Obiettivo 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. Campo scientifico natural sciencesbiological sciencesmicrobiologybacteriologynatural scienceschemical sciencesinorganic chemistryalkali metalsnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsmedical and health sciencesbasic medicinepharmacology and pharmacypharmaceutical drugsantibioticsnatural scienceschemical sciencesorganic chemistryamines Programma(i) H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions Main Programme H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility Argomento(i) MSCA-IF-2014-EF - Marie Skłodowska-Curie Individual Fellowships (IF-EF) Invito a presentare proposte H2020-MSCA-IF-2014 Vedi altri progetti per questo bando Meccanismo di finanziamento MSCA-IF-EF-ST - Standard EF Coordinatore THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD Contribution nette de l'UE € 183 454,80 Indirizzo WELLINGTON SQUARE UNIVERSITY OFFICES OX1 2JD Oxford Regno Unito Mostra sulla mappa Regione South East (England) Berkshire, Buckinghamshire and Oxfordshire Oxfordshire Tipo di attività Higher or Secondary Education Establishments Collegamenti Contatta l’organizzazione Opens in new window Sito web Opens in new window Partecipazione a programmi di R&I dell'UE Opens in new window Rete di collaborazione HORIZON Opens in new window Costo totale € 183 454,80
