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Author:
Date:
2014-06-05
[Abstract] This protocol describes a robust technique for the measurement of pseudokinase-ligand interaction by a fluorescence-based Thermal Shift Assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulators of signal transduction and may therefore represent a novel class of drug targets. Unlike kinases, the catalytic efficiency of pseudokinases is rather poor or non existent, making it difficult to dissect the function of their nucleotide binding sites. Thermal denaturation-based methods have proven to be a powerful method for determining ligand binding capacity ...
[摘要] 该协议描述了通过基于荧光的热漂移测定（TSA）测量假激酶 - 配体相互作用的鲁棒技术。假激酶是激酶样蛋白，其最近作为信号转导的关键调节剂出现，因此可以代表一类新型的药物靶标。与激酶不同，假激酶的催化效率相当差或不存在，使得难以解析其核苷酸结合位点的功能。已证明基于热变性的方法是确定与纯化的假激酶的配体结合能力的有力方法，并且可以告知核苷酸结合位点的潜在可药物性。该测定利用了当荧光染料（在这种情况下为SYPROOrange）与当蛋白质经历热变性时暴露的疏水性贴片结合时产生的荧光变化。已知配体结合蛋白质增加其热稳定性，其通过在未配对蛋白质和配体蛋白质之间的热变性曲线中观察到的移动来反映。这一广义协议也可以适合于其他蛋白家族。此外，基于热变性的方法可用于鉴定可提高蛋白质稳定性的最佳缓冲液条件
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