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Measuring Effective Concentrations Enforced by Intrinsically Disordered Linkers

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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.

Titel Intrinsically Disordered Proteins : Methods and protocols
RedaktørerBirthe B. Kragelund, Karen Skriver
Antal sider14
UdgivelsesstedNew York
ForlagHumana Press
ISBN (trykt)978-1-0716-0523-3
ISBN (Elektronisk)978-1-0716-0524-0
StatusUdgivet - 2020
SerietitelMethods in Molecular Biology

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