Visualizing AMPK drug binding sites through crystallization of full-length phosphorylated α2β1γ1 heterotrimer
Book chapter
Langendorf, Christopher G., Oakhill, Jonathan S. and Kemp, Bruce E.. (2018). Visualizing AMPK drug binding sites through crystallization of full-length phosphorylated α2β1γ1 heterotrimer. In In D. Neumann and B. Viollet (Ed.). AMPK: Methods and protocol pp. 15 - 27 Humana Press, Inc.. https://doi.org/10.1007/978-1-4939-7598-3_2
Authors | Langendorf, Christopher G., Oakhill, Jonathan S. and Kemp, Bruce E. |
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Editors | D. Neumann and B. Viollet |
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. |
Keywords | AMPK heterotrimer; protein crystallization; allosteric activation; recombinant protein expression and purification |
Page range | 15 - 27 |
Year | 2018 |
Book title | AMPK: Methods and protocol |
Publisher | Humana Press, Inc. |
Place of publication | United States of America |
Series | Methods in molecular biology |
ISBN | 9781493975976 |
Digital Object Identifier (DOI) | https://doi.org/10.1007/978-1-4939-7598-3_2 |
Scopus EID | 2-s2.0-85042697528 |
Research Group | Mary MacKillop Institute for Health Research |
Publisher's version | File Access Level Controlled |
https://acuresearchbank.acu.edu.au/item/87619/visualizing-ampk-drug-binding-sites-through-crystallization-of-full-length-phosphorylated-2-1-1-heterotrimer
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