title: Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli.
abstract: Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from the aqueous phase are susceptible to cross-linking. This approach is now extended to most of the remaining positions in the molecule. Of an additional 143 single-Cys mutants studied, homodimer formation is observed with both a 5-A- and a 21-A-long crosslinking agent containing bis-methane thiosulfonate reactive groups in 33 mutants and exclusively with the 21-A-long reagent in 43 mutants. Furthermore, intermolecular cross-linking has little or no effect on transport activity, thereby providing further support for the argument that lactose permease is functionally, as well as structurally, a monomer in the membrane. In addition, evidence is presented indicating that reentrance loops are unlikely in this polytopic membrane transport protein.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California, Los Angeles, CA 90095-1662, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ermolova
authors.forename: Lan
authors.initials: L
authors.lastname: Guan
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D027981:Symporters
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; C021499:lactose permease; D007785:Lactose