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] indi cates that one face contains positions where Cys replacement or Cys replace ment followed by treatment with N-ethylmaleimide (NEM) significantly inacti vates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [C-14]NEM labeling of 13 s ingle-Cys mutants, including the nine NEM-sensitive Cys replacements, in ri ght-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 an d 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. Bindi ng of substrate leads to a marked increase in labeling of Cys residues at p ositions 45, 49, or 53 in the periplasmic half of helix II and a slight dec rease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) sho w that positions 45 and 53 are accessible to solvent in the presence of lig and only, while positions 46, 49, 67, and 70 are accessible to solvent in t he absence or presence of ligand. Position 60 is also exposed to solvent, a nd substrate binding causes a decrease in solvent accessibility. The findin gs demonstrate that the NEM-sensitive face of helix II participates in liga nd-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membran e. 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 he lix X.