Mutational and Topological Analysis of the Escherichia coli BamA ProteinReport as inadecuate

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The multi-protein β-barrel assembly machine BAM of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins OMPs into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated POTRA domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.

Author: Douglas F. Browning , Sophie A. Matthews, Amanda E. Rossiter, Yanina R. Sevastsyanovich, Mark Jeeves, Jessica L. Mason, Timothy J



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