ArticleProceedings of the National Academy of Sciences of the United States of America2009
Probing of the rates of alternating access in LacY with Trp fluorescence.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 37 papers.
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Who cites it
37 citing papers in PubMed, 61 citations in OpenAlex.
- Iron Acquisition Systems of Gram-negative Bacterial Pathogens Define TonB-Dependent Pathways to Novel Antibiotics.Chemical reviews · 2021Review
- Article
- The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance.The Journal of biological chemistry · 2019Article
- It takes two to tango: The dance of the permease.The Journal of general physiology · 2019Review
- Structural Basis of HStructure (London, England : 1993) · 2019Article
- Engineered occluded apo-intermediate of LacY.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Crystal Structure of a ligand-bound LacY-Nanobody Complex.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.Scientific reports · 2018Article
- Oversized galactosides as a probe for conformational dynamics in LacY.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
- Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY.Biochemistry · 2016Article
- Transient conformers of LacY are trapped by nanobodies.Proceedings of the National Academy of Sciences of the United States of America · 2015Article
- A chemiosmotic mechanism of symport.Proceedings of the National Academy of Sciences of the United States of America · 2015Article
- Outward-facing conformers of LacY stabilized by nanobodies.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Concerted loop motion triggers induced fit of FepA to ferric enterobactin.The Journal of general physiology · 2014Article
- Real-time conformational changes in LacY.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Galactoside-binding site in LacY.Biochemistry · 2014Article
- Flexible gates generate occluded intermediates in the transport cycle of LacY.Journal of molecular biology · 2014Article
- Structure of sugar-bound LacY.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
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Authors and funding
4 authors at 1 institution in 2 countries.
Funding
Abstract
Sugar/H(+) symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H(+) binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pK(a) of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is approximately 100 s(-1). On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140-->Trp/Phe-334-->His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK(a) = 8), but remarkably, the rate of sugar-induced quenching is only approximately 0.4 s(-1). The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.
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