Probing Protein Kinase-ATP Interactions Using a Fluorescent ATP Analog
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.
Key wordsKinase Nucleotide ATP binding CASK Pseudokinase TNP-ATP Fluorescence
The work was supported by startup funds to KM from VTCRI. Experiments were designed and performed by KM. The manuscript was conceived and written by LL, SS, and KM. KM and his laboratory are supported by R01EY024712 from NEI.
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