title: Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach.
abstract: The β-barrel assembly machinery (BAM) is involved in folding and insertion of outer membrane proteins in Gram-negative bacteria, a process that is still poorly understood. With its 790 residues, BamA presents a challenge to current NMR methods. We utilized a "divide and conquer" approach in which we first obtained resonance assignments for BamA's periplasmic POTRA domains 4 and 5 by solution NMR. Comparison of these assignments to solid-state NMR (ssNMR) data obtained on two BamA constructs including the transmembrane domain and one or two soluble POTRA domains suggested that the fold of POTRA domain 5 critically depends on the interface with POTRA 4. Using specific labeling schemes we furthermore obtained ssNMR resonance assignments for residues in the extracellular loop 6 that is known to be crucial for BamA-mediated substrate folding and insertion. Taken together, our data provide novel insights into the conformational stability of membrane-embedded, non-crystalline BamA.
authors.affiliation: NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Tessa
authors.initials: T
authors.lastname: Sinnige
authors.forename: Klaartje
authors.initials: K
authors.lastname: Houben
authors.forename: Iva
authors.initials: I
authors.lastname: Pritisanac
authors.forename: Marie
authors.initials: M
authors.lastname: Renault
authors.forename: Rolf
authors.initials: R
authors.lastname: Boelens
authors.forename: Marc
authors.initials: M
authors.lastname: Baldus
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins