School of Life Science, Northeast Agricultural University, Xiangfang District, Harbin 150030, Heilongjiang, China.
Sensors (Basel). 2013 Apr 8;13(4):4553-70. doi: 10.3390/s130404553.
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach.
Förster 共振能量转移(FRET)技术已广泛应用于生物和生物医学研究。这种强大的工具可以阐明无论是在动态还是稳态下的蛋白质相互作用。我们最近开发了一系列基于 FRET 的技术来确定蛋白质相互作用的离解常数,并用于 SUMOylation 的高通量筛选测定。SUMO(小泛素样修饰物)通过酶级联反应与底物结合。这种重要的翻译后蛋白质修饰对多种生物过程至关重要。Sentrin/SUMO 特异性蛋白酶(SENPs)作为内切酶来处理前 SUMO,或作为异肽酶从其底物上除去 SUMO。在这里,我们描述了一种用于确定 SENP1 动力学的新型定量 FRET 蛋白酶测定法。我们的策略基于在 FRET 受体发射波长处荧光发射信号的定量分析和区分。这些荧光发射信号由三个组成部分组成:FRET 信号以及供体(CyPet)和受体(YPet)的荧光发射。与我们之前的方法不同,该方法通过标准曲线排除供体和受体的直接发射,而是在 SENP 消化过程中从一个样品中定量确定这三个荧光发射。开发了新的数学算法,可直接从 FRET 信号和供体/受体直接发射中确定消化底物的浓度。衍生出 SENP1 催化结构域对预 SUMO1/2/3 的催化参数 kcat、KM 和催化效率(kcat/KM)。重要的是,这种基于 FRET 的蛋白酶动力学测定的新型定量方法的一般原理可以应用于其他蛋白酶,以稳健和系统生物学的方法。