Purification of Gαi3 mutants for biochemical studies

Human Gαi3 was expressed from the same plasmid as described for the NMR studies and purified as an uncleaved His-tagged protein. Pelleted bacteria from 1 l of culture were resuspended in 25 ml of buffer (50 mM NaH₂PO₄, pH 7.4, 300 mM NaCl, 10 mM imidazole, 25 μM GDP and 1% (v:v) Triton X-100 supplemented with protease inhibitor cocktail (leupeptin 1 μM, pepstatin 2.5 μM, aprotinin 0.2 μM and phenylmethylsulfonyl fluoride 1 mM)). After sonication (four cycles, with pulses lasting 20 s and with 1 min interval between pulses to prevent heating), lysates were centrifuged at 12,000g for 20 min at 4 °C. Solubilized proteins were affinity-purified on HisPur Cobalt Resin (Pierce) and eluted with lysis buffer supplemented with 250 mM imidazole. The buffer was exchanged for 20 mM Tris-HCl, pH 7.4, 20 mM NaCl, 1 mM MgCl₂, 1 mM DTT, 10 μM GDP and 5% (v/v) glycerol using a HiTrap Desalting column (GE Healthcare). Protein samples were stored at −80 °C.