Site-directed alkylation of cysteine replacements in the lactose permease of Escherichia coli: helices I, III, VI, and XI.
To complete a study on site-directed alkylation of Cys replacements in the lactose permease of Escherichia coli (LacY), the reactivity of single-Cys mutants in helices I, III, VI, and XI, as well as some of the adjoining loops, with N-[14C]ethylmaleimide (NEM) or methanethiosulfonate ethylsulfonate (MTSES) was studied in right-side-out membrane vesicles. With the exception of several positions in the middle of helix I, which either face the bilayer or are in close proximity to other helices, the remaining Cys replacements react with the membrane-permeant alkylating agent NEM. In helices III and XI, most Cys replacements are also alkylated by NEM except for positions that face the bilayer. The reactivity of Cys replacements in helix VI is noticeably lower and only 45% of the replacements label. Binding of sugar leads to significant increases in the reactivity of Cys residues that are located primarily at the same level as the sugar-binding site or in the periplasmic half of each helix. Remarkably, studies with small, impermeant MTSES show that single-Cys replacements in the cytoplasmic portions of helices I and XI, which line the inward-facing cavity, are accessible to solvent from the periplasmic surface of the membrane. Moreover, addition of ligand results in increased accessibility of Cys residues to the aqueous milieu in the periplasmic region of the helices, which may reflect structural rearrangements leading to opening of an outward-facing cavity. The findings are consistent with the X-ray structure of LacY and with the alternating access model [Abramson, J., Smirnova, I., et al. (2003) Science 301, 610-615].
Department of Physiology and Microbiology, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Natalia
N
Ermolova
Roshni Vyas
RV
Madhvani
H Ronald
HR
Kaback
D000478:Alkylation; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005033:Ethylmaleimide / Q000737:chemistry; D008081:Liposomes; D008698:Mesylates / Q000737:chemistry; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D027981:Symporters / Q000737:chemistry* / Q000235:genetics
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D008698:Mesylates; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C109153:(2-sulfonatoethyl)methanethiosulfonate; D003545:Cysteine; D005033:Ethylmaleimide