Abstract
Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to understanding its biological function and role in a number of diseases related to energy metabolism including type 2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods for its crystallization in complex with small molecule activators. Quality control of the purified protein by functional and biophysical analysis was an essential part of the process enabling the achievement of crystals of the full-length protein capable of being used for high-resolution structure determination by X-ray diffraction. X-ray structures have been determined of both phosphorylated and non-phosphorylated forms of full-length human AMPK α1β1γ1.
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Acknowledgments
This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council and the Wellcome Trust. Julia Hubbard is a recipient of a Daphne Jackson Research Fellowship, which is funded by the Royal Society of Chemistry and the UK Medical Research Council. We greatly acknowledge Diamond Light Source for access to synchrotron time under proposal MX9826.
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Hubbard, J.A., Xiao, B., Wilson, J.R. (2018). Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1β1γ1). 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_1
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DOI: https://doi.org/10.1007/978-1-4939-7598-3_1
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