In a functional lactose permease mutant from Escherichia coli (LacY) devoid of native Cys residues, almost every residue was replaced individually with Cys and tested for reactivity with the permeant alkylating agent N-ethylmaleimide in right-side-out membrane vesicles. Here we present the results in the context of the crystal structure of LacY. Engineered Cys replacements located near or within the inward-facing hydrophilic cavity or at other solvent-accessible positions in LacY react well with this alkylating agent. Cys residues facing the low dielectric of the membrane or located in tightly packed regions of the structure react poorly. Remarkably, in the presence of ligand, increased reactivity is observed with Cys replacements located predominantly on the periplasmic side of the sugar-binding site. In contrast, other Cys replacements largely on the cytoplasmic side of the binding site exhibit decreased reactivity. Furthermore, both sets of Cys replacements in the putative cavities are located at the periplasmic (increased reactivity) and cytoplasmic (decreased reactivity) ends of the same helices and distributed in a pseudosymmetrical manner. The results are consistent with a model in which the single sugar-binding site in the approximate middle of the molecule is alternately exposed to either side of the membrane due to opening and closing of cytoplasmic and periplasmic hydrophilic cavities. membrane proteins ͉ membranes ͉ permease ͉ symport ͉ transport T he lactose permease of Escherichia coli (LacY) is encoded by the lacY gene and catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H ϩ . As such, LacY is a paradigm for membrane proteins that transduce free energy stored in an electrochemical ion gradient into a solute concentration gradient or vice versa. LacY has been solubilized from the membrane, purified to homogeneity in a completely functional state (reviewed in ref.

Site-directed N-ethylmaleimide labeling was studied with Glu-126 and͞or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed f luorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 3 Cys was also studied. In marked contrast to Val-331 3 Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 3 Ala͞Val-331 3 Cys, Arg-144 3 Ala͞Val-331 3 Cys, or Arg-144 3 Lys͞Val-331 3 Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 3 Ala or Arg-144 3 Ala and, to a lesser extent, Arg-144 3 Lys cause a red-shift in the emission spectrum and render the f luorophore more accessible to I ؊ . The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H ؉ translocation pathway at the interface between helices IX and X.The lactose permease (lac permease) of Escherichia coli is representative of secondary active transport proteins that convert free energy stored in electrochemical ion gradients into work in the form of a concentration gradient (reviewed in ref. 1). This hydrophobic, polytopic, cytoplasmic membrane protein catalyzes the coupled stoichiometric translocation of ␤-galactosides and H ϩ . The lacY gene that encodes the permease has been cloned and sequenced, and the product of the lacY gene has been solubilized, purified, reconstituted into proteoliposomes, and shown to be solely responsible for ␤-galactoside transport as a monomer (see ref.2). All available evidence indicates that the permease consists of 12 hydrophobic, membrane-spanning, ␣-helical domains connected by hydrophilic loops with both the N and C termini on the cytoplasmic face of the membrane (Fig. 1) (reviewed in refs. 3 and 4).Site-directed mutagenesis of wild-type permease and Cysscanning mutagenesis of a functional mutant devoid of Cys residues (C-less permease) reveals that 4 of 417 residues in the protein are irreplaceable with respect to coupling between lactose and H ϩ translocation-Glu-269 (helix VIII), Arg-302 (helix IX), His-322 (helix X), and Glu-325 (helix X) (reviewed in refs. 3-5). Although the permease has not been crystallized, application of a battery of site-directed biochemical and biophysical te...

Helix X in the lactose permease of Escherichia coli contains two residues that are irreplaceable with respect to active transport, His322 and Glu325, as well as Lys319, which is charge-paired with Asp240 in helix VII. Structural and dynamic features of transmembrane helix X are investigated here by site-directed thiol modification of 14 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in right-side-out membrane vesicles. Permease mutants with a Cys residue at position 326, 327, 329, 330, or 331 in the cytoplasmic half of the transmembrane domain are alkylated by NEM at 25 degrees C, a mutant with Cys at position 315 at the periplasmic surface is labeled in the presence of substrate exclusively, and mutants with Cys at positions 317, 318, 320, 321, 324, 328, 332, or 333 do not react with NEM under the conditions tested. Binding of substrate causes increased labeling of a Cys residue at position 315 and decreased labeling of Cys residues at positions 326, 327, and 329. Studies with methanethiosulfonate ethylsulfonate indicate that Cys residues at positions 326, 329, 330, and 331 in the cytoplasmic half are accessible to the aqueous phase from the periplasmic face of the membrane. Ligand binding results in clear attenuation of solvent accessibility of Cys at position 326 and a marginal increase in accessibility of Cys at position 327 to solvent. The findings indicate that the cytoplasmic half of helix X is more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that reflect ligand-induced conformational changes are located on the same face of helix X as Lys319, His322, and Glu325.