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.

Results suggest that eating pistachio nuts instead of other dietary fat calories can improve lipid profiles, thereby decreasing coronary risk. Further studies will be required to confirm these results and to determine the mechanism of this effect.

Helices IV and V in the lactose permease of Escherichia coli contain the major determinants for substrate binding [Glu126 (helix IV), Arg144 (helix V), and Cys148 (helix V)]. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 48 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in the absence or presence of ligand. In right-side-out membrane vesicles, Cys residues in the cytoplasmic halves of both helices react with NEM in the absence of ligand, while Cys residues in the periplasmic halves do not. Five Cys replacement mutants at the periplasmic end of helix V and one at the cytoplasmic end of helix V label only in the presence of ligand. Interestingly, in addition to native Cys148, a known binding-site residue, labeling of mutant Ala122 --> Cys, which is located in helix IV across from Cys148, is markedly attenuated by ligand. Furthermore, alkylation of the Ala122 --> Cys mutant blocks transport, and protection is afforded by substrate, indicating that Ala122 is also a component of the sugar binding site. Methanethiosulfonate ethylsulfonate, an impermeant thiol reagent shown clearly in this paper to be impermeant in E. coli spheroplasts, was used to identify substituted Cys side chains exposed to water and accessible from the periplasmic side. Most of the Cys mutants in the cytoplasmic halves of helices IV and V, as well as two residues in the intervening loop, are accessible to the aqueous phase from the periplasmic face of the membrane. The findings indicate that the cytoplasmic halves of helices IV and V are more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that exhibit ligand-induced changes are located for the most part in the vicinity of the residues directly involved in substrate binding, as well as the cytoplasmic loop between helices IV and V.

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.