Application Notes

©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 5 APPLICATION NOTE Discussion The ribosomal P-protein play a major role in functional ribosome serving as a docking site for variety of translational factors. This acceptor platform is also a target for variety of RIP protein, which utilize it to gain access to SRL and its depurination. Here, we show that the individual CTDs of P1 and P2 heterodimers directly interact with RTA. In addition, different mutants of P1 and P2 proteins were used to analyze the importance of CTD region. Interestingly, CTD of P1 protein is more critical than the CTD of P2 protein for efficient interaction of the stalk complex with RTA. These variations in binding capacity of P1-P2 dimer and CTD mutants could be explained by the unique behavior of the disordered nature of the CTD of P1 and P2 proteins, especially the hinge region [13, 14]. Previous studies showed that P2 forms a homodimer in solution, while in the absence of P2, P1 forms high-mass oligomeric aggregates. The formation of P1-P2 heterodimer is a favorable spontaneous process in which the less stable P2 homodimer is displaced by P1 to form a more stable P1-P2 heterodimer [15, 17]. We also show that MST was the only method that allowed us to characterize in solution the interactions between the trimeric complex (P1-P2 and RTA) and P1-P2 mutants with RTA that exhibit lower affinities. In addition, MST required less material which was essential as purification of these recombinant proteins and in-vitro reconstitution of dimeric P1-P2 complexes is extremely challenging and time consuming. Materials and Methods Protein Expression and Purification Expression, purification of recombinant human P1, P2 proteins, truncated forms and preparation of the complexes were performed according to the procedure established previously [11, 12] (9,10). The P1-P2 heterocomplex was prepared following the denaturation/renaturation procedure established for the yeast P protein complex [15] (13). RTA was expressed in Escherichia coli BL21(DE3) RIL cells and N-terminal 10xHis-tagged recombinant RTA was purified using Ni- NTA agarose from QIAGEN (Valencia, CA, USA). MicroScale Thermophoresis Purified RTA was labeled with amine reactive protein labeling kit RED-NHS Monolith (NanoTemper Technologies MO-L001) using red fluorescent dye NT- 647-NHS. 60µM of the dye was mixed in ratio 3:1 with 20µM of RTA in total volume of 200µL. Free dye was removed by buffer-exchange column chromatography into MST reaction buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05 % Tween-20 (assay buffer). The concentration of labeled RTA was adjusted to approximately 5–20 nM in 200µL of assay buffer that was supplemented with 1mg/mL of bovine serum albumin. 16 twofold dilutions starting from 50 or 100 μM of reconstituted P1-P2 dimers were prepared in a final volume of 10 μL of assay buffer and mixed with 10µL of previously adjusted labelled RTA. Thermophoresis was measured using a Monolith NT.115pico instrument (NanoTemper) at an ambient temperature of 25 °C. The

• Cover