Ni nta resin

Assembly of lipid nanodiscs containing A_2AAR followed protocols from previous studies^{47 (link)–49 (link)} optimized to facilitate samples prepared with a wider range of lipid compositions. The scaffold protein MSP1D1 was expressed and purified similarly as described in previous studies^{47 (link),48} . 100 mM stock solutions of all lipids were prepared in a cholate buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 200 mM sodium cholate). To initiate nanodisc assembly, 27 μM of purified A_2AAR in DDM/CHS micelles was mixed with purified MSP1D1 and detergent-solubilized lipids in a molar ratio of 1:5:250, respectively. The mixture was incubated for 1-2 h at 4 °C and incubated overnight with pre-washed bio-beads (Bio-Rad Laboratories) at 4 °C. Following the overnight incubation, the bio-beads were removed, and the resulting mixture was incubated with Ni-NTA resin (GoldBio) for 24 h at 4 °C. The Ni-resin was collected after 24 h and washed with 2 CV of a wash buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, and 10 mM imidazole). Nanodiscs containing A_2AAR were eluted with buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, 300 mM imidazole and ligand) and exchanged into a final buffer used for all experiments (25 mM HEPES pH 7.0, 75 mM NaCl, 100 μM TFA and ligand) using a PD-10 desalting column (Cytiva). All ligand-containing buffers were prepared with a saturating concentration of ligand.

Expi293F (Thermo Fisher) cells were transiently transfected with the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher) according to the manufacturer’s recommendations and grown in Expi293™ expression medium (Thermo Fisher). Incubation with agitation was performed at 37 °C and 8% CO₂ for five days. For proteins containing Histag, cell broth was collected after five days and clarified by centrifugation, followed by overnight dialysis against a NiNTA A buffer (50 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 8) with dialysis tubes and a 6–8 kDa cutoff (Spectrum Laboratories, Rancho Dominguez, CA, USA). Samples were purified using NiNTA resin (Goldbio, St Louis, MO, USA) and eluted with 250 mM imidazole in NiNTA A buffer. For proteins containing strep-tag, cell broth was also collected after five days and clarified by centrifugation, followed by purification, using Strep-trap (Cytiva, Marlborough, MA, USA), according to the manufacturer’s recommendations. After affinity isolation, all samples were dialyzed against 20 mM Tris, 150 mM NaCl, pH 7.5.

The TEV protease was produced following the above-described protocol, encompassing ultrasonication, Ni-NTA and SEC chromatography.
TEV protease was used for performing the cleavage of the 8xHis-tag in the case of the monomeric proteins SBP1_9.a, SBP2_9.a, SBP1_9.b and SBP2_9.b before mixing the two subunits, whereas cleavage of the proteins SBP1₁₁ and SBP2₁₁ was initiated only after mixing them in equimolar ratio.
Proteins subjected to controlled proteolysis were incubated overnight at 4 °C with the addition of 50 µg of TEV protease per mg of target protein (~50–200 molar excesses of target protein). Subsequently, in order to promote dissociation from the cleaved products (consisting of only affinity tags or tagged 2-helix-long segments as in the case of SBP1₁₁ and SBP2₁₁) the sample was incubated at 37 °C for 15 min and the mixture was flown through 2.5 ml of Ni-NTA resin (Goldbio, MO, USA) previously conditioned in IEX buffer; the eluted sample was then collected for further analysis.

Ni-NTA resin (GoldBio) was prepared by washing (addition of water/buffer, incubating/rotating for 5 min at 4 °C, centrifugation at 1500g for 5 min, removal of supernatant) twice with water and then with buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM β -ME). Twenty microliters of the prepared resin was added into individual centrifuge tubes, and a total of 0.36 mg of Prx (with a His-tag) was added to each. Following incubation for 30 min at 4 °C, and two wash steps with buffer, 40 μl at 1 mg/ml (0.4 mg total) of each ComR ortholog (without a His-tag) was added to individual Prx saturated resin samples. Protein/resin mixtures were mixed for 1 h at 4 °C, then washed twice with buffer. Next 15 μl of 4× Laemmli sample buffer was loaded directly into each sample, which was then boiled at 93 °C for 5 min, and centrifuged at 21,000g for 3 min. Five microliters of each sample was then run on a 15% SDS-PAGE gel. The No-Prx controls (ComR background) were performed alongside and identically to the pull-downs, with buffer added rather than Prx. ComR-only controls, of similar amounts of the ComR added to the pull-down experiment, were also run on an SDS-PAGE gel to ensure sample purity and consistent protein concentration measurements.