Characterization of ligand binding to pseudokinases using a thermal shift assay
- Author(s)
- Lucet, IS; Murphy, JM;
- Journal Title
- Methods Mol Biol
- Publication Type
- Journal Article
- 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.
- Publisher
- Springer
- Research Division(s)
- Chemical Biology; Cell Signalling And Cell Death
- PubMed ID
- 28730475
- Publisher's Version
- https://doi.org/10.1007/978-1-4939-7154-1_7
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2017-08-30 02:22:38
Last Modified: 2018-01-10 09:21:08