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Measuring Effective Concentrations Enforced by Intrinsically Disordered Linkers.

Charlotte S Sørensen^1,2, Magnus Kjaergaard^3,4,5

¹Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

Methods in Molecular Biology (Clifton, N.J.)

|July 23, 2020

Summary

Intrinsically disordered linkers are crucial for protein function. This study introduces a competition assay using a FRET biosensor to accurately measure the effective concentrations these linkers enforce in protein complexes.

Keywords:

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

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Area of Science:

Biochemistry
Molecular Biology
Protein Science

Background:

Intrinsically disordered linkers play vital roles in multidomain protein functions, including avidity, auto-inhibition, catalysis, and phase separation.
These linkers establish effective concentrations that influence the kinetics and equilibrium of intramolecular reactions within proteins.

Purpose of the Study:

To develop and present an experimental protocol for quantifying the effective concentrations imposed by intrinsically disordered linkers.
To enable mechanistic studies on the role of linkers in supramolecular complexes.

Main Methods:

A competition assay utilizing a Förster Resonance Energy Transfer (FRET) biosensor.
Titration of the FRET biosensor with a competitor peptide to measure linker-enforced effective concentrations.
Parallel analysis of multiple protein constructs using a fluorescent plate reader.

Main Results:

The described assay provides a robust method for measuring effective concentrations enforced by various synthetic disordered linkers.
The protocol has been successfully applied to study hundreds of different linker constructs.
Enabled quantitative assessment of linker-mediated molecular interactions.

Conclusions:

The developed competition assay is a valuable tool for understanding the functional impact of intrinsically disordered linkers.
Accurate measurement of effective concentrations is essential for deciphering linker mechanisms in protein function.
This method facilitates high-throughput analysis of disordered linkers in complex biological systems.