Fluorescent AMP-Kinase Biosensors
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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.
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Citations
49 Claims
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1-29. -29. (canceled)
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30. :
- A heterotrimeric AMP-activated protein kinase (AMPK) construct comprising;
an AMPK α
-subunit or a mutant or fragment thereof,an AMPK β
-subunit or a mutant or fragment thereof,an AMPK γ
-subunit or a mutant or fragment thereof, anda fluorescent dye pair tagging at least one of said α
-subunit, β
-subunit, γ
-subunit, mutants or fragments, said fluorescent dye pair being placed to allow detection of conformational changes within the AMPK construct. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
- A heterotrimeric AMP-activated protein kinase (AMPK) construct comprising;
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48. :
- A method for identifying an AMPK allosteric interactor in a sample, the method comprising the steps of;
(a) contacting the sample with a heterotrimeric AMPK construct comprising; an AMPK α
-subunit or a mutant or fragment thereof,an AMPK β
-subunit or a mutant or fragment thereof,an AMPK γ
-subunit or a mutant or fragment thereof, anda fluorescent dye pair tagging at least one of said α
-subunit, β
-subunit, γ
-subunit, mutants or fragments, said fluorescent dye pair being placed to allow detection of conformational changes within the AMPK construct; 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.
- A method for identifying an AMPK allosteric interactor in a sample, the method comprising the steps of;
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49. :
- 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 AMPK construct comprising; an AMPK α
-subunit or a mutant or fragment thereof,an AMPK β
-subunit or a mutant or fragment thereof,an AMPK γ
-subunit or a mutant or fragment thereof, anda fluorescent dye pair tagging at least one of said α
-subunit, β
-subunit, γ
-subunit, mutants or fragments, said fluorescent dye pair being placed to allow detection of conformational changes within the AMPK construct;(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.
- An ex vivo method of screening for an AMPK allosteric interactor, the method comprising the steps of;
Specification