Application Notes

3 Conclusion Our results show that the Prometheus NT.48 by NanoTemper Technologies is suitable for monitoring fluorescence emission changes of membrane proteins in the presence of detergent micelles. The nanoDSF method allowed to identify DDM as the best suited detergent for PA2949 stabilization. This result was confirmed with an enzymatic activity assay. Moreover, nanoDSF showed that SDS and SB3-16 completely denature PA2949 in agreement with a complete loss of esterase activity of PA2949 measured enzymatically. Apparently, the nanoDSF method can be used for the fast and reliable identification of detergents suitable for the solubilization and storage of membrane proteins. These results demonstrate the potential of the Prometheus NT.48 for determination of detergent- protein compatibility, which is particularly interesting for proteins which cannot be assayed enzymatically. Material and Methods Protein Preparation PA2949 was expressed in P. aeruginosa PA01 carrying pBBRplbFH6 expression plasmid [3] cultivated in LB media supplemented with 100 µg/ml tetracycline at 37 °C. The cells were harvested when the culture reached an optical density of 1 by centrifugation for 10 min at 3000g and 4°C. Cells suspended in 100 mM Tris-HCl (pH 8.0) were disrupted by French press and total cell membranes were isolated by ultracentrifugation for 1 h at 200,000 g and 4 °C. The total membrane fraction containing PA2949 was solubilized with Tris-HCl (100 mM, pH 8.0) containing 1 % (v/v) Triton X-100 by gentle agitation overnight at 4 °C. PA2949 was purified from the solubilized membranes by immobilized metal affinity chromatography using Ni-NTA material [3]. Elution buffer was exchanged to Tris- HCl (100 mM, pH 8) containing 0.22 mM DDM by PD-10 desalting column (GE Healthcare, Solingen, Germany) according to the manufacturers´ protocol. This PA2949 sample was furthermore purified by anion exchange chromatography using UNOsphere Q material (BioRad, Hercules, CA, USA). Thermal unfolding experiments The samples for thermal unfolding experiments were prepared by combining the same volume of the pure PA2949 sample (6 µM) and Tris-HCl buffer (100 mM, pH 8) containing the respective detergent. The final concentrations of each detergent given in the table 1 were above the CMC. PA2949 was incubated with detergent for 30 min at room temperature. The same PA2949 samples were used for determination of fluorescence and enzymatic activity. Fluorescence based thermal unfolding experiments were performed using the Prometheus NT.48. The capillaries containing 10 µl PA2949 samples were inserted into the machine, the temperature was increased by a rate of 1 °C/min from 20 °C to 90 °C and the fluorescence at emission wavelengths of 330 nm and 350 nm was measured. Enzyme activity based thermal unfolding experiments were performed by measuring the residual esterase activity of purified PA2949 samples incubated at temperatures from 30 °C to 70 °C. Enzyme assay was performed in a 96-well microplate by combining 10 µl of the enzyme sample with 150 µL of p-nitrophenyl butyrate substrate [5]. References [1] Ghisaidoobe, A.B.T. and S.J. Chung, Intrinsic tryptophan fluorescence in the detection and analysis of proteins. Int. J. Mol. Sci. 2014. 15:22518-22538. [2] Lakowicz, J.R., Principles of fluorescence spectroscopy. 3rd ed. 2006, New York: Springer. XXVI, 954:16-19. [3] Kovacic, F., et al., A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli. FEBS Open Bio, 2016. 6:484-493. [4] Ondetti, M.A., B. Rubin, and D.W. Cushman, Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. Science, 1977. 196: 441-444. [5] Jaeger, K.E. and F. Kovacic, Determination of lipolytic enzyme activities. Methods Mol Biol, 2014. 1149:111-134. This application note was prepared in a collaboration project of NanoTemper Technologies and the Forschungszentrum Jülich.

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