Park Chiwook, Marqusee Susan
Purdue University, West Lafayette, Indiana.
University of California, Berkeley, California.
Curr Protoc Protein Sci. 2006 Dec;Chapter 20:20.11.1-20.11.14. doi: 10.1002/0471140864.ps2011s46.
Pulse proteolysis exploits the difference in proteolytic susceptibility between folded and unfolded proteins for facile but quantitative determination of protein stability. The method requires only common biochemistry and molecular biology lab equipment. Pulse proteolysis also can be used to determine the affinity of a ligand to its protein target by monitoring the change in protein stability upon ligand binding. The Basic Protocol describes the detailed procedure for determining protein stability using pulse proteolysis. For pulse proteolysis to be used for determining a protein's stability, the protein should not be digested significantly by pulse proteolysis when it is in the folded conformation. The Support Protocol describes a procedure for determining whether a protein satisfies this requirement. The principles of protein stability determination using denaturant and pulse proteolysis are also discussed.
脉冲蛋白酶解利用折叠态和未折叠态蛋白质在蛋白酶解敏感性上的差异，以简便且定量的方式测定蛋白质稳定性。该方法仅需常规的生物化学和分子生物学实验室设备。脉冲蛋白酶解还可通过监测配体结合后蛋白质稳定性的变化，用于测定配体与其蛋白质靶点的亲和力。基本方案描述了使用脉冲蛋白酶解测定蛋白质稳定性的详细步骤。要使用脉冲蛋白酶解来测定蛋白质的稳定性，该蛋白质在折叠构象时不应被脉冲蛋白酶解显著消化。支持方案描述了一种确定蛋白质是否满足此要求的程序。还讨论了使用变性剂和脉冲蛋白酶解测定蛋白质稳定性的原理。
