Determine Protein Interaction Affinity without Purification Reveals a Two-enzyme Co-recognition of SUMOylation Substrate using Quantitative FRET (qFRET) Technology

Protein-protein interaction is the most fundamental process in life, and the quantification and manipulation of protein interaction activation or inhibition are critical for a comprehensive understanding of physiological processes and the development of new medicines that modulate protein-protein interactions. Due to its essential roles in physiology and pathology, many methodologies and technologies have been developed to determine the interaction events and parameters of interaction affinity. However, due to the nature of these methodologies, interactive partner needs to be purified, and large quantities of proteins are needed. This significantly limits quantitatively understanding of protein interaction and its application in therapeutics development. Recently, a quantitative FRET(qFRET) method was developed to determine purified protein-protein interaction affinity for purified proteins in vitro. Here, for the first time, we report the development of a high-throughput approach to determining the protein interaction affinity between two un-purified proteins using a quantitative FRET assay(qFRET). Two proteins, SUMO E2 conjugating enzyme, Ubc9, and RanGap1c, were first employed for the methodology development. We then determined the interaction affinities of Ubc9 and PIAS1 with influenza A virus NS1 protein without purifying two interactive partners. We found that both enzymes have high affinities to interaction affinity to the NS1, and this provides a kinetics explanation for the SUMOylation two-enzyme co-recognition mechanism to achieve high substrate specificity in vivo. The protein interaction affinity determination without purification provides several advantages over the current methods, including high accuracy, no tedious protein purification process, and high-throughput assay. The novel approach, so-called High-throughput determination of protein interaction affinity, KD, without purification using quantitative FRET assay, and scientific discoveries, representing a significant advancement in technology and scientific discovery, holds great potential for large-scale protein-protein interaction affinity determinations in vitro and in vivo.