Department of Systems Analysis and Computer Simulation, Belarusian State University, 220030 Minsk, Belarus.
Laboratory of Biochemistry, Microspectroscopy Research Facility, Wageningen University and Research, PO Box 8128, 6700 ET Wageningen, The Netherlands.
Methods Appl Fluoresc. 2022 Aug 24;10(4). doi: 10.1088/2050-6120/ac8911.
Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores.
通过将 EGFP 与两亲嵌段共聚物以等电荷比混合，可以在复杂凝聚核胶束（C3M）中封装增强型绿色荧光蛋白（EGFP）。以前已经表明，这种封装系统具有高度的动态性，这意味着存在不同的群体；在溶液中游离的 GFP 或与聚合物（小复合物）复合的 GFP 以及包封在 C3M 中的 EGFP。我们进行了时间分辨荧光各向异性实验，使用三种不同的荧光各向异性衰减分析方法来确定包封在 C3M 中的 EGFP 的相对群体。首先，使用最大熵法（MEM）数据分析了与暗荧光蛋白混合的五种不同浓度的 C3M 中的 EGFP（分别为 10、20、30、40 和 50% EGFP）。在所有情况下，0.1 和 100 ns 之间的相关时间分布（在对数时标上）都清晰可见，显示出双峰分布。0.1 和 2.0 ns 之间的分布是由于胶束中包装的 EGFP 分子之间的同分子荧光能量转移，8 和 30 ns 之间的分布与溶液中游离 EGFP 的相关时间分布一致。同分子荧光能量转移分布的分数随相对胶束 EGFP 浓度的增加呈线性增加。这些 MEM 结果得到了两种不同分析方法的证实：共聚物中所有五个荧光各向异性衰减的整体群体分析，以及自由 EGFP 的一个（各向异性的直接分析）和通过拟合平行和垂直荧光衰减分量对各向异性的全局关联群体分析。与全局分析方法相反，MEM 方法直接揭示了相关时间的分布，而无需有关样品的任何先验信息。然而，通过拟合平行和垂直荧光衰减分量对各向异性的全局关联分析是唯一能够准确估计溶液中游离荧光团和包封荧光团分数的方法。
