Membrane proteins constitute about 30 % of all prokaryotic and eukaryotic proteomes. They are responsible for ion conduction, chemical transport, energy conversion, signal transduction, hormone- and photo-reception, cell adhesion, and many other functions. Only a little more than 1% of all structures currently deposited in the Protein Data Bank are those of membrane proteins, i.e. the structural biology of membrane proteins still lags far behind that of soluble proteins. To fully understand their function, not ony the structure, but also the dynamics of membrane proteins should be known on multiple time-scales. Therefore, this project develops and uses tools of solution NMR spectroscopy for structure determination and dynamical characterization of membrane proteins in multiple lipid environments. Communication to the outside world occurs in Gram-negative bacteria through two membranes where the outer membrane forms the first barrier that nutrients and antibiotics have to cross to gain access to the cell. Pseudomonas aeruginosa is a serious Gram-negative pathogen with a particularly tough outer membrane that is difficult to penetrate by nutrients and antibiotics. Compared to E. coli, Pseudomonas harbors no general and only a relatively small number of specific porins in its outer membrane. In this project, we will determine the dynamic structure, substrate specificity, and the mechanism of transport of the P. aeruginosa OprG porin, which has been hypothesized to be responsible for the uptake of hydrophobic compounds and antibiotics. We will also determine the molecular interactions between OprH and lipopolysaccarides in the outer membrane that contribute to the mechanical strength, high antibiotic resistance, and strong tendency of P. aeruginosa to form biofilms in the lungs of pneumonia patients. Finally, using a combination of NMR and electrophysiology-based approaches, we will engineer the monomeric porin OmpG of E. coli into a practically useful and, compared to previous designs, superior nanopore platform for single molecule biosensing that may be used in the future to detect neurotransmitters, nucleotides, rare metals, or second messengers.
Infections by Gram-negative bacteria cause many difficult to treat diseases. For example, Pseudomonas aeruginosa is a clinically serious pathogen in hospital-acquired infections and the leading cause of death in patients with cystic fibrosis. Antibiotic resistance of P. aeruginosa is partially due to the extreme difficulty for drugs to penetrate its outer membrane. In this research, we will characterize the structures, functions, and drug interactions of three outer membrane proteins from P. aeruginosa and E. coli. The E. coli protein will also be developed into a novel nanopore platform for single molecule biosensing.
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