The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. We hope the information summarized in this article can serve as technical references for further understanding the regulation of plant adaptation to abiotic stress at the protein level. This review concludes classic PPI approaches in plant response to abiotic stresses and their limitations for identifying complex network of regulatory proteins of plant abiotic stresses, and introduces the working mechanism of TurboID-based PL, as well as its feasibility and advantages in plant abiotic stress research. TurboID-based PL has been successfully applied in animal, microorganism and plant systems, particularly to screen transient or weak protein interactions, and detect spatially or temporally restricted local proteomes in living cells. TurboID, a biotin ligase produced by directed evolution, has the advantages of non-toxicity, time-saving and high catalytic efficiency compared to other classic protein-labeling enzymes. Biotin-based proximity labeling (PL) is a recently developed technique to label proximal proteins of a target protein. Protein-protein interaction (PPI) approaches can be used to screen stress-responsive proteins and reveal the mechanisms of protein response to various abiotic stresses. In general, our studies allow high-throughput determination of protein interacting affinity without any purification, and both the approach and scientific discoveries can be applied to proteins that are difficult to be expressed in general.Ībiotic stresses are major environmental conditions that reduce plant growth, productivity and quality. We then applied this approach to determine the interaction affinities of SUMO E3 PIAS1 and Ubc9 with a SUMO substrate, influenza virus protein, NS1, and, for the first time, the SUMO E3 ligase-substrate interaction affinity is determined, which enables us to provide a kinetics explanation for the two-enzyme substrate recognition mode. The interaction affinities from two purified proteins and two un-purified proteins in the presence of BSA, bacterial extracts or two mixtures are all in excellent agreement with that obtained from the SPR measurement. We developed this approach first using SUMO E2 conjugating enzyme, Ubc9, and SUMO substrate, RanGap1c. Here, we present a development of high-throughput approach to determine protein interaction affinity for un-purified interacting proteins using quantitative FRET assay. In this article, an overview is given of the methodologies available for analysis of protein-protein interactions.Īlthough various technologies can determine protein-protein interaction affinity, Kd, current approaches require at least one interacting protein to be purified. Currently available techniques vary with respect to accuracy, reliability, reproducibility and throughput and their performances range from a mere qualitative demonstration of binding to a quantitative characterization of affinities. It is made even more demanding by the need to determine the intensity of interactions quantitatively in order to properly understand protein interplay. However, this does not meet the challenge of investigating the highly complex interaction patterns in cellular systems. Until recently, interactions of only few protein partners could be analyzed in a single experiment. Many protein isoforms that result from mutations, splice-variations and post-translational modifications also come into play. Far less information, however, exists about protein-protein interactions, which are required and responsible for cellular activities and their control. In consequence, the basic set of human proteins is generally known. Genome sequencing has led to the identification of many proteins, which had not been recognized before.
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