Issue link: https://resources.nanotempertech.com/i/1050641
2 TECHNICAL NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. In Figure 2, the interaction between maltose-binding protein (MBP) and maltose is shown. The presence of maltose triggers a shi in the inflection temperature from 56.3 to 58.5 °C. Also observed is a change in the initial ratio value measured which can likely be attributed to the marked structural changes of the protein upon binding of maltose. In the final example shown in Figure 3, the interaction between lysozyme and the bacterial cell wall component 3'NAG was analyzed. Besides a shi of the unfolding transition by 1.8 °C, the initial ratio of lysozyme in the presence of 3'NAG is much lower than in its absence. Reports suggest that binding of the ligand covers several otherwise exposed tryptophan residues and likely results in a shi of the tryptophan fluorescence towards lower wavelengths which translates into a lower initial ratio. 1 changes in the protein's stability as monitored by fluorescence properties can be derived to conclude binding. Thus, the Tycho NT.6 system allows for evaluating protein functionality within minutes, and can be integrated in purification workflows or assay development routines with ease. Results In this study, the unfolding profiles of a series of proteins were examined in the absence and presence of ligands known to bind to their target proteins. Figures 1A and 1B show the binding of p38 kinase to Mg 2+ ions and a small molecule inhibitor, respectively. Both interactions induce a shi of the unfolding transition to higher inflection temperatures (T i ), indicating that an interaction is occurring. In the case of the small molecule inhibitor (SB203580) binding also changes the ratio of the folded state (initial ratio), which is likely caused by effects of the small molecule on tryptophan fluorescence emission. 30 40 50 60 70 80 90 100 0.70 0.75 0.80 0.85 0.90 0.95 Temperature (°C) 30 40 50 60 70 80 90 100 0.70 0.75 0.80 0.85 0.90 Temperature (°C) p38 kinase Ratio 350 nm / 330 nm Ratio 350 nm / 330 nm p38 kinase + 5 mM MgCl2 p38 kinase p38 kinase + 500 nM SB203580 A B Figure 1: Monitoring p38 kinase binding activity using a label-free thermal shi analysis. A) Interaction of 4 µM p38 kinase with 5 mM Mg2+ ions. B) Interaction of 4 µM p38 kinase with 500 nM SB203580. Vertical lines indicate inflection temperatures (T i ), colored circles on the y-axis indicate initial ratio values.