Jaijun Wang successfully defended his PhD at Jacobs University on May 18, 2017
A facilitated Method to Characterize Rapid Substrate Binding to Membrane Channels
The bacterial outer membrane porins are regarded as one of the main pathways for hydrophilic antibiotic molecules to penetrate through the bacterial outer membrane. To understand the interaction mechanisms of antimicrobial molecules with the porins, the latter are extracted from their natural environment, reconstituted into free standing artificial lipid bilayer and characterized by electrophysiology. A broad range of general diffusion porins, i.e. OmpF and OmpC from E. coli, and their orthologs Omp35, Omp36 from E. aerogenes; OmpK35, OmpK36 from K.pneumoniae; OmpE35, OmpE36 from E. cloacae were characterized to study the biophysical properties of porins in different bacterial species. Electrophysiology together with MIC (Minimum Inhibitory Concentration) determination, high-resolution protein crystal structure and all atom MD (Molecular Dynamic) simulation were used to study the interaction mechanisms of ten ß-lactam molecules. A further research question was about the effect of naturally abundant divalent ions like calcium and magnesium on fluoroquinolone molecules. Although these molecules easily chelate in presence of divalent ions, the chelation appears to be unstable at the porin lumen and the single fluoroquinolone follow the electrostatic properties at the porin lumen.
Within this thesis we have in particular applied a relatively rapid screening approach by means of membranes-on-chip. This provides the potential to study the molecule-porin interaction at single molecule level in a higher throughput manner. β-lactamase inhibitors, though have little intrinsic antibacterial activity, inhibit the activity of massive plasmid-mediated β-lactamases. These agents are normally dosed in combination with β-lactams to tackle with MDR (Multi-Drug Resistant) bacteria. We found that the combinations of β-lactam and β-lactamase inhibitor combination gave a lower interaction rate than pure ß-lactam substrates via electrophysiology characterization.
With respect to data analysis of our approach we need to break the data acquisition limitation (10 kHz). We introduced a physical model dedicated to extract the signal out of noise up to 2 MHz for the interaction between OmpF and meropenem at 40 oC. A software analysis package with user interface has been developed for the easy proceeding of the bilayer electrophysiology recordings.