NMR-Based Conformational Ensembles Explain pH-Gated Opening and Closing of OmpG Channel

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 mecha...

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Veröffentlicht in:Journal of the American Chemical Society 2013-10, Vol.135 (40), p.15101-15113
Hauptverfasser: Zhuang, Tiandi, Chisholm, Christina, Chen, Min, Tamm, Lukas K
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Sprache:eng
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Zusammenfassung: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.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja408206e