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Vacariu, Condurache

University of British Columbia

The periplasmic pore protein AmpG, normally responsible for the transport of peptidoglycan fragments from the periplasm to the cytosol of Gram-negative bacteria, has been implicated in the biochemical pathway for AmpC β-lactamase induction. Consequently, strains of pathogenic Gram-negative bacteria such as Pseudomonas aeruginosa have developed resistance to a wide swath of antibiotics bearing the β-lactam functional group (i.e. antibiotics of the penicillin family). One approach to combat this growing threat would be to prevent the induction of AmpC’s in the target organisms before treating them with antibiotics. AmpG is ubiquitous and highly conserved among Gram-negative bacteria; it therefore serves as a potential drug target. Unfortunately, relatively little is known about AmpG’s structure and substrate preference owing to the difficulty in both the expression and isolation of this integral membrane protein. Working in collaboration with the Strynadka lab at UBC, who are experts in membrane protein characterization, we sought to elucidate the structure of AmpG – in particular, its glycopeptide substrates’ recognition site – in the hopes of eventually designing an irreversible inhibitor specific to it. Towards this end, we chemically synthesized multimilligram quantities of various putative AmpG substrates – including a large glycopeptide bearing the unnatural meso-oxa-diaminopimelic acid – for our collaborators to test against recombinant AmpG that was purified and reconstituted either in detergent micelles or in peptide nanodiscs. The affinities of our putative substrates towards AmpG are currently being evaluated via isothermal calorimetry. In addition, we synthesized a dendron-based putative inhibitor of AmpG in which a bulky “plug” is attached to an AmpG substrate via a flexible linker: this putative inhibitor will be tested using a lipid planar bilayer or fluorescence assay experiment. Finally, we synthesized a putative AmpG substrate with a compact photoaffinity tag as a probe for the substrate recognition site. We undertook in vitro photolabeling experiments on the AmpG provided to us – unfortunately, we were unable to specifically tag AmpG’s substrate recognition site using our probe.

Text

2024-07-22

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88674

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/