Division of Genetics, Brigham and Women's Hospital, Department of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland.
Curr Protoc. 2021 Jan;1(1):e14. doi: 10.1002/cpz1.14.
Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.
蛋白质标记策略已经被探索了几十年，用于研究蛋白质结构、功能和调节。蛋白质的荧光标记可通过微尺度热泳（MST）等生物物理方法研究蛋白质-蛋白质相互作用。MST 测量荧光标记的蛋白质对微观温度梯度的定向运动，并且可以使用蛋白质的热迁移率来确定结合亲和力。然而，荧光标记的化学计量和位点特异性难以控制，异质标记会导致结合测量的不准确性。在这里，我们描述了一种易于应用的方案，用于通过 NHS 酯将蛋白质的 N 末端进行高化学计量、位点特异性标记，作为通过 MST 测量蛋白质-蛋白质相互作用亲和力的一种手段。该方案包括 NHS 酯标记、荧光标记蛋白纯化和使用标记蛋白进行 MST 测量的指南。作为整个工作流程的一个例子，我们还提供了一个标记泛素 E3 酶并测试泛素 E2-E3 酶结合亲和力的方案。这些方法具有高度的适应性，可以扩展用于各种生物和生化情况下的蛋白质相互作用研究。 © 2021 Wiley Periodicals LLC. 基本方案 1：通过逐步反应在蛋白质的 N 末端用 NHS 酯进行标记 备选方案 1：通过一锅法在蛋白质的 N 末端用 NHS 酯进行标记 基本方案 2：纯化 N 末端荧光标记的蛋白质并确定其浓度和标记效率 基本方案 3：使用 MST 确定 N 末端荧光标记蛋白质与结合伴侣的结合亲和力。 备选方案 2：使用 MST 确定 N 末端荧光标记的蛋白质与结合伴侣的结合亲和力。 基本方案 4：泛素 E3 连接酶 WWP2 的 NHS 酯标记和通过 MST 结合测定法测量 WWP2 与 E2 连接酶之间的结合亲和力。
