Fellow: Jiajun Wang
Supervisor: Niels Fertig (Nanion Technologies Munich)
Co-Supervisor: Mathias Winterhalter (Jacobs University)
OmpF, OmpC from E. coli and their porin homologs from E. aerogenes, E. cloacae and K. pneumoniae are invested at single molecule level.. In my studies, 12 antibiotics including carbapenems, cephalosporins and penicillin groups of antibiotics are tested to study the biophysical properties of porin homologs and to serve the data base for the novel drug design. As one of the major cation in vivo, the effect of magnesium ions to the molecule-porin interaction has also been investigated profoundly Combining with structural information (St. Andrews) our data will be used in all atom modelling (Cagliari) to predict the pathway of antibiotic penetration.
Screening of antibiotic permeation through porins towards low throughput <100 recordings/day. Nanion developed a unique planar patch clamp chip used for the electrophysiological analysis of mammalian cells. Planar bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles or bacteria. This technique offers substantial advantages as compared to traditional patch clamp and BLM recording, in terms of facile handling and improved sensitivity. Particularly the enhanced sensitivity will improve the time resolution of the measurements.
First we did complementary measurements to ESR1 but on the chip. Reconstitution of MOMP/OMP50 and testing for a larger compound library.
The investigation of the penetration strategies of molecules through the bacteria cell membrane at the basis of influx and efflux is the aim of the whole project. In this study, the interactions of outer membrane proteins (OMPs) with antibiotic molecules were analyzed on the single molecular level. Omp35 and Omp36 from Enterobacter aerogenes are the porin homologs of OmpF and OmpC found in Escherichia coli and shares high similarities in gene sequence. In my studies, enrofloxacin, cefepime, imipenem gave different association rate between OmpF and its homologue Omp35. Magnesium ions also gave different modification to the interaction of norfloxacin with OmpC and its homologue Omp36. With the aid of crystallization and computerization, the function of residue groups and antibiotic interaction strategies would be addressed.
Instrument for bilayer study with improved sensitivity and time resolution designed by Nanion Technologies GmbH such as port-a-patch (solvent free), Orbit 16 (automatic bilayer station) and Orbit mini (with temperature control) have been used. The interaction between purified protein (OmpF) and antibiotics are recorded by bilayer stations (Orbit 16 and Orbit mini) and compared to BLM techniques. The results are published as Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms. The purified proteins are reconstituted with the planar lipid bilayer. The bilayer stations from Nanion technologies have great advantages to achieve rapid screening of bilayers at single molecule level. Along with the easy handling temperature system, electrophysiology study on antibiotic-porin interaction could be studied at host body temperature to step closer to the in vivo environment.
C. Weichbrodt, H. Bajaj, G. Baaken, J. Wang, S. Guinot, M. Kreir, J. C. Behrends, M. Winterhalter and N. Fertig, Analyst, 2015, DOI: 10.1039/C4AN02335H