Application Notes

©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 6 APPLICATION NOTE initial fluorescent measurement and MST-ON time was set to 5 and 30 sec respectively. The LED power and MST power were set to 90 % and 20 % respectively. The data were analyzed using NanoTemper's NT analysis so ware (version 1.5.41). Titration of the non-fluorescent ligand resulted in a gradual change in MST signal, which was plotted as ΔFnorm to yield a binding curve, which was fitted to derive the binding constants. Circular Dichroism Spectroscopy and Mass Spectrometry CD spectra were recorded with application of a Chirascan Plus Spectrometer (Applied Photophysics, UK.). The purified stalk dimers and their truncated variants were dialyzed against 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2. Spectra were recorded in the range 200–250 nm, at 25 °C temperature, with a 1 nm resolution. The scan rate was 60 nm/min. Protein samples for CD were scanned three times and averaged. The averaged baseline spectra were then subtracted from the averaged sample spectra and converted to molar ellipticity. The recorded spectra were analyzed with application of the Grams/AI so ware from Thermo Scientific (USA). All complexes were analyzed using SYNAPT G2-Si High Definition Mass Spectrometer (Waters, Manchester, U.K.). For the analysis, all protein solutions were buffer exchanged into 200 mM ammonium acetate (pH 7.5) using Micro Bio-Spin chromatography columns (Bio-Rad). Aliquots (~2 μL) were introduced into the mass spectrometer via nanoflow capillaries using the following conditions: capillary voltage 1.2 kV, sampling cone 120 V, source offset 20 V. The source temperature was set up for 25 °C. The collision voltage was adjusted for the optimal signal level. Maximum entropy (MaxEnt, Waters) deconvolution was applied to electrospray data to recalculate the gas phase existing masses. Acknowledgments The data presented in this application note were published in the article Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with aprominent role for P1 C-terminus by by Grela, P. et al. 19 . We would like to thank Dr. Przemyslaw Grela from University of Marie-Curie Sklodowska in Lublin for sharing his research findings and his support in the preparation and revision of this application note. References 1 Hashimoto, K. and Panchenko A. R. Mechanisms of protein oligomerization, the critical role of insertions and deletions in maintaining different oligomeric states. PNAS 107 (47) 20352-20357 (2010) 2 Protein Dimerization and Oligomerization in Biology (Advances in Experimental Medicine and Biology). Edited by Jacqueline M. Matthews. Landes Bioscience and Springer Science+Business Media, LLC (2012) 3 Choi, A. K., Wong, E. C., Lee, K. M. & Wong, K. B. Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes. Toxins (Basel) 7, 638–647 (2015)

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