Fluorescence Lifetime Phasor Analysis of the Decamer-Dimer Equilibrium of Human Peroxiredoxin 1.
Fluorescence Lifetime Phasor Analysis of the Decamer-Dimer Equilibrium of Human Peroxiredoxin 1.
复制标题
DOI：
10.3390/ijms23095260
复制
发表时间：
2022-05-09
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白质自组装是生物学中的一个共同特征，通常是无数基本过程所必需的，如酶活性、信号转导、溶质跨膜运输等。有几种技术可以发现和评估蛋白质中同质寡聚物的形成。当然，所有这些方法都有其局限性，这意味着至少需要两种或更多不同的方法来描述案例研究。在此，我们提出了一种新的方法来研究蛋白质与相量的内在荧光寿命。在这种情况下，该方法被用于确定人过氧化物酶1 （hPrx1）的平衡解离常数（KD）， hPrx1是一种高效的半胱氨酸依赖过氧化物酶，具有由五个头对尾非共价排列在十聚体中的四元结构。hPrx1寡聚状态不仅影响其活性，而且影响其与其他蛋白质的结合。hPrx1的激发态寿命在高浓度和低浓度下有不同的值，表明存在两种不同的物质。hPrx1发射寿命的相量分析允许在不同蛋白质浓度下对hPrx1十聚体、二聚体及其混合物进行鉴定和定量。利用相量代数计算了不同浓度下hPrx1十聚体的比例，得到了十聚体-二聚体平衡的KD （1.1 × 10−24 M4）和C0.5 （1.36 μM）值。结果与粒径排除色谱法进行了验证和比较。此外，光谱相量提供了类似的结果，尽管发射光谱作为hPrx1浓度的函数差异很小。相量方法被证明是一种高度敏感和定量的方法来评估蛋白质寡聚化，是生物物理学家工具箱中一个有吸引力的补充。
Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitations, meaning that at least two or more different approaches are needed to characterize a case study. Herein, we present a new method to study protein associations using intrinsic fluorescence lifetime with phasors. In this case, the method is applied to determine the equilibrium dissociation constant (KD) of human peroxiredoxin 1 (hPrx1), an efficient cysteine-dependent peroxidase, that has a quaternary structure comprised of five head-to-tail homodimers non-covalently arranged in a decamer. The hPrx1 oligomeric state not only affects its activity but also its association with other proteins. The excited state lifetime of hPrx1 has distinct values at high and low concentrations, suggesting the presence of two different species. Phasor analysis of hPrx1 emission lifetime allowed for the identification and quantification of hPrx1 decamers, dimers, and their mixture at diverse protein concentrations. Using phasor algebra, we calculated the fraction of hPrx1 decamers at different concentrations and obtained KD (1.1 × 10−24 M4) and C0.5 (1.36 μM) values for the decamer–dimer equilibrium. The results were validated and compared with size exclusion chromatography. In addition, spectral phasors provided similar results despite the small differences in emission spectra as a function of hPrx1 concentration. The phasor approach was shown to be a highly sensitive and quantitative method to assess protein oligomerization and an attractive addition to the biophysicist’s toolkit.