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Biophysical Interactions of Direct AMPK Activators
Ravi G Kurumbail1, Graham M West2, Venkatasubramanian Dharmarajan3
1Worldwide Research and Development, Pfizer Inc., Groton, CT, USA. ravi.g.kurumbail@pfizer.com.
Methods in Molecular Biology (Clifton, N.J.)
|February 27, 2018
Summary
This study details methods for generating AMP-activated protein kinase (AMPK) and analyzing its interactions with activators using biophysical techniques. These methods provide insights into enzyme activation mechanisms and ligand binding.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Protein-ligand interactions are crucial for biological processes.
- Understanding AMP-activated protein kinase (AMPK) function requires detailed characterization of its interactions with activators.
- Various biophysical methods can be employed to study these interactions.
Purpose of the Study:
- To describe experimental methods for generating AMPK protein reagents.
- To characterize the interactions between AMPK and its direct synthetic activators.
- To elucidate the mechanism of enzyme activation by activators.
Main Methods:
- Recombinant heterotrimeric AMPK complexes expressed in insect cells or Escherichia coli.
- Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to probe conformational changes and binding sites.
- X-ray crystallography to determine near-atomic resolution structures of AMPK with bound ligands.
- Enzyme kinetic studies using radioactive 33P-ATP-based filter assay to assess kinetic parameters (Km, Vmax).
- Surface Plasmon Resonance (SPR) to determine equilibrium dissociation constants (KD) and ligand binding kinetics (on/off rates).
Main Results:
- Successful generation of recombinant heterotrimeric AMPK complexes.
- HDX-MS identified conformational changes and potential activator binding sites on AMPK.
- X-ray crystallography revealed detailed molecular interactions between AMPK activators and the enzyme.
- Enzyme kinetics demonstrated the effects of activators on kinetic parameters.
- SPR provided quantitative data on ligand binding affinity and kinetics.
Conclusions:
- A comprehensive set of biophysical methods has been established for studying AMPK and its activators.
- These methods provide multi-faceted insights into the molecular mechanisms of AMPK activation.
- The characterized interactions and kinetic parameters are vital for understanding AMPK's role in cellular energy homeostasis.
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