Orientation of the Escherichia coli Outer Membrane Protein OmpX in Phospholipid Bilayer Membranes Determined by Solid-State NMR †

Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry (Impact Factor: 3.02). 06/2008; 47(25):6531-8. DOI: 10.1021/bi800362b
Source: PubMed


The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.

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Available from: Radhakrishnan Mahalakshmi, Apr 11, 2014
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    • "Unfortunately the experimental data necessary to perform such studies are not yet available . However, progress is being made towards collecting NMR data from membrane proteins in bilayers (solid-state NMR) (Mahalakshmi and Marassi 2008) and bicelles (liquid NMR) (Lee et al. 2008) and towards collecting crystallographic data in environments more closely related to physiological conditions (Long et al. 2007), which will hopefully soon lead to new experimental structures. Computational studies can then be expected to contribute new insights into the extent to which data from membrane proteins in micelle or other detergent milieu can relate to the situation in the physiological membrane environment. "
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    ABSTRACT: The bacterial outer membrane protein OmpX from Escherichia coli has been investigated by molecular dynamics simulations when embedded in a phospholipid bilayer and as a protein-micelle aggregate. The resulting simulation trajectories were analysed in terms of structural and dynamic properties of the membrane protein. In agreement with experimental observations, highest relative stability was found for the β-barrel region that is embedded in the lipophilic phase, whereas an extracellular protruding β-sheet, which is a unique structural feature of OmpX that supposedly plays an important role in cell adhesion and invasion, shows larger structure fluctuations. Additionally, we investigated water permeation into the core of the β-barrel protein, which contains a tight salt-bridge and hydrogen-bond network, so that extensive water flux is unlikely. Differences between the bilayer and the micellar system were observed in the length of the barrel and its position inside the lipid environment, and in the protein interactions with the hydrophilic part of the lipids near the lipid/water interface. Those variations suggest that micelles and other detergent environments might not offer a wholly membrane-like milieu to promote adoption of the physiological conformational state by OmpX.
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    • "Finally, sparse solid-state NMR data have been used to complement X-ray structures to orient β-barrels within lipid bilayers (Mahalakshmi and Marassi 2008). "
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