Mechanism of outer membrane protein assembly by the Bam complex
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CitationTomasek, David. 2020. Mechanism of outer membrane protein assembly by the Bam complex. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
Abstractβ-barrel integral membrane proteins perform important roles in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. In Gram-negative bacteria, the β-barrel assembly machine (Bam) complex accelerates the folding and membrane integration of these proteins. Structural and biochemical studies of the Bam complex have led to hypotheses of how BamA, the central component of the complex that is itself a β-barrel protein, may interact with substrates to promote their assembly. However, an understanding of how this molecular machine accelerates folding in the absence of an energy source remains unclear. Characterization of folding intermediates trapped on the Bam complex is needed to develop a physical picture for how catalysis occurs.
This thesis describes biochemical and structural experiments aimed at revealing how the Bam complex promotes the assembly of outer membrane proteins. In Chapter 2, we use mutant substrates that become trapped on the machine during folding to show that folding is catalyzed within the interior of the BamA β-barrel. In Chapter 3, we generate and characterize a larger series of Bam complex substrates that are trapped at different stages of folding. These experiments demonstrate that folding occurs in a stepwise fashion from the C-terminus to the N-terminus of the substrate, with early stages occurring outside the membrane and late stages occurring within the membrane. In Chapter 4, the ability to trap substrate folding intermediates is exploited to obtain a structure of a substrate-bound Bam complex by cryo-electron microscopy. This structure reveals a network of interactions between BamA and the substrate, suggesting that BamA templates substrate folding and that intramolecular interactions within the substrate trigger its release once folding has finished.
These results provide insight into how the Bam complex assembles its substrates in an energy-independent manner. The findings here may guide further structural studies with substrate folding intermediates and may facilitate development of antibiotics that target Gram-negative pathogens by preventing proper function of the Bam complex.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365985
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