Quantitative FRET (qFRET) Technology for the Determination of Protein-Protein Interaction Affinity in Solution

Protein-protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Forster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein-protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein-protein interaction affinity (K-D), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.

Automated summary

Protein-protein interactions are vital in life, and FRET is widely used to detect molecular interactions with high sensitivity and efficiency. Attempts to develop FRET into a quantitative measurement for interaction affinity have faced challenges, but the method shows great potential for studying difficult-to-express proteins and interactions in living cells.