title: Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility.
abstract: The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.
authors: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Tessa
authors: T
authors: Sinnige
authors: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Markus
authors: M
authors: Weingarth
authors: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Marie
authors: M
authors: Renault
authors: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Lindsay
authors: L
authors: Baker
authors: Department of Molecular Microbiology, Institute of Biomembranes, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Jan
authors: J
authors: Tommassen
authors: NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Electronic address: m.baldus@uu.nl.
authors: Marc
authors: M
authors: Baldus
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D011487:Protein Conformation
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins
keywords: electron microscopy; outer membrane protein; protein dynamics; protein–lipid interactions; β-barrel assembly