A precise and general FRET-based method for monitoring structural transitions in protein self-organization

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.