NMR studies of the BamA complex proteins at high resolution

The β-barrel assembly machinery (BAM) complex is essential for the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria, with the membrane protein BamA acting as a catalyst for folding of OMPs in the outer membrane. Recently, structures of the BAM complex have been solved, displaying the molecular organization of the five proteins of the complex (BamABCDE). However, the mechanism by which BamA completes its insertase role is unclear. This PhD thesis focuses on the optimization of sample preparation and backbone assignment of the BamA β-barrel domain for solution NMR spectroscopy. Initial NMR spectra of the BamA β-barrel showed broad peaks with a low signal-to-noise ratio. This was likely due to a dynamic nature of the gate-region, as revealed by cysteine-scanning experiments. Therefore, as a first step to obtain a sample of BamA β-barrel suitable for NMR spectroscopy, buffer conditions were optimized. Then, in order to reduce the dynamics of the BamA β-barrel, a construct was designed with a C-terminal extension by nine residues. In addition to this extension, the G433A mutation in the gate-region was identified as to improve the quality of the NMR spectra. At that point, a combination of specific isotopic labeling and unlabeling in auxotrophic strains, triple-resonance experiments and 3D NOESY experiments allowed to obtain sequence-specific NMR resonance assignments of a large portion of the BamA β-barrel in LDAO micelles. The assignments revealed that some residues of the BamA β-barrel were found in different conformations that can be stabilized by the formation of a disulfide bond or by the C-terminal extension. Moreover, the crystal structure of the extended BamA β-barrel was determined, revealing a longer, and therefore more stable β-strand formed between the first and last strand of the barrel, explaining the stabilizing effect observed in its NMR spectrum. Additional work was performed on the soluble proteins of the BAM complex (BamBCDE). The expression and purification of BamB, BamC BamD and BamE was optimized and NMR spectra were recorded. BamD was found to be unstable once purified, and quickly precipitated, preventing to reach a molar concentration suitable for NMR spectroscopy. As a way to circumvent this issue, a hybrid construct of BamCD was prepared. The protein was able to reach high concentrations while keeping its stability. The fingerprint spectrum of BamCD was recorded and the peaks belonging to BamD overlapped with the peaks measured from a sample of individual BamD. This stabilized sample opens the possibility to obtain the sequence-specific assignments of BamD. Overall, this work resulted in the assignment of a large portion of the BamA β-barrel. As BamA is a potential target for new antibiotics, this assignment opens a way to perform NMR studies on BamA with substrates and ligands and understand the mechanical implications of their binding. Additionally, the dynamic nature of the BamA β-barrel was demonstrated by observing multiple conformations with solution-state NMR spectroscopy. In combination with the available assignment, it will be possible to observe the effects of binding molecules, mutations, or of the molecular environment on the conformational ensemble of BamA.