Lessons From Lactose Permease

Guan, Lan; Kaback, H. Ronald

doi:10.1146/annurev.biophys.35.040405.102005

Cited by 315 publications

(452 citation statements)

References 119 publications

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“…The sm-FRET findings with wild-type LacY are consistent with extensive site-directed alkylation studies (10) that support an alternating access model for galactoside/H ϩ symport (1,6). By this means, the single sugar-binding site in LacY in the approximate middle of the molecule is alternately exposed to either side of the membrane due to opening and closing of cytoplasmic and periplasmic hydrophilic cavities.…”

Section: Mutantssupporting

confidence: 82%

“…The crystal structure of wild-type LacY exhibits an inwardfacing conformation much like the C154G mutant (1,8). Furthermore, ligand binding markedly increases the apparent ac- Fig.…”

Section: Mutantsmentioning

confidence: 97%

“…Conversely, in the absence of ⌬ Hϩ , LacY utilizes free energy released from downhill translocation of galactosides in either direction to drive uphill translocation of H ϩ with generation of ⌬ Hϩ (reviewed in ref. 1). …”

mentioning

confidence: 99%

“…Notably, wild-type LacY has the same global fold (1,8). The symporter contains 12 transmembrane ␣-helices organized in two pseudosymmetrical bundles.…”

mentioning

confidence: 99%

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Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Majumdar

Смирнова²,

Kasho³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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lactose permease ͉ major facilitator superfamily ͉ single-molecule spectroscopy ͉ transporters T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily, utilizes free energy stored in an electrochemical H ϩ gradient (⌬ Hϩ ) to drive active transport by coupling the downhill, stoichiometric translocation of H ϩ with ⌬ Hϩ to the uphill accumulation of galactopyranosides. Conversely, in the absence of ⌬ Hϩ , LacY utilizes free energy released from downhill translocation of galactosides in either direction to drive uphill translocation of H ϩ with generation of ⌬ Hϩ (reviewed in ref.

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Section: Mutantssupporting

confidence: 82%

“…The crystal structure of wild-type LacY exhibits an inwardfacing conformation much like the C154G mutant (1,8). Furthermore, ligand binding markedly increases the apparent ac- Fig.…”

Section: Mutantsmentioning

confidence: 97%

mentioning

confidence: 99%

“…Notably, wild-type LacY has the same global fold (1,8). The symporter contains 12 transmembrane ␣-helices organized in two pseudosymmetrical bundles.…”

mentioning

confidence: 99%

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Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Majumdar

Смирнова²,

Kasho³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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141

178

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“…LacY, a member of the major facilitated superfamily (3), catalyzes the coupled stoichiometric transport of an H + and a galactopyranoside (galactoside/H + symport) across the cytoplasmic membrane (reviewed in ref. 4). With an abundance of biochemical, spectroscopic, and crystallographic data regarding structure and function, LacY is arguably the most extensively studied symport protein at the present time (reviewed in ref.…”

mentioning

confidence: 99%

Real-time conformational changes in LacY

Смирнова¹,

Kasho²,

Kaback

2014

Proc. Natl. Acad. Sci. U.S.A.

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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. LacY is organized into two pseudosymmetrical bundles, each containing six transmembrane helices, most of which are irregular, with the N and C termini on the cytoplasmic side of the membrane. WT LacY and a conformationally restricted mutant exhibit an inward-facing conformation with a tightly sealed periplasmic side and a water-filled cavity open to the cytoplasm (6-9), which is the conformation present in the membrane in the absence of sugar (10). Another conformation has been observed recently with a double-Trp mutant (11) that exhibits an occluded galactoside molecule with a narrow opening on the periplasmic side and a tightly sealed cytoplasmic aspect (12) (Fig. 1A).LacY is highly dynamic and exhibits multiple conformations at any given time, and galactoside binding triggers a shift between conformers (1). Thus, measurement of interspin distances with nitroxide-labeled Cys pairs in LacY reveals that sugar binding induces a decrease in distances on the cytoplasmic side and a corresponding increase in distances on the periplasmic side (13,14). Site-directed alkylation of single Cys LacY mutants in either right-side-out membrane vesicles (15, 16) or dodecyl-β-D-maltopyranoside (DDM) micelles (10), as well as single-molecule fluorescence (17) and thiol cross-linking (18), also indicates that sugar binding increases the probability of opening on the periplasmic side and closing on the cytoplasmic side.Recently, conformational changes in LacY have been studied dynamically by site-directed Trp fluorescence quenching by a protonated His or Lys residue (19). Sugar binding leads to unquenching of Trp fluorescence in periplasmic LacY mutants N245W (helix VII) or F378...

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Monoclonal antibody 4B1 influences the pK_a of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS

et al. 2020

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The monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic side of lactose permease (LacY) of Escherichia coli and inhibits H+/lactose symport and lactose efflux under nonenergized conditions. At the same time, ligand binding and translocation reactions that do not involve net H+ translocation remain unaffected by 4B1. In this study, surface‐enhanced infrared absorption spectroscopy applied to the immobilized LacY was used to study the pH‐dependent changes in LacY and to access in situ the effect of the 4B1 antibody on the pKa of Glu325, the primary functional H+‐binding site in LacY. A small shift of the pK value from 10.5 to 9.5 was identified that can be corroborated with the inactivation of LacY upon 4B1 binding.

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Lessons From Lactose Permease

Cited by 315 publications

References 119 publications

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Real-time conformational changes in LacY

Monoclonal antibody 4B1 influences the pK_a of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS

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Lessons From Lactose Permease

Cited by 315 publications

References 119 publications

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Real-time conformational changes in LacY

Monoclonal antibody 4B1 influences the pKa of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS

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Product

Resources

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Monoclonal antibody 4B1 influences the pK_a of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS