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Analysis of protein-ligand interactions by fluorescence polarization

Abstract

Quantification of the associations between biomolecules is required both to predict and understand the interactions that underpin all biological activity. Fluorescence polarization (FP) provides a nondisruptive means of measuring the association of a fluorescent ligand with a larger molecule. We describe an FP assay in which binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP3) to N-terminal fragments of IP3 receptors can be characterized at different temperatures and in competition with other ligands. The assay allows the standard Gibbs free energy (ΔG°), enthalpy (ΔH°) and entropy (ΔS°) changes of ligand binding to be determined. The method is applicable to any purified ligand-binding site for which an appropriate fluorescent ligand is available. FP can be used to measure low-affinity interactions in real time without the use of radioactive materials, it is nondestructive and, with appropriate care, it can resolve ΔH° and ΔS°. The first part of the protocol, protein preparation, may take several weeks, whereas the FP measurements, once they have been optimized, would normally take 1–6 h.

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Figure 1: Saturable binding of a ligand (D) to its target (R).
Figure 2: Ligand binding analyzed by fluorescence polarization.
Figure 3: Structure of the IP3 receptor and the ligands used.
Figure 4: Experimental design flow chart.
Figure 5: Expression, purification and quantification of NT fragments of IP3Rs.
Figure 6: Typical 96-well plate layout for the FP saturation binding assay.
Figure 7: Typical results from FP analysis of equilibrium ligand binding to N-terminal IP3 receptor fragments.

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Acknowledgements

This work was supported by the Wellcome Trust (085295) and Biotechnology and Biological Sciences Research Council (BB/H009736). A.M.R. is a fellow of Queens' College, Cambridge. We thank Z. Ding (University of Cambridge), B.V.L. Potter and A.M. Riley (both University of Bath) for their contributions to our development of FP analyses18, and B. Luisi (University of Cambridge) for advice and providing access to equipment during our preliminary FP analyses.

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Correspondence to Colin W Taylor.

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Rossi, A., Taylor, C. Analysis of protein-ligand interactions by fluorescence polarization. Nat Protoc 6, 365–387 (2011). https://doi.org/10.1038/nprot.2011.305

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