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Abstract
Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. 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. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.
Highlights
Protein-protein interactions (PPIs) are essential to cellular biology and both high- and low-affinity interactions are required to maintain robust and dynamic responses in biological circuits [23, 29]
In the mechanically transduced immunosorbent (METRIS) assay, rollers are placed in a Helmholtz coil inspired apparatus where an externally rotating magnetic field is applied at a constant frequency, ω
Friction is determined by the strength and density of PPIs between the roller and the coated substrate
Summary
Protein-protein interactions (PPIs) are essential to cellular biology and both high- and low-affinity interactions are required to maintain robust and dynamic responses in biological circuits [23, 29]. NMR is the gold standard method to measure weak interactions [26, 46], in addition to requiring copious amounts of materials, the proteins must be isotopically labeled, a single affinity measurement requires substantial instrument time and complex data analysis, and of all the methods mentioned is the lowest throughput Another difficulty arising when measuring similar strength interactions, e.g., 3-5 fold differences. Several factors contribute to this limitation, but determining the active fraction of protein is significant because, for most fitting techniques, the calculated affinity is a dependent variable of the protein concentration [21, 22] Another factor in differentiating similar strength interactions is that most binding measurements have low statistical power due to the resource intensiveness of performing multiple replicates. A method where binding strength can be measured independent of protein concentration and that has high statistical power would be valuable
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