The DNA fragment encoding the PUB domain (residue 4–179) of RNF31 from Homo sapiens was obtained by PCR from Homo sapiens brains genomic DNA using the two primers 5’-GGGAATTCCATATGGAACGCGCGTTTCTG-3’ and 5’- CCGCTCGAGTTATTCCAGCAGTTG-3’ and cloned into the NdeI/XhoI-cleaved vector PGEX-4T-1. Recombinant vector was introduced into Escherichia coli BL21. The recombinant PUB domain possesses 176 amino acids. To mark the PUB domain with 13C, 15N, cells were cultured at 37℃ in M9 minimal medium (containing 2.5 g/L 99% 13C-glucose and 0.5 g/L 99% 15NH4Cl as the sole carbon and nitrogen source, respectively) to an OD600 of 1.0, and induced with 0.5 mM IPTG for 24 h. The induced cells were harvested by centrifugation at 5,000 g for 10 min, and resuspended in 35 mL Tris–NaCl buffer (20 mM Tris, 1 M NaCl, pH 7.3). Cells were lysed by high pressure and centrifuged at 12,000 rpm for 30 min at 4℃. GSTSep glutathione agarose resin was added to the supernatant and incubated for 24 h. Protein impurity is released at the end of incubation.30 mL Tris-NaCl (20 mM Tris, 300 mM NaCl, pH 7.3) suspension was then added to GSTSep glutathione agarbose resin, followed by TEV enzyme and 0.2 mM DTT, digested at 4℃ for 20 hours. The target protein was washed with 20 ml with buffer (20 mM Tris,300 mM NaCl, pH 7.3) and further purified by gel filtration with superdex 75 column (Amersham). The buffer passing through the gel column contains 200 mM NaCl and 20 mM Na2HPO4 (pH 7.0). The 0.5 mM prepared protein sample was dissolved in final NMR buffer containing 20 mM phosphate (pH 6.0) and 200 mM NaCl in 10:90% D2O: H2O.
All NMR experiments were recorded at 298 K on a Bruker AvanceIII 600 spectrometer equipped with a cryoprobe. The following spectra were recorded to obtain backbone and side-chain resonance assignments: 1H-15N HSQC, 1H-13C HSQC, CBCA(CO)NH, CBCANH, HNCA, HN(CO)CA, HNCO, HN(CA)CO, H(CCO)NH-TOCSY, HBHA(CO)NH-TOCSY, (H)C(CO)NH-TOCSY, HCCH-COSY and HCCH-TOCSY. NMR data processing was achieved using NMRpipe software (Delaglio et al. 1995), and then analyzed with Sparky (Goddard and Kneller 1993).
15N relaxation measurements were also performed on the Bruker AvanceIII 600 spectrometer using 15N-labeled PUB domain with a concentration of 0.6 mM at 298 K. The 1H-15N heteronuclear NOE experiment was recorded in an interleaved fashion, alternately with and without proton presaturation in the recovery delay. The proton saturation time, D1, was set to 5 s. 15N longitudinal relaxation times (T1) and transverse relaxation times (T2) were derived from eight spectra with different values for the relaxation delay (11, 61, 142, 243, 362, 523, 753, and 1147 ms) and seven relaxation delays (0, 17.6, 35.2, 52.8, 70.4, 105.6, and 140.8 ms), respectively. T1 and T2 values were extracted and fitted using a curve-fitting subroutine included in the program Sparky3 (Goddard et al. 1993).