#	Pagina
attuale pagina	/open-h2020/projects/195902/index.html

Opendata, web and dolomites

FLUOROKEF

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

Total Cost €

EC-Contrib. €

Partnership

Views

Outcomes and
results

FLUOROKEF project word cloud

Explore the words cloud of the FLUOROKEF project. It provides you a very rough idea of what is the project "FLUOROKEF" about.

monitor codon antibiotics electrophilic hypothesis coli denitrificans inside f448tfmf nmr shewanella conformation kd protects efflux employ cell soluble terminal f441 f448 data probe molecular indicates maintaining residue physiologically domain homeostasis sdkefctd allowed diffusion detrimental kirby lack escherichia crystallographic cells compounds affinities screen mechanism activators potassium amber tfmf vital function truncated conserved assays ray biophysical stop gain understand binding drugs library generation wish sdkef extend construct 441 dynamics employed vitro trifluromethyl replace bauer length phenylalanine disc antibiotic kef transformants cytoplasm kill inhibitors bacteria cellular direct dynamic channel quantitatively full 19f activation ligands acidification eckefctd

Project "FLUOROKEF" data sheet

The following table provides information about the project.

Coordinator	THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD Organization address address: WELLINGTON SQUARE UNIVERSITY OFFICES city: OXFORD postcode: OX1 2JD website: www.ox.ac.uk contact info title: n.a. name: n.a. surname: n.a. function: n.a. email: n.a. telephone: n.a. fax: n.a.
Coordinator Country	United Kingdom [UK]
Total cost	183˙454 €
EC max contribution	183˙454 € (100%)
Programme	1. H2020-EU.1.3.2. (Nurturing excellence by means of cross-border and cross-sector mobility)
Code Call	H2020-MSCA-IF-2014
Funding Scheme	MSCA-IF-EF-ST
Starting year	2015
Duration (year-month-day)	from 2015-10-01 to 2017-09-30

Partnership

Take a look of project's partnership.

#	participants	country	role	EC contrib. [€]
1	THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD Organization address address: WELLINGTON SQUARE UNIVERSITY OFFICES city: OXFORD postcode: OX1 2JD website: www.ox.ac.uk contact info title: n.a. name: n.a. surname: n.a. function: n.a. email: n.a. telephone: n.a. fax: n.a.	UK (OXFORD)	coordinator	183˙454.00

Map

Project 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.

Publications

List of publications.
year	authors and title	journal	last update
2017	Christos Pliotas, Samuel C. Grayer, Silvia Ekkerman, Anthony K. N. Chan, Jess Healy, Phedra Marius, Wendy Bartlett, Amjad Khan, Wilian A. Cortopassi, Shane A. Chandler, Tim Rasmussen, Justin L. P. Benesch, Robert S. Paton, Timothy D. W. Claridge, Samantha Miller, Ian R. Booth, James H. Naismith, Stuart J. Conway Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System published pages: 4219-4234, ISSN: 0006-2960, DOI: 10.1021/acs.biochem.7b00300	Biochemistry 56/32	2019-06-18

Are you the coordinator (or a participant) of this project? Plaese send me more information about the "FLUOROKEF" project.

For instance: the website url (it has not provided by EU-opendata yet), the logo, a more detailed description of the project (in plain text as a rtf file or a word file), some pictures (as picture files, not embedded into any word file), twitter account, linkedin page, etc.

Send me an email (fabio@fabiodisconzi.com) and I put them in your project's page as son as possible.

Thanks. And then put a link of this page into your project's website.

The information about "FLUOROKEF" are provided by the European Opendata Portal: CORDIS opendata.