Abstract
Here, we describe the crystallization protocol for AMPK, including protein production and purification. AMPK can be readily crystallized in the presence of PEG to give diffracting crystals to a resolution of between 2.5 and 3.5 Å using synchrotron radiation. This method allows for visualization of drugs or small molecules that bind to the ADaM site, CBS sites, ATP binding site, and the newly identified C2 binding sites in the γ-subunit via co-crystallization with phosphorylated AMPK (pT172) α2β1γ1 isoform or α2/1β1γ1 chimera. Drugs with binding affinities above 500 nM fail to co-crystallize with AMPK using these parameters.
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Acknowledgments
These authors are supported by grants from the National Health and Medical Research Council, the Australian Research Council, the Victorian Government Operational Infrastructure Support Scheme, and the Jack Brockhoff Foundation (JBF-4206, 2016), and C.G.L. is an E.H. Flack Research fellow. B.E.K. and J.S.O. are NHMRC and ARC Research fellows, respectively.
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Langendorf, C.G., Oakhill, J.S., Kemp, B.E. (2018). Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylated α2β1γ1 Heterotrimer. In: Neumann, D., Viollet, B. (eds) AMPK. Methods in Molecular Biology, vol 1732. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7598-3_2
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DOI: https://doi.org/10.1007/978-1-4939-7598-3_2
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