title: NMR-based conformational ensembles explain pH-gated opening and closing of OmpG channel.
abstract: The outer membrane protein G (OmpG) is a monomeric 33 kDa 14-stranded β-barrel membrane protein functioning as a nonspecific porin for the uptake of oligosaccharides in Escherichia coli. Two different crystal structures of OmpG obtained at different values of pH suggest a pH-gated pore opening mechanism. In these structures, extracellular loop 6 extends away from the barrel wall at neutral pH but is folded back into the pore lumen at low pH, blocking transport through the pore. Loop 6 was invisible in a previously published solution NMR structure of OmpG in n-dodecylphosphocholine micelles, presumably due to conformational exchange on an intermediate NMR time scale. Here we present an NMR paramagnetic relaxation enhancement (PRE)-based approach to visualize the conformational dynamics of loop 6 and to calculate conformational ensembles that explain the pH-gated opening and closing of the OmpG channel. The different loop conformers detected by the PRE ensemble calculations were validated by disulfide cross-linking of strategically engineered cysteines and electrophysiological single channel recordings. The results indicate a more dynamically regulated channel opening and closing than previously thought and reveal additional membrane-associated conformational ensembles at pH 6.3 and 7.0. We anticipate this approach to be generally applicable to detect and characterize functionally important conformational ensembles of membrane proteins.
authors.affiliation: Department of Molecular Physiology and Biological Physics and Center for Membrane Biology, University of Virginia , Charlottesville, Virginia 22903, United States.
authors.forename: Tiandi
authors.initials: T
authors.lastname: Zhuang
authors.forename: Christina
authors.initials: C
authors.lastname: Chisholm
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Lukas K
authors.initials: LK
authors.lastname: Tamm
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D008823:Micelles; C114347:OmpG protein, E coli; D018272:Porins; D010767:Phosphorylcholine; C028810:dodecylphosphocholine
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