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Title: The Roles of Activation and Interaction of Essential Proteins in the β-Barrel Assembly Machinery in Escherichia coli
Authors: Adetunji, Funmi
Advisors: Silhavy, Thomas J.
Department: Molecular Biology
Class Year: 2016
Abstract: The outer membrane (OM) of Gram-negative bacteria serves as a robust permeability barrier, protecting the cell from toxic agents in the environment. The OM contains integral β-barrel proteins (OMPs), which are assembled by the β-barrel assembly machine (Bam) complex through an unknown mechanism. In Escherichia coli, the Bam complex consists of the essential OMP BamA and four associated lipoproteins BamBCDE, of which only BamD is essential. The fifth polypeptide transport-associated (POTRA 5) domain of BamA is required for stable interaction with BamCDE. Molecular dynamics simulations reveal a dynamic network of interchanging electrostatic interactions within POTRA 5, which may play an important role in BamA-BamD interaction and OMP assembly. Previously, two mutations in the POTRA 5 domain affecting the electrostatic network, bamAE373K and bamAR366A, were found to prevent BamAB-CDE complex formation in vivo. We report the characterization of mutations in residue K351 of POTRA 5 that alter the electrostatic network involving E373 and R366 and restore BamA-CDE interactions. By restoring interaction with BamD, the mutation bamAK351E suppresses the lethality, but not the OM permeability defects of bamAE373K and suppresses the growth and permeability defects of bamAR366A bamDR197L. In contrast, a previously identified suppressor of bamAR366A bamDR197L, bamAD362A, fails to restore BamA-BamD interaction, instead activating BamA through altering the POTRA 5 electrostatic network. These results indicate an important role for POTRA 5 electrostatic interactions in BamA activation and interaction with BamD. Our results further support a model for OMP assembly in which communication between BamA and BamD enables activation of both components, although the need for interaction can be bypassed through activating mutations in BamA and BamD.
Extent: 105 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Molecular Biology, 1954-2016

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