Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification.
By using site-directed chemical labeling of lactose permease, conformational changes induced by ligand binding are observed in the native membrane of Escherichia coli. Membranes containing permease mutants with a single-Cys residue and a biotin-acceptor domain were labeled with radioactive N-ethylmaleimide (NEM) in the presence or absence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or a proton electrochemical gradient, followed by solubilization in n-dodecyl beta-D-maltopyranoside and adsorption to avidin. TDG-induced enhancement of the reactivity of membrane-embedded Val315-->Cys (helix X) permease is observed, while the reactivity of Val331-->Cys (helix X) permease is inhibited by ligand binding or imposition of a proton electrochemical gradient. In contrast, the reactivity of permease with a single native Cys residue at position 148 (helix V) is blocked by TDG, but unaffected by the proton electrochemical gradient. Furthermore, as shown with right-side-out and inside-out membrane vesicles, the accessibility of Cys148 to either NEM or impermeant methanethiosulfonate derivatives is comparable from both sides of the membrane. On the other hand, TDG protects Cys148 from alkylation more effectively in right-side-out vesicles (apparent KD = 20-50 microM) than inside-out vesicles (apparent KD ca. 1.0 microM). The findings provide strong support for the conclusion that the permease retains close to native conformation in n-dodecyl-beta-D-maltopyranoside. In addition, the results are consistent with the idea that lactose permease has two binding sites: one with higher affinity on the periplasmic surface of the membrane and another with lower affinity on the cytoplasmic surface.
Frillingos
S
S
Howard Hughes Medical Institute, University of California, Los Angeles, 90024-1570, USA.
Kaback
H R
HR
D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds; D027981:Symporters; D013862:Thiogalactosides / Q000494:pharmacology*; D014633:Valine
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; D013862:Thiogalactosides; D001710:Biotin; C031803:thiodigalactoside; C021499:lactose permease; D014633:Valine; D003545:Cysteine; D005033:Ethylmaleimide