Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands.
Laboratory of Biochemistry, Wageningen University & Research, 6708 WE Wageningen, The Netherlands.
Int J Mol Sci. 2021 Jul 7;22(14):7300. doi: 10.3390/ijms22147300.
Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.
蛋白质二聚化在调节许多生物过程中起着至关重要的作用。然而，在细胞环境中检测蛋白质二聚体仍然是一个挑战。在这里，我们提出了一种使用荧光相关光谱（FCS）和单颜色荧光波动数据的光子计数直方图（PCH）分析相结合的方法，来测量活细胞中 GFP 标记蛋白二聚化的程度。我们将这种分析方法命名为亮度和扩散全局分析（BDGA），并将其应用于生物学目的。使用含有不同 GFP 和串联二聚体 GFP（diGFP）比例的细胞裂解物，我们表明每个粒子的平均亮度与二聚体的存在分数成正比。我们进一步将这种方法应用于活细胞，并能够区分 GFP、diGFP 以及 FKBP12（FK506 结合蛋白 12）-GFP 的配体诱导二聚化。虽然其他分析方法偶尔也被用于研究活细胞中的二聚化，但可能容易出错，而本文提供了一种使用单颜色荧光波动光谱研究任何细胞质蛋白的稳健方法。
