title: Evidence for an intermediate conformational state of LacY.
abstract: LacY mutant Cys154 → Gly exhibits a periplasmic-closed crystal structure identical to the WT, but is periplasmic-open in the membrane. The mutant hardly catalyzes transport, but binds galactosides from either side of the membrane with the same affinity and is resistant to site-directed proteolysis relative to the pseudo-WT. Site-directed alkylation was also applied to 11 single-Cys mutants in Cys154 → Gly LacY in right-side-out membrane vesicles or after solubilization and purification in dodecyl-β-D-maltopyranoside (DDM). Unlike the pseudo-WT, Cys replacements on the periplasmic side of the Cys154 → Gly mutant label rapidly in the membrane without sugar, but labeling decreases markedly after the mutant proteins are purified. Thus, Cys154 → Gly LacY likely favors a higher-energy intermediate periplasmic-open conformation in situ, but collapses to a lower-energy periplasmic-closed conformation in DDM after purification. Notably, branched-chain or neopentyl glycol maltoside detergents stabilize Cys154 → Gly LacY in the membrane-embedded form.
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.forename: Lan
authors.initials: L
authors.lastname: Guan
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Zhou
authors.forename: Wen-Xu
authors.initials: WX
authors.lastname: Hong
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Zhang
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D002241:Carbohydrates / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008024:Ligands; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D027981:Symporters / Q000378:metabolism*
chemical_list: D002241:Carbohydrates; D003902:Detergents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C411362:dodecyl maltopyranoside; D008320:Maltose; C021499:lactose permease; D003545:Cysteine