Chapter title |
Characterization of Ligand Binding to Pseudokinases Using a Thermal Shift Assay
|
---|---|
Chapter number | 7 |
Book title |
Kinase Signaling Networks
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-7154-1_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7152-7, 978-1-4939-7154-1
|
Authors |
Isabelle S. Lucet, James M. Murphy, Lucet, Isabelle S., Murphy, James M. |
Abstract |
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. |
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