title: Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix VII.
abstract: Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.
authors.affiliation: 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.
authors.forename: P
authors.initials: P
authors.lastname: Venkatesan
authors.forename: I
authors.initials: I
authors.lastname: Kwaw
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003080:Cold Temperature; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000737:chemistry; D006358:Hot Temperature; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics*; D008698:Mesylates / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D027981:Symporters
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C109153:(2-sulfonatoethyl)methanethiosulfonate; C021499:lactose permease; D003545:Cysteine; D005033:Ethylmaleimide