Date of Award

Spring 1-1-2018

Document Type


Degree Name

Doctor of Philosophy (PhD)

First Advisor

Marcelo C. Sousa

Second Advisor

Natalie Ahn

Third Advisor

Joseph Falke

Fourth Advisor

Deborah Wuttke

Fifth Advisor

Shelley Copley


BamA is an essential component of the β-­‐barrel assembly machine that folds and inserts outer membrane proteins (OMPs) into the outer membrane of Gram-­‐negative bacteria. BamA is an OMP itself comprised of two domains, a soluble periplasmic N-­‐terminal domain consisting of five polypeptide transport associated (POTRA) repeats and a C-­‐terminal 16-­‐stranded β-­‐barrel domain. The mechanism of BamA is poorly understood but structural data suggests BamA is a dynamic protein with respect to the POTRA domains and barrel. The POTRA domains, numbered 1-5 from the N- to C- termini, can be divided into two sub-domains formed by POTRAs 1-2 and 3-5 connected by a flexible linker between POTRAs 2-3. The flexibility between POTRAs 2-3 allows the POTRA domains to adopt a range of conformations from extended to bent. To determine if flexibility at POTRA 2-3 is important for BamA function, we introduced two sets of cysteine pairs in POTRA 2 and 3 such that formation of a disulfide bond would restrict flexibility and stabilize the POTRAs in an extended or bent conformation. Using a cell viability assay, we found that both sets of cysteine mutants impaired BamA function, indicating flexibility in this region is important.

Structural data has indicated that the barrel seam of BamA formed by strands β1 and 16 is destabilized. This has lead to a mechanistic hypothesis in which the seam constitutes a lateral gate that may open to accept β-hairpins from a nascent OMP such that a new barrel can bud into the membrane. Consistent with this, disulfide cross-linking to lock β1-16 is lethal in E. coli. Here, we tested the ability of BamA to catalyze folding of a model OMP, OmpX into liposomes when β1-16 are disulfide cross-linked and found that it does not affect BamA activity. We also demonstrated via disulfide cross-linking that β1-16 are highly dynamic, able to undergo shifts in register more than ~14Å up and down the plane of the membrane in liposomes as well as cells. We concluded that BamA does not use a barrel budding mechanism in vitro but likely folds OMPs through a series of parallel pathways in vivo.