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Incorporation of unnatural fluorinated amino acids to probe the function of the bacterial efflux system Kef in a cellular setting.

Objective

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Its vital role in maintaining cell homeostasis makes Kef a promising target for antibiotics, but such drugs can be developed only with a molecular understanding of Kef activation. Our work indicates that phenylalanine residue 441 (F441) in Escherichia coli is crucial for the activation of K+ efflux. This mechanism is conserved, and F448 of Shewanella denitrificans Kef (SdKef) has the same function. This work employed X-ray crystallographic studies using a truncated construct of the E. coli Kef soluble C-terminal domain (EcKefCTD) and biophysical studies conducted on the SdKef C-terminal domain (SdKefCTD). While the crystallographic studies have been essential in hypothesis generation, they lack the dynamic aspect that is required to understand the mechanism of Kef activation. Although the use of a truncated construct has allowed development of biophysical assays, we wish to extend our work to focus on the full-length channel. To achieve these aims we will employ whole cell 19F NMR to probe the function of Kef.

To determine if 19F NMR can be used to monitor F448 conformation, we will employ amber stop codon technology to replace F448 of SdKef with 4-trifluromethyl-L-phenylalanine (tfmF). We will monitor the dynamics of F448tfmF by 19F NMR upon binding of activators and inhibitors, using our library of SdKef ligands. To gain an understanding of Kef activation in cells, we will quantitatively determine binding affinities of the Kef ligands to SdKef(F448tfmF), in E. coli transformants using in-cell 19F NMR. This direct determination of binding inside the cells will be more physiologically relevant than the in vitro KD data. To assess the antibiotic activity of compounds that show cellular activity against Kef, we will employ the Kirby-Bauer disc diffusion method to screen for compounds that can kill bacteria.

Call for proposal

H2020-MSCA-IF-2014
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Funding Scheme

MSCA-IF-EF-ST - Standard EF
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Coordinator

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
Address
Wellington Square University Offices
OX1 2JD Oxford
United Kingdom
Activity type
Higher or Secondary Education Establishments
EU contribution
€ 183 454,80