Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding
Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding
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DOI：
10.1038/nmeth740
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发表时间：
2005-03-01
期刊：
影响因子：
48
通讯作者：
Marqusee, S
中科院分区：
文献类型：
--
作者：
Park, CW;Marqusee, S
热力学稳定性是蛋白质生物学的基础。有关蛋白质稳定性的信息对于研究蛋白质的结构和折叠是必不可少的，也可以间接用于监测蛋白质-配体或蛋白质-蛋白质的相互作用。虽然蛋白质稳定性的实验测定显然很有价值，但通常需要生物物理仪器和大量纯化的蛋白质，这限制了这项技术作为一般实验室方法的使用。我们报道了一种用不同浓度变性剂的脉冲蛋白水解法测定蛋白质稳定性的简单新方法。脉冲蛋白水解法被设计为只消化折叠和未折叠蛋白质的平衡混合物中的未折叠蛋白质，该混合物的松弛时间长于蛋白质降解脉冲。我们用这种方法研究了大肠杆菌核糖核酸酶H及其变异体的稳定性，包括纯化形式和直接从细胞裂解物中提取的。用该技术测得的DeltaG(Unf)度值与传统方法测得的值一致。我们还成功地利用这种方法来监测麦芽糖结合蛋白与麦芽糖的结合，以及快速筛选该蛋白的同源配体。脉冲蛋白质组分的简单性表明，在蛋白质工程和药物开发应用中，脉冲蛋白质组分是高通量测定蛋白质稳定性的极佳策略。
Thermodynamic stability is fundamental to the biology of proteins. Information on protein stability is essential for studying protein structure and folding and can also be used indirectly to monitor protein-ligand or protein-protein interactions. While clearly valuable, the experimental determination of a protein's stability typically requires biophysical instrumentation and substantial quantities of purified protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining protein stability by using pulse proteotysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The DeltaG(unf)degrees values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding protein to maltose, as well as to rapidly screen cognate ligands for this protein. The simplicity of pulse proteotysis suggests that it is an excellent strategy for the high-throughput determination of protein stability in protein engineering and drug discovery applications.