Development of protein probes to monitor and regulate protein oligomerization in cells

Abstract

Cell surface receptor oligomerization is an attractive target process for drug screening. However, Chapter 1 shows that simple but reliable (thus high-throughput) visualization methods for receptor oligomerization are still lacking. Chapter 2 describes the development of a new single-construct Homo-molecular Fluorescence Complementation (Homo-FC) probe, which shows strong fluorescence signals by oligomerization of fused receptors in living cells with unexpectedly low background signals. Importantly, this high signal-to-noise ratio was not affected by expression level variations of fused receptors. The Homo-FC probe was developed by optimized flopped fusion of split fragments of superfolder green fluorescence protein and subsequent surface charge engineering. Homo-FC reliably visualized oligomerization of diverse natural receptors such as GPCR, EGFR, and even cytosolic DAI. By combining Homo-FC probe and photoactivatable proteins, Chapter 3 provides novel tools to monitor and regulate protein oligomerization in phase-separated condensates. Membrane-less organelles or compartments are considered as dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated, biomolecular behaviors inside the compartments are largely unexplored. I quantitatively monitored protein interaction changes when client proteins were recruited into membrane-less compartments formed in living cells. Fluorescent protein (FP) probes, which generate fluorescence signals upon self-interaction or -complementation, were used as clients. I organized cellular compartment formation by tandemly repeating or clustering intrinsically disordered proteins (IDPs), which are primary scaffold proteins to form membrane-less organelles in cells. Client probe recruitment into these IDP compartments was controlled by fusing different IDPs to clients, where IDP-fused clients can be enriched inside compartments via diverse IDP-IDP interactions. I observed vastly increased FP probe interactions inside IDP compartments, although inner probe enrichment was only marginal (2-3 fold), suggesting that inner compartment nature such as percolated protein network structures can accelerate protein interactions. Real-time imaging of compartment formation and subsequent client interactions indicated that IDP-mediated client recruitment can be an instant process, where client interaction signals reached maximums within 4 min. I also observed that client recruitment and interactions inside compartments greatly varied by client-fused IDPs, suggesting a nature’s potential selection strategy to enrich specific sets of biomolecules inside membrane-less organelles.