Authors

E M Cardew¹;  ¹ School of Biological and Biomedical Sciences, Durham University

Discussion

A thermal shift assay (TSA) is a high-throughput fluorescence-based biophysical technique for assessing protein stability based on protein melting temperature (Tm). A non-covalent dye, namely SPYRO Orange, is used; upon binding the hydrophobic core of thermally denatured protein, a fluorescence signal is emitted at a time-point that corresponds to the Tm. Two screens, namely the Durham Salt and Durham pH screens, have been developed and are currently being commercialised by Molecular Dimensions Ltd. The screens can be used in a TSA to assess the thermal stability of a protein in a broad range of solutions. Utilising the Durham Screens offers the chance to improve your protein purification and discover potential crystallisation conditions. Protein-ligand interactions can also be explored using compound libraries in a TSA, where ligand binding is indicated by a positive shift in Tm. NAMI is a GUI-based Python programme for automatic TSA data analysis and interpretation (Groftehauge et al., 2015). NAMI facilitates the rapid determination of Tm values and generates a colour-coded table of all results, allowing simple visual analysis. Overall, combining the use of the Durham Screens and compound libraries in TSAs with data analysis in NAMI generates a powerful tool for the assessment of protein stability for structural studies and structure-based drug design.