Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: N-ethylmaleimide-sensitive face of helix II.
Cys-scanning mutagenesis of helix II in the lactose permease of Escherichia coli [Frillingos, S., Sun, J. et al. (1997) Biochemistry 36, 269-273] indicates that one face contains positions where Cys replacement or Cys replacement followed by treatment with N-ethylmaleimide (NEM) significantly inactivates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [(14)C]NEM labeling of 13 single-Cys mutants, including the nine NEM-sensitive Cys replacements, in right-side-out membrane vesicles is examined. Permease mutants with a single-Cys residue in place of Gly46, Phe49, Gln60, Ser67, or Leu70 are alkylated by NEM at 25 degrees C in 10 min, and mutants with Cys in place of Thr45 and Ser53 are labeled only in the presence of ligand, while mutants with Cys in place of Ile52, Ser56, Leu57, Leu62, Phe63, or Leu65 do not react. Binding of substrate leads to a marked increase in labeling of Cys residues at positions 45, 49, or 53 in the periplasmic half of helix II and a slight decrease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) show that positions 45 and 53 are accessible to solvent in the presence of ligand only, while positions 46, 49, 67, and 70 are accessible to solvent in the absence or presence of ligand. Position 60 is also exposed to solvent, and substrate binding causes a decrease in solvent accessibility. The findings demonstrate that the NEM-sensitive face of helix II participates in ligand-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membrane. In the following paper in this issue [Venkatesan, P., Hu, Y., and Kaback, H. R. (2000) Biochemistry 39, 10656-10661], the approach is applied to helix X.
Venkatesan
P
P
Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California 90095-1662, USA.
Liu
Z
Z
Hu
Y
Y
Kaback
H R
HR
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D003080:Cold Temperature; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000737:chemistry*; D005998:Glycine / Q000235:genetics; D006358:Hot Temperature; D007930:Leucine / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D008698:Mesylates / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000201:enzymology / Q000235:genetics; D017433:Protein Structure, Secondary / Q000187:drug effects / Q000235:genetics; D012997:Solvents; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D008698:Mesylates; D009004:Monosaccharide Transport Proteins; D012997:Solvents; D013439:Sulfhydryl Reagents; D027981:Symporters; C109153:(2-sulfonatoethyl)methanethiosulfonate; C021499:lactose permease; D007930:Leucine; D003545:Cysteine; D005033:Ethylmaleimide; D000409:Alanine; D005998:Glycine