title: Characterization of the insertase BamA in three different membrane mimetics by solution NMR spectroscopy.
abstract: The insertase BamA is the central protein of the Bam complex responsible for outer membrane protein biogenesis in Gram-negative bacteria. BamA features a 16-stranded transmembrane β-barrel and five periplasmic POTRA domains, with a total molecular weight of 88 kDa. Whereas the structure of BamA has recently been determined by X-ray crystallography, its functional mechanism is not well understood. This mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and NMR spectroscopy is thus a method of choice for its elucidation. Here, we report solution NMR studies of different BamA constructs in three different membrane mimetic systems: LDAO micelles, DMPC:DiC7PC bicelles and MSP1D1:DMPC nanodiscs. The impact of biochemical parameters on the spectral quality was investigated, including the total protein concentration and the detergent:protein ratio. The barrel of BamA is folded in micelles, bicelles and nanodiscs, but the N-terminal POTRA5 domain is flexibly unfolded in the absence of POTRA4. Measurements of backbone dynamics show that the variable insertion region of BamA, located in the extracellular lid loop L6, features high local flexibility. Our work establishes biochemical preparation schemes for BamA, which will serve as a platform for structural and functional studies of BamA and its role within the Bam complex by solution NMR spectroscopy.
authors: Biozentrum, University of Basel, Basel, Switzerland.
authors: Leonor
authors: L
authors: Morgado
authors: Kornelius
authors: K
authors: Zeth
authors: Björn M
authors: BM
authors: Burmann
authors: Timm
authors: T
authors: Maier
authors: Sebastian
authors: S
authors: Hiller
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008823:Micelles
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