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

Proteins assemble into a variety of dynamic and functional structures. Their structural transitions are often challenging to distinguish inside cells, particularly with a high spatiotemporal resolution. Here, we present a fluorescence resonance energy transfer (FRET)-based method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor self-assembly of three proteins. We show that the mutant Huntingtin exon1 (mHttex1) first forms less-ordered assemblies, which develop into fibril-like aggregates, and demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1. We also monitor the structural changes in fused in sarcoma (FUS) condensates. This method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation.

Automated summary

This study presents a fluorescence resonance energy transfer (FRET)-based method for monitoring protein self-assembly with continuous and high-throughput capabilities, allowing for the observation of transient intermediate states. The approach utilizes intermolecular FRET between donor and acceptor proteins on the same target protein, providing high sensitivity and straightforward ratiometric imaging. The method was successfully applied to monitor the self-assembly of three different proteins, revealing the regulatory role of the chaperone protein DNAJB6b.