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

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.

How Far Are We from the Completion of the Human Protein Interactome Reconstruction?

“…New experiments enrich the network with new PPIs between existing protein nodes and it was observed that, in this way, previously remote protein neighborhoods become connected, thus decreasing the network diameter and the number of independent connected subnetworks. We envisage that the human protein interactome may be substantially enriched by the development and application of advanced methodologies including (i) the highly sensitive quantitative FRET technology for the determination of PPI affinity in high-throughput assays, especially for proteins that are difficult to be expressed and for PPIs in living cells [ 38 ]; (ii) the functional protein microarrays, especially for the quantitation of PPIs of membrane proteins for receptor interactions [ 39 , 40 ]; and (iii) the application and evaluation of advanced, more quantitative mass spectrometry technologies [ 41 ]. These technologies could assist in the validation of the currently identified PPIs as their vast majority is presently supported by a single publication.…”

Human SUMOylation Pathway Is Critical for Influenza B Virus

“…We did not observe too much FRET signal increase after the addition of PIAS1 to the reaction, which agreed with the general common knowledge (Figure 3A No PIAS1 blue). However, after a careful quantification of the FRET signal through our qFRET method, the SUMOylation reaction produced higher signals than the one without PIAS1 (Figure 3B) [47]. The SUMOylation reaction monitored with the FRET signal was a convenient method for following the reaction and obtaining results.…”

“…We applied our FRET-based KD determination technology to the determinations of affinities between Ubc9 or PIAS1 to the IBV M1 protein [42,47,53]. The Ubc9 or PIAS1 genes were first fused with the FRET donor, CyPet, and IBV M1 was fused with the FRET acceptor, YPet.…”

“…The KD values for Ubc9-M1 or PIAS1-M1 interactions were 0.20 µ M and 0.22 µ M, respectively, indicating a very high affinity of both SUMOylation enzymes E2 and E3 for the IBV M1. These KD values are equivalent or lower than We applied our FRET-based K D determination technology to the determinations of affinities between Ubc9 or PIAS1 to the IBV M1 protein [42,47,53]. The Ubc9 or PIAS1 genes were first fused with the FRET donor, CyPet, and IBV M1 was fused with the FRET acceptor, YPet.…”

“…Then, in a fixed concentration of the FRET donor, the CyPet-Ubc9 or the CyPet-PIAS1, different concentrations of the FRET acceptor, the YPet-M1, were titrated. We have developed an algorithm to extract the absolute FRET signal, which corresponded to the interactions, from the total fluorescence signal [47]. The titrated absolute fluorescence signal (Em FRET ) for Ubc9 and PIAS1 with M1 showed good sigmoidal curves (Figure 4A,B).…”