Biophysical and biochemical studies of the outer membrane proteins OprG and OprH from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common Gram-negative bacterium that can be found in many different environments. It is also an opportunistic human pathogen and the most common cause of lung infections in cystic fibrosis patients. P. aeruginosa infections are very challenging to treat as this bacterium displays high intrinsic resistance to a wide range of antibiotics. This is mainly caused by the low permeability of its outer membrane, which on the outer surface is densely packed with lipopolysaccharide molecules. The outer membrane also contains multiple embedded proteins, which provide the only effective passage for the bacterial cell to exchange matter with the environment. About 70 different outer membrane proteins transport a variety of compounds across the outer membrane of P. aeruginosa. Some of them have been studied in some detail, but the exact substrate specificity and the mechanism of transport of many of them remains unknown.
In this dissertation I present a detailed structural and functional characterization of two outer membrane proteins from P. aeruginosa – OprG and OprH. OprG is a member of the OmpW family of proteins whose function as an antibiotic-sensitive porin has been controversial and not well defined. Circumstantial evidence led to the proposal that OprG might transport hydrophobic compounds by using a lateral gate in the barrel wall thought to be lined by three conserved prolines. To test this hypothesis and to find the physiological substrates of OprG, the purified protein was reconstituted into liposomes and was found to facilitate the transport of small amino acids such as glycine, alanine, valine, and serine, which was confirmed by Pseudomonas growth assays. The structures of the wild-type protein and a critical proline mutant were determined by nuclear magnetic resonance spectroscopy in dihexanoyl-phosphatidylcholine micellar solutions. Both proteins formed eight-stranded β-barrels with flexible extracellular loops. The interfacial prolines did not form a lateral gate in these structures, but loop 3 exhibited restricted motions in the inactive P92A mutant, but not in wild-type OprG.
The function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. The NMR solution structure of OprH reveals an eight-stranded β-barrel with four extracellular loops of unequal size. Based on NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, I concluded that the interaction is predominantly electrostatic and localized to charged regions near upper rim of the barrel. By applying site-directed mutagenesis and ELISA I was able to identify OprH residues that are essential for the interaction with LPS. The results of this study provide a more definitive molecular model for the binding of LPS to OprH and offer new insight into protein-lipid interactions that likely contribute to the antibiotic resistance during P. aeruginosa infections.