Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay
Isabelle Lucet,
Joanne Hildebrand,
Peter Czabotar,
Jian-Guo Zhang,
Nicos Nicola,
John Silke,
Jeffrey Babon,
James Murphy
Affiliations
Isabelle Lucet
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, and The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
Joanne Hildebrand
Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
Peter Czabotar
Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
Jian-Guo Zhang
Cancer and Haematology Division and Department of Medical Biology, The Walter and Eliza Hall Institute of Medical Research and The University of Melbourne, Melbourne, Australia
Nicos Nicola
Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
John Silke
Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
Jeffrey Babon
Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
James Murphy
Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Melbourne, Australia
This protocol describes a robust technique for the measurement of pseudokinase-ligand interaction by a fluorescence-based Thermal Shift Assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulators of signal transduction and may therefore represent a novel class of drug targets. Unlike kinases, the catalytic efficiency of pseudokinases is rather poor or non existent, making it difficult to dissect the function of their nucleotide binding sites. Thermal denaturation-based methods have proven to be a powerful method for determining ligand binding capacity to purified pseudokinases and can inform on the potential drugability of the nucleotide binding site. This assay takes advantage of a change in flurorescence arising when a flurorescence dye, in this instance SYPRO® Orange, binds to hydrohobic patches that become exposed when a protein undergoes thermal denaturation. Ligand binding to a protein is known to increase its thermal stability which is reflected by a shift observed in the thermal denaturation curve between the unliganded protein and the liganded protein. This generalized protocol can also be tailored to other protein families. In addition, thermal denaturation-based methods can be used to identify optimal buffer conditions that may increase protein stability.
