Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis

Hopkins, Alex Hunt

Graduate Thesis Or Dissertation

Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/jd472w56b

Abstract

The outer membranes of Gram-negative bacteria are essential to their survival and have a unique asymmetric structure that features an outer leaflet of lipopolysaccharide. Bacterial outer membranes are populated by integral membrane proteins with amphipathic transmembrane β-barrel domains called outer membrane proteins (OMPs). OMPs are translated in the cytoplasm and translocated across the inner membrane and periplasm before being inserted into the outer membrane. The β-barrel Assembly Machine (BAM), a hetero-pentameric protein complex at the outer membrane, recognizes, folds, and releases OMPs into the membrane. However, the functional contributions of individual BAM subunits, the architecture of the BAM, and the molecular mechanism by which the BAM recognizes and folds its substrates are unknown. To test whether the non-essential BAM subunits have OMP-specific functions or function generally in OMP biogenesis, the outer membrane proteomes of BAM subunit knockout strains were compared to wild-type via mass spectrometry. The general decrease of OMP abundance observed in the ΔbamB strain and very minor effects observed in the ΔbamC and ΔbamE strains suggest they play general roles in OMP biogenesis. To better understand the complex interface and provide a structural model of the entire BAM, the structural characterization of a BamA-BamD fusion protein was undertaken and the observed crystallographic interface validated in vivo. The model of BamABCD revealed a periplasmic ring formed by BamD and the POTRA domains of BamA that is proposed to bind nascent OMPs prior to membrane insertion. To enable experiments to analyze OMP biogenesis in vivo, OMP translocation intermediates were generated and characterized by periplasmic cysteine modification and OMP folding assays. To show that these intermediates can report on OMP biogenesis events in the periplasm, the intermediates were further characterized via site-specific photo-activatable cross-linking. The OMP translocation intermediates yielded site-specific cross-links that are modulated by both the location of the cross-linker in the β-barrel and the length of a linker domain that controls how far the β-barrel domain can extend across the periplasm. Therefore, the OMP translocation intermediates are a tool that can be used to track OMP biogenesis in vivo, test proposed OMP targeting mechanisms, and capture OMP folding intermediates.

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