Chapter title |
Probing Protein Kinase-ATP Interactions Using a Fluorescent ATP Analog
|
---|---|
Chapter number | 11 |
Book title |
Proteomics for Drug Discovery
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7201-2_11 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7200-5, 978-1-4939-7201-2
|
Authors |
Leslie E. W. LaConte, Sarika Srivastava, Konark Mukherjee |
Abstract |
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. |
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