Proteins assemble into a tremendous variety of dynamic and functional structures. Sensitive measurements directly in cells with a high spatiotemporal resolution are needed to distinguish these different assemblies. Here, we demonstrate precise and continuous monitoring of cytoplasmic protein self-assemblies and their structural transitions. Intermolecular FRET with both the donor and acceptor protein at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We measure different assembly structures, transient intermediate states' kinetics, and assembly formation resolved in space and time. Specifically, the method recapitulates that i) the mutant Huntingtin exon1 (mHttex1) protein first forms low-FRET and presumably less ordered assemblies in yeast and human cells, which develop into high-FRET aggregates, ii) the chaperone DNAJB6b prevents low-FRET mHttex1 assemblies, yet coassembles with mHttex1 aggregates, and iii) FUS' condensates have mutation-dependent nanoscopic structures. FACS measurements allow assembly measurement in a high-throughput manner crucial for screening efforts, while fluorescence microscopy provides spatiotemporally-resolved measurements on the single-condensate level during a cell's lifetime to assess the biological consequences. Implementation in other native or non-native proteins could provide insight into many studies involving protein condensation or aggregation.

中文翻译：

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一种精确的，基于FRET的通用方法，用于监测蛋白质自组织中的结构转变

蛋白质组装成各种各样的动态和功能结构。需要直接在具有高时空分辨率的单元中进行灵敏的测量，以区分这些不同的程序集。在这里，我们展示了细胞质蛋白自组装及其结构转变的精确连续监控。具有相同目标蛋白上的供体和受体蛋白的分子间FRET提供了高灵敏度，同时保留了直接比例成像的优势。我们测量不同的组装结构，瞬态中间态的动力学以及在空间和时间上解决的组装形式。具体来说，该方法概述了：i）突变的Huntingtin外显子1（mHttex1）蛋白首先在酵母和人类细胞中形成低FRET且组装顺序可能较不规则，它们会发展成高FRET的聚集体； ii）分子伴侣DNAJB6b阻止了低FRET的mHttex1聚集体，但与mHttex1聚集体共组装，并且iii）FUS的缩合物具有依赖突变的纳米结构。FACS测量允许以高通量的方式进行组装测量，这对于筛选工作至关重要，而荧光显微镜则可在细胞的生命周期内提供单个冷凝物水平的时空分辨测量，以评估生物学后果。在其他天然或非天然蛋白质中的实施可以提供对涉及蛋白质浓缩或聚集的许多研究的洞察力。FACS测量允许以高通量的方式进行组装测量，这对于筛选工作至关重要，而荧光显微镜则可在细胞的生命周期内提供单个冷凝物水平的时空分辨测量，以评估生物学后果。在其他天然或非天然蛋白质中的实施可以提供对涉及蛋白质浓缩或聚集的许多研究的洞察力。FACS测量允许以高通量的方式进行组装测量，这对于筛选工作至关重要，而荧光显微镜则可在细胞的生命周期内提供单个冷凝物水平的时空分辨测量，以评估生物学后果。在其他天然或非天然蛋白质中的实施可以提供对涉及蛋白质浓缩或聚集的许多研究的洞察力。