Fluorescent AMP-kinase biosensors
AssigneesUniversite Grenoble Alpes, European Molecular Biology Laboratory
InventorsImre Berger, Martin Pelosse, Uwe Schlattner
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Abstract
The present invention deals with heterotrimeric AMP-activated protein kinase (AMPK) comprising a fluorophore pair wherein the conformational change can be measured by FRET. It represents an advanced tool to screen and identify AMPK interactors in vitro and in cells in vivo. Such invention can also be considered as a reporter of the cellular energy status as it allows the spatiotemporal monitoring, in situ, of fluctuations in the ratio of AMP and ADP versus ATP.
Claims15
A heterotrimeric mammalian AMP-activated protein kinase (AMPK) construct having AMPK activity and the capacity of being allosterically activated, the construct comprising a complex consisting of a first component, a second component, and a third component that together provide the construct's AMPK activity and capacity of being allosterically activated, wherein the first component comprises a mammalian AMPK α-subunit or a mutant or fragment thereof, the second component comprises a mammalian AMPK β-subunit or a mutant or fragment thereof, and the third component comprises a mammalian AMPK γ-subunit or a mutant or fragment thereof, wherein the first component is tagged with a first fluorophore, and either the second component or third component is tagged with a second fluorophore such that conformational changes within the AMPK construct changes the Förster Resonance Energy Transfer between the first fluorophore and the second fluorophore.
A method for identifying an AMPK allosteric interactor in a sample, the method comprising the steps of:
- (a) contacting the sample with a heterotrimeric mammalian
- AMP-activated protein kinase (AMPK) construct having AMPK activity and the capacity of being allosterically activated, the construct comprising a complex consisting of a first component, a second component, and a third component that together provide the construct's AMPK activity and capacity of being allosterically activated, wherein the first component comprises a mammalian AMPK α-subunit or a mutant or fragment thereof, the second component comprises a mammalian AMPK β-subunit or a mutant or fragment thereof, and the third component comprises a mammalian AMPK γ-subunit or a mutant or fragment thereof, wherein the first component is tagged with a first fluorophore, and either the second component or third component is tagged with a second fluorophore such that conformational changes within the AMPK construct changes the Förster Resonance Energy Transfer between the first fluorophore and the second fluorophore;
- and
- (b) detecting, in the sample, a modification of the FRET fluorescence by fluorescence techniques;
- wherein the detecting of the modification of the FRET fluorescence in the sample indicates the presence of an AMPK allosteric interactor in the sample.
An ex vivo method of screening for an AMPK allosteric interactor, the method comprising the steps of:
- (a) providing a cell culture comprising cells expressing a heterotrimeric mammalian
- AMP-activated protein kinase (AMPK) construct having AMPK activity and the capacity of being allosterically activated, the construct comprising a complex consisting of a first component, a second component, and a third component that together provide the construct's AMPK activity and capacity of being allosterically activated, wherein the first component comprises a mammalian AMPK α-subunit or a mutant or fragment thereof, the second component comprises a mammalian AMPK β-subunit or a mutant or fragment thereof, and the third component comprises a mammalian AMPK γ-subunit or a mutant or fragment thereof, wherein the first component is tagged with a first fluorophore, and either the second component or third component is tagged with a second fluorophore such that conformational changes within the AMPK construct changes the Förster Resonance Energy Transfer between the first fluorophore and the second fluorophore;
- (b) providing candidate AMPK allosteric interactor;
- (c) contacting the cells in the cell culture with said candidate AMPK allosteric interactor;
- and
- (d) detecting a modification of the FRET fluorescence in the cells by fluorescent techniques;
- wherein detecting of the modification of the FRET fluorescence in the cells indicates that the candidate AMPK allosteric interactor is an AMPK allosteric interactor.
Dependencies are inferred from the claim text.