Application Notes

4 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Assessing long-term stability of integral membrane proteins Next, we asked whether this approach can even be used for long-term storage tests of detergent-solubilized membrane proteins. For this, we used the slow-anion channel protein HiTeha [3] and incubated the protein at 4 °C and at RT, respectively. Then, we determined initial F350/F330 ratios and measured thermal unfolding curves over a period of 34 days. Again, a clear storage temperature- and incubation time-dependent shi in the F350/F330 ratio could be observed, while the T m - values of the thermal unfolding curve did not change significantly. The results show that HiTehA is rather stable, but that it tends to degrade slowly when stored at room temperature, and that the F350/F330 ratio can serve as a measure of the amount of unfolded protein in solution. -2 0 2 4 6 8 10 12 14 0 5 10 15 20 25 30 35 40 % unfolded # incubation days incubation at RT incubation at 4°C 30 40 50 60 70 80 0.55 0.60 0.65 0.70 0.75 0.80 0.85 25 26 27 28 29 30 31 0.60 F350/F330 Temperature (°C) membrane protein stability 1 day 5 days 14 days 20 days -2 0 2 4 6 8 10 12 14 0 5 10 15 20 25 30 35 40 % unfolded # incubation days incubation at RT incubation at 4°C 30 40 50 60 70 80 0.55 0.60 0.65 0.70 0.75 0.80 0.85 25 26 27 28 29 30 31 0.60 F350/F330 Temperature (°C) membrane protein stability 1 day 5 days 14 days 20 days Forced denaturation of the drug target MEK1 Last, we asked whether this approach can also be applied to evaluate the effects of forced degradation on proteins. Such an approach can be valuable to identify critical handling steps during protein purification, storage, or setup of biochemical assays. Information about the stability of a protein under different conditions is particularly important for biophysical screening campaigns, in which multiple biochemical assays are applied. We chose the popular drug target kinase MEK1, which already has been used in a large number of different drug discovery screening campaigns, including an MST-based fragment screen [1, 3, 4]. MEK1 solutions were subjected to a number of thermal and physical stresses, namely incubation at different temperatures, heavy vortexing, and repeated Figure 3: Long-term storage test on HiTehA. Aliquouts of the integral membrane protein HiTehA were stored at 4 °C and at RT, respectively, and thermal unfolding curves were measured over a time period of 34 days. % unfolded protein was calculated based on the F350/F330 ratio.

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