FRETting about the affinity of bimolecular protein-protein interactions

Abstract

Fluorescence resonance energy transfer (FRET) is a powerful tool to study macromolecular interactions such as protein-protein interactions (PPIs). Fluorescent protein (FP) fusions enable FRET-based PPI analysis of signaling pathways and molecular structure in living cells. Despite FRET's importance in PPI studies, FRET has seen limited use in quantifying the affinities of PPIs in living cells. Here, we have explored the relationship between FRET efficiency and PPI affinity over a wide range when expressed from a single plasmid system in Escherichia coli. Using live-cell microscopy and a set of 20 pairs of small interacting proteins, belonging to different structural folds and interaction affinities, we demonstrate that FRET efficiency can reliably measure the dissociation constant (K_D ) over a range of mM to nM. A 10-fold increase in the interaction affinity results in 0.05 unit increase in FRET efficiency, providing sufficient resolution to quantify large affinity differences (> 10-fold) using live-cell FRET. This approach provides a rapid and simple strategy for assessment of PPI affinities over a wide range and will have utility for high-throughput analysis of protein interactions.

Keywords: FRET efficiency; PPI; affinity; fluorescent proteins.

Figure 1

Relationship between FRET efficiency (E) and affinity (pK _D): (A) live‐cell imaging of E. coli cells to quantify E (filled circles) as a function of pK _D (bottom). The colored symbols represent pair of proteins belonging to different categories as different‐fold (black), same‐fold (red and green) and the non‐interacting pairs as negative control (blue). (B) E for the interaction between histone‐readers (KDM5B‐PHD1 and KDM5B‐PHD1) and N‐terminal unmodified histone H3 (top). Interaction between KDM5B‐PHD1 and histone H3 mutants (bottom) quantified by FRET.