Characterization of peptide-fused protein assemblies in living cells

Assembly of de novo peptides designed from scratch is in a semi-rational manner and creates artificial supramolecular structures with unique properties. Considering that the functions of various proteins in living cells are highly regulated by their assemblies, building artificial assemblies within cells holds the potential to simulate the functions of natural protein assemblies and engineer cellular activities for controlled manipulation. How can we evaluate the self-assembly of designed peptides in cells? The most effective approach involves the genetic fusion of fluorescent proteins (FPs). Expressing a self-assembling peptide fused with an FP within cells allows for evaluating assemblies through fluorescence signal. When µm-scale assemblies such as condensates are formed, the peptide assemblies can be directly observed by imaging. For sub-µm-scale assemblies, fluorescence correlation spectroscopy analysis is more practical. Additionally, the fluorescence resonance energy transfer (FRET) signal between FPs is valuable evidence of proximity. The decrease in fluorescence anisotropy associated with homo-FRET reveals the properties of self-assembly. Furthermore, by combining two FPs, one acting as a donor and the other as an acceptor, the heteromeric interaction between two different components can be studied through the FRET signal. In this chapter, we provide detailed protocols, from designing and constructing plasmid DNA expressing the peptide-fused protein to analysis of self-assembly in living cells.