Intrinsically disordered linkers control avidity, auto-inhibition, catalysis, and liquid-liquid phase separation in multidomain proteins. Linkers enforce effective concentrations that directly affect the kinetics and equilibrium positions of intramolecular reactions. Mechanistic understanding of the role of linkers thus requires measurements of the effective concentrations in supramolecular complexes. Here, we describe an experimental protocol for measuring the effective concentrations enforced by a linker using a competition assay. The experiment uses a FRET biosensor that is titrated by a competitor peptide. The assay is designed for parallel analysis of several constructs in a fluorescent plate reader and has been used to study hundreds of synthetic disordered linkers.
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This work was supported by grants to M.K. from the “Young Investigator Program” of the Villum Foundation, Independent Research Fund Denmark (FTP), and PROMEMO—Center for Proteins in Memory – a center of excellence funded by the Danish National Research Foundation (grant number DNRF133).
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