Solid-State NMR Studies of Full-Length BamA in Lipid Bilayers Suggest Limited Overall POTRA Mobility

•Full-length BamA was reconstituted in liposomes at low lipid-to-protein ratio.•ssNMR indicates limited mobility of the periplasmic POTRA domains.•BamA can accommodate to lipid bilayers of different hydrophobic widths.•BamA causes disruptions of the lipid bilayer as seen in electron micrographs.

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.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (303 K)Download as PowerPoint slide