Structure-function characterisation of Chlamydia pneumoniae's major outer membrane protein

Amy Elizabeth Danson
2019
Chlamydia pneumoniae is a Gram negative bacterium responsible for a number of human respiratory diseases and linked to some chronic inflammatory diseases. The major outer membrane protein (MOMP) of Chlamydia is a conserved immunologically dominant protein located in the outer membrane, which together with its surface exposure and abundance, has led to MOMP being the main focus for vaccine and antimicrobial studies in recent decades. In the absence of structural data for MOMP, the fatty acid
more » ... sporter of E. coli, FadL, was used for the development of a plausible homology model, which suggested a 14-stranded b-barrel. X-ray crystallography has revealed a lowresolution crystal structure at 4 Å, from non-optimised crystals, that showed a β-barrel structure with an occluded pore. Initial research focussed on producing recombinant stable and active MOMP for higher resolution structural studies. In this work, MOMP's yield was greatly improved through the use of SB3-14 for solubilisation, identified through detergent screening using FSEC and Western blotting. Optimisation of the purification procedure led to crystallisation screening of DDM exchanged MOMP. The resulting crystals were analysed using a synchrotron radiation source. Diffraction patterns indicated that a small selection of crystals were protein, yielding new crystallisation conditions that can be optimised in future research. Confirmed as a β-barrel protein, MOMP is often considered to have a porin-like function. Homology modelling with FadL, as well as the low resolution structure, indicate an occluded barrel and is suggestive of a more active transport role for MOMP, namely for fatty acids which are known to be scavenged from the host. The ability of MOMP to transport fatty acids was assessed using a novel functional growth assay in E. coli, developed in this work. Although MOMP was shown to be expressed in the outer membrane, using fluorescence microscopy, interestingly, transport of fatty acids was not observed. However, MOMP has a more widely accepted [...]
doi:10.48683/1926.00084914 fatcat:zf4cm7aaxzethhg7bgys44hp74