Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa. Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE. Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å. / Chemistry and Chemical Biology

β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli , the β-barrel component of the OM LPS translocon is one of only two essential β-barrels, the other being the central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion into the OM by the Bam machinery. An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery. Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE templates the folding of LptD. / Chemistry and Chemical Biology