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FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK
Roland Abi Nahed1, Martin Pelosse1, Francesco Aulicino2
1Univ. Grenoble Alpes, INSERM U1055, Laboratory of Fundamental and Applied Bioenergetics (LBFA), 2280 Rue de la Piscine, Domaine Universitaire Gières, Grenoble, France.
Methods in Molecular Biology (Clifton, N.J.)
|February 24, 2025
Summary
AMP-activated protein kinase (AMPK) senses cellular energy levels. A new sensor, AMPfret, translates AMPK
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis.
- AMPK activation is triggered by increased cellular AMP/ATP and ADP/ATP ratios, indicating energy stress.
- AMPK activation involves a conformational change within its heterotrimeric complex.
Purpose of the Study:
- To develop and describe a novel AMPK-based sensor, AMPfret.
- To enable the analysis of direct AMPK activation by small molecules.
- To facilitate the estimation of cellular energy state changes.
Main Methods:
- Engineering of an AMPK-based sensor (AMPfret) utilizing Förster Resonance Energy Transfer (FRET).
- Measurement of AMPK activation in vitro using a fluorimeter.
- Assessment of cellular energy state changes using fluorescence and confocal microscopy.
Main Results:
- AMPfret translates the activating conformational switch of AMPK into a measurable fluorescence signal.
- The sensor allows for in vitro analysis of direct AMPK activation by small molecules.
- AMPfret can be used to estimate changes in cellular energy states.
Conclusions:
- AMPfret is a valuable tool for studying AMPK activation mechanisms.
- The sensor facilitates the discovery of novel AMPK modulators.
- AMPfret aids in understanding cellular responses to energy stress.
