Characterization of ligand binding to pseudokinases using a thermal shift assay

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The protocol herein describes a robust and proven method for the measurement of pseudokinase-ligand interaction using a fluorescence-based thermal shift assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulatory modules of signal transduction pathways and may well represent a novel class of drug targets. However, unlike kinases, the regulatory activity of pseudokinases is mainly conferred through protein-protein interactions. Understanding the mechanisms that underlie pseudokinase conformational changes through ligand binding and how such conformational changes can tune signaling pathways is a necessary step to unravel their biological functions. Thermal denaturation-based methods have proven to be a powerful method for determining the capacity of purified recombinant pseudokinases to bind ligands and can simultaneously inform on the potential druggability of the nucleotide-binding site. This assay takes advantage of a change in fluorescence arising when the dye, SYPRO Orange, binds to hydrophobic patches that become exposed when a protein undergoes thermal unfolding. Ligand binding to a protein is known to increase its thermal stability, which is reflected by a shift between the thermal denaturation curves of the unliganded protein and the liganded protein. Here, we illustrate the utility of the method with the pseudokinases, ErbB3/HER3, ILK, ROP5Bi, JAK1, JAK2, TYK2, MLKL, STRAD, TRIB1, VRK3, and ROR1. This method can also be used to determine optimal buffer conditions that may increase protein stability and can be tailored to other protein families.

Original languageEnglish
Title of host publicationKinase Signaling Networks
EditorsAik-Choon Tan, Paul H. Huang
Place of PublicationNew York NY USA
PublisherHumana Press
Number of pages14
ISBN (Electronic)9781493971541
ISBN (Print)9781493971527, 9781493984008
Publication statusPublished - 2017
Externally publishedYes

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • ATP mimetics
  • Kinase inhibitors
  • Kinases
  • Nucleotides
  • Pseudokinases
  • Thermal stability shift assay

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