MRBLE-pep Measurements Reveal Accurate Binding Affinities for B56, a PP2A Regulatory Subunit

Signal transductionpathways rely on dynamic interactions betweenprotein globular domains and short linear motifs (SLiMs). The weakaffinities of these interactions are essential to allow fast rewiringof signaling pathways and downstream responses but also pose technicalchallenges for interaction detection and measurement. We recentlydeveloped a technique (MRBLE-pep) that leverages spectrally encodedhydrogel beads to measure binding affinities between a single proteinof interest and 48 different peptide sequences in a single small volume.In prior work, we applied it to map the binding specificity landscapebetween calcineurin and the PxIxIT SLiM (Nguyen, H. Q. et al. Elife 2019, 8). Here, usingpeptide sequences known to bind the PP2A regulatory subunit B56 & alpha;,we systematically compare affinities measured by MRBLE-pep or isothermalcalorimetry (ITC) and confirm that MRBLE-pep accurately quantifiesrelative affinity over a wide dynamic range while using a fractionof the material required for traditional methods such as ITC.