title: Real-time conformational changes in LacY.
abstract: Galactoside/H(+) symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein. Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20-30 s(-1), independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity.
authors.affiliation: Departments of Physiology and.
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.affiliation: Departments of Physiology and.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kasho
authors.affiliation: Departments of Physiology andMicrobiology, Immunology, and Molecular Genetics, andMolecular Biology Institute, University of California, Los Angeles, CA 90095-7327 rkaback@mednet.ucla.edu.
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011510:Proteolipids / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D029968:Escherichia coli Proteins; D005697:Galactosides; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C021149:bimanes; C020485:proteoliposomes
keywords: major facilitator superfamily; membrane transport proteins