Abstract
Protein–protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster 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 (KD), 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.
Highlights
According to the mass action lawprotein of molecular interaction, to determine protein signal resulting from the interactive complex, to elucidate the KDthe from the Förster resonance energy transfer (FRET)
We systematically summarized the novel developments of both the donor quenching-based and the acceptor emission-based methods for KD determination
FRET measurement for interactive proteins is carried out in a solution, and this condition mimics the physiological environment of living cells
Summary
D) Determination accuracy andPrinciples robustness of of these approaches for KD determinations in According to the mass action lawprotein of molecular interaction, to determine protein signal resulting from the interactive complex, to elucidate the KDthe from the FRET interaction equilibrium constant (KD ) an essential step is to quantify the absolute FRET [33,35] (Figure 1). Several effortsatwere to elucidate the absolute including the three‐cube method [28,36] These methods need toincluding determine the FRET signal from the fluorescence emissionHowever, at the acceptor emission wavelength, the three-cube [28,36]. Thesedistance, methods and needFRET to determine the quantum quantum yield ofmethod fluorophores, fluorophore efficiency, which heavily yield of fluorophore distance, and FRET efficiency, which heavily depends FRET depends onfluorophores, instruments and assumptions.
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