 12 to 14 transmembrane segments and catalyze transport by an alternating access mechanism (1-3). The lactose permease of Escherichia coli (LacY) is the most intensively studied representative of the MFS and embodies a paradigm for understanding general transport mechanisms throughout the superfamily.LacY catalyzes a symport reaction-the coupled translocation of a H þ and a galactopyranoside (galactoside∕H þ symport). Because translocation is obligatorily coupled, sugar accumulation against a concentration gradient is achieved by using the free energy released from the downhill movement of H þ with the electrochemical H þ gradient (Δμ H þ ; interior negative and/or alkaline). Conversely, downhill sugar translocation by LacY drives uphill translocation of H þ with the generation of Δμ H þ , the polarity of which depends on the direction of the sugar concentration gradient (4).Several crystal structures of LacY have been resolved, from both a conformationally restricted mutant C154G (5, 6) and the wild-type protein (7), all of which exhibit the same overall architecture. The protein is composed of 12 transmembrane helices organized in two pseudosymmetrical six α-helical bundles surrounding a large hydrophilic cavity open solely to the cytoplasm representing an inward-facing conformation. The sugar-binding site and the residues involved in H þ translocation are near the apex of the cavity, approximately in the middle of the molecule. For the most part, residues involved in sugar recognition are confined to the N-terminal bundle, while those important for H þ translocation are located in the C-terminal bundle. Systematic mutagenesis of each residue in LacY has identified less than 10 irreplaceable residues absolutely required for lactose∕H þ symport. E126 (helix IV) and R144 (helix V) are essential for substrate r...
Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C in ) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C out ) to C in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescencedetection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C out topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.
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