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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.

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AMP-activated protein kinase (AMPK) senses cellular energy levels. A new sensor, AMPfret, translates AMPK

Keywords:
ADPAMPAMP-activated protein kinaseATPCellular energy stateFluorescence resonance energy transferGenetically encoded sensorsLarge cargo vectors

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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.