Protein Expression and Purification
To obtain GTP-bound nGTPase, residues 1 to 180 of RhoT2 (Miro2) cDNA (Sinobiological) were PCR amplified and subcloned into the Nde1/XhoI site of the pET28a expression vector, creating an N-terminal His6-tag for purification. The plasmid was transformed into Escherichia coli BL21 (DE3) competent cells for overexpression. Cultures were grown in Luria broth at 37° C to OD600=0.6. Cells were harvested at 3000 rpm and resuspended in labeled media for expression. Double labeled (13C-15N) protein was expressed in M9 media containing 1 g/L 15NH4Cl and 2 g/L 13C-glucose, and 2H-13C-15N labeled protein was expressed in M9 media containing 1 g/L 15NH4Cl and 2 g/L 13C-glucose in 99.9% D2O. Cultures were grown at 37 °C to OD600 = 0.8. Protein expression was induced by adding 0.125 mM isopropyl 1-thio-beta-D-galactopyranoside (IPTG), and cells were grown for 6 hrs at 18 °C. Cells were harvested by centrifugation at 5000 rpm for 15 min at 4 °C and resuspended in 50 mM HEPES, 500 mM NaCl, 1 mM DTT, 1 mM MgCl2, 5% sucrose, 10 mM imidazole, 0.2 mM GTP, pH 7.5 and lysed by sonication on ice. The lysate was clarified by centrifugation at 15,000 rpm for 20 min. The soluble fraction was loaded onto Ni-NTA column (GE Pharma), equilibrated with 50 mM HEPES, 500 mM NaCl, 1 mM DTT, 1 mM MgCl2, 5% sucrose, 10 mM imidazole, pH 7.5. The Ni-NTA resin was washed using a step gradient by increasing the imidazole concentration from 10 mM to 25 mM and then to 50 mM. The protein was eluted in the same buffer containing 250 mM imidazole and diluted 5-fold in 25 mM HEPES 1 mM DTT 1 mM MgCl2, pH 7.4. The protein was then loaded onto a HiTrap Q column (GE Pharma) and eluted using a linear gradient against 25 mM HEPES, 1 M NaCl, 1 mM DTT, 1 mM MgCl2, pH 7.4. Protein was concentrated and further purified by size exclusion chromatography (Superdex 75, GE Healthcare) with buffer containing 25 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM MgCl2, 5% sucrose, pH 7.5. Protein purity was assessed by SDS-PAGE to be greater than 95%. Protein concentration was determined by UV absorbance at 280 nm using a NanoDrop (Thermofisher) and a molar extinction coefficient of 20,050 M-1 cm-1 for a 1:1 complex with GTP.
NMR Spectroscopy
For NMR measurements, the protein was concentrated to 0.3-0.4 mM, and 0.2 mM GTP, 5 mM DTT, and 10% D2O (v/v) were added immediately prior to data acquisition. NMR experiments were performed at 25 ˚C on a Bruker Avance Neo 600 MHz equipped with a cryoprobe. Assignments of the protein main-chain atoms were made using sensitivity enhanced versions of 2D 1H/15N-HSQC (Kay et al. 1992), 3D HNCO (Grzesiek et al. 1992b, Muhandiram et al. 1994), 3D HNCACO (Clubb et al. 1992), 3D HNCACB, CBCA(CO)NH (Grzesiek et al. 1992a) and (H)CC(CO)NH (Grzesiek et al. 1993). of protonated samples and 3D HN(CO)CA, HN(COCA)CB and HNCACB (Grzesiek et al. 1992b, Yamazaki et al. 1994) of per-deuterated samples.
All 3D experiments were collected using non-uniform sampling methods (Barna et al. 1987) using the Poisson-gap sampling schemes implemented by Hyberts et al. (Hyberts et al. 2010) and with a sampling density of 35-40%. Data were processed using NMRpipe (Delaglio et al. 1995) and NUS data were reconstructed using SMILE (Hyberts et al. 2012, Hyberts et al. 2014), and resonance assignments were determined using Ccpnmr Analysis v 2.4.2 (Vranken et al. 2005).