Measuring Effective Concentrations Enforced by Intrinsically Disordered Linkers

Charlotte S Sørensen, Magnus Kjaergaard

Research output: Contribution to book/anthology/report/proceedingBook chapterResearchpeer-review

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology : Methods and protocols
EditorsBirthe B. Kragelund, Karen Skriver
Number of pages14
Place of publicationNew York
PublisherHumana Press
Publication date2020
Pages505-518
ISBN (Print)978-1-0716-0523-3
ISBN (Electronic)978-1-0716-0524-0
DOIs
Publication statusPublished - 2020
SeriesMethods in Molecular Biology
Volume2141
ISSN1064-3745

Keywords

  • Effective concentration
  • Fluorescent biosensor
  • Intrinsically disordered protein (IDP)
  • Linker
  • Polymer physics

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