Mutational and Topological Analysis of the Escherichia coli BamA Protein
| Autor: | Ian R. Henderson, Mark Jeeves, Michael Overduin, Sophie A. Matthews, Douglas F. Browning, Timothy J. Wells, Amanda E. Rossiter, Timothy J. Knowles, Catherine A. Wardius, Jessica L. Mason, Vassiliy N. Bavro, Adam F. Cunningham, Yanina R. Sevastsyanovich |
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| Rok vydání: | 2013 |
| Předmět: |
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Molecular Blotting Western Fluorescent Antibody Technique lcsh:Medicine Sequence alignment Linker-Scanning Mutagenesis Biology medicine.disease_cause Protein structure Bama Escherichia coli medicine lcsh:Science Multidisciplinary Escherichia coli Proteins lcsh:R Periplasmic space Protein Structure Tertiary Cell biology Biochemistry Membrane protein Mutagenesis lcsh:Q Bacterial outer membrane Research Article Bacterial Outer Membrane Proteins Plasmids |
| Zdroj: | PLoS ONE, Vol 8, Iss 12, p e84512 (2013) PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0084512 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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