Application Notes

5 Moreover, the unfolding curves revealed that some detergents greatly reduced the unfolding onset temperature. As an example, Figure 2B shows normalized unfolding curves of HiTehA in presence of DDM and C6E3, the Tm-values of which differ by only ~3.8 °C. However, the unfolding onset temperature was much reduced in presence of C6E3 compared to DDM, with a difference of 11 °C. Since some membrane proteins can only be purified in very limited amounts, we tested whether it is possible to perform thermal unfolding experiments at very low protein concentrations. For this, a serial dilution of HiTehA in Tris buffer containing 0.02 % DDM was prepared and subjected to thermal unfolding. Notably, samples spanning a concentration range from 120 µg/ml to 1.9 µg/ml could be analyzed in a single run, showing that protein fluorescence intensities can differ by as much as ~70 fold and still allow for robust Tm determination in one experiment (Figure 3A and B). Interestingly, Tm-values decreased slightly with decreasing protein concentration, pointing towards a dilution-induced destabilization of the membrane protein (Figure 3C). Owed to the high sensitivity of the Prometheus NT.48, the entire detergent screen with 22 different detergents could have been performed with less than 0.5 µg of protein, demonstrating the efficiency of the Prometheus NT.48 for thermal unfolding experiments. Finally, we aimed to evaluate the precision of Tm determination by comparing nanoDSF results to the thermal unfolding of HiTehA by DSC. Importantly, DSC is very sensitive to detergents which can produce aberrant signals thus precluding any Tm analysis. For these reasons, only few detergents can be used for DSC experiments. We therefore chose DDM, which shows acceptable compatibility with DSC experiments. Tm values determined by DSC and nanoDSF were compared directly (Figure 4) and the obtained values demonstrate an excellent agreement with 62.1 °C and 62.7 °C, respectively. These data show that nanoDSF can provide Tm values with calorimetric precision. On top of that nanoDSF allows for Tm determination without buffer limitations consuming only a fraction of time and sample when compared to traditional methods. Figure 4: Comparison of HiTehA thermal stability measurements by DSC and nanoDSF. 1 mg/ml HiTehA in 50 mM Tris HCl pH 8, 200 mM NaCl and 0.02 % DDM, were subjected to thermal ramps and Tm-values were calculated. Conclusions Our results show that monitoring of tryptophan fluorescence emission shifts during thermal unfolding using the Prometheus NT.48 is a perfect approach to determine the optimal conditions for membrane protein purification and storage. Since maximal stability of membrane proteins is a prerequisite for subsequent biophysical experiments and interaction studies, this approach enables the thorough and precise characterization of membrane proteins, which is particularly important in pharmacological screenings to identify novel drugs. Notably, micelle formation, autofluorescence or other secondary effects which are common for detergents, and which often interfere with biophysical characterization of membrane protein stability, do not impede measurements employing the Prometheus NT.48. Moreover, the high precision and sensitivity of the instrument provides DSC-grade data and allows for

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