LaConte Leslie E W, Srivastava Sarika, Mukherjee Konark
Virginia Tech Carilion Research Institute, Roanoke, 2 Riverside Circle, Roanoke, VA, 24016, USA.
Department of Biological Sciences, Virginia Tech, Blacksburg, VA, 24060, USA.
Methods Mol Biol. 2017;1647:171-183. doi: 10.1007/978-1-4939-7201-2_11.
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.
真核蛋白激酶是一类受到深入研究的酶，因其作为药物靶点的实用性而备受关注。确定ATP与蛋白激酶结合的调控不仅对于理解细胞环境中的功能至关重要，而且对于设计激酶特异性分子抑制剂也很关键。在此，我们提供了一种表征ATP与真核蛋白激酶结合的通用方法。该方案可用于确定特定激酶被激活的条件。该方法简单，仅需一种荧光ATP类似物，如TNP-ATP或MANT-ATP，以及一台监测荧光变化的仪器。尽管激酶与给定ATP类似物之间的相互作用动力学可能与天然ATP不同，但该检测方法操作简便且易于解读，弥补了这一缺点。重要的是，它可以进行优化，以探究激酶-核苷酸结合可能受到影响的各种条件。
