title: Site-directed alkylation and the alternating access model for LacY.
abstract: 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.
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095-1662, USA. rkaback@mednet.ucla.edu
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
authors.forename: R
authors.initials: R
authors.lastname: Dunten
authors.forename: S
authors.initials: S
authors.lastname: Frillingos
authors.forename: P
authors.initials: P
authors.lastname: Venkatesan
authors.forename: I
authors.initials: I
authors.lastname: Kwaw
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: Natalia
authors.initials: N
authors.lastname: Ermolova
mesh_terms: D000477:Alkylating Agents; D000478:Alkylation; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide; D008024:Ligands; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000477:Alkylating Agents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; D003545:Cysteine; D005033:Ethylmaleimide