Eukaryotic protein kinases are an intensely investigated class of enzymes which have garnered attention due to their usefulness as drug targets. Determining the regulation of ATP binding to a protein kinase is not only critical for understanding function in a cellular context but also for designing kinase-specific molecular inhibitors. Here, we provide a general procedure for characterizing ATP binding to eukaryotic protein kinases. The protocol can be adapted to identify the conditions under which a particular kinase is activated. The approach is simple, requiring only a fluorescent ATP analog such as TNP-ATP or MANT-ATP and an instrument to monitor changes in fluorescence. Although the interaction kinetics between a kinase and a given ATP analog may differ from that of native ATP, this disadvantage is offset by the ease of performing and interpreting this assay. Importantly, it can be optimized to probe a large variety of conditions under which the kinase-nucleotide binding might be affected.
CITATION STYLE
LaConte, L. E. W., Srivastava, S., & Mukherjee, K. (2017). Probing protein kinase-ATP interactions using a fluorescent ATP analog. In Methods in Molecular Biology (Vol. 1647, pp. 171–183). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7201-2_11
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