Chemicals and Reagents Butyrolactone I and other compounds were provided by Dr

Chemicals and Reagents Butyrolactone I and other compounds were provided by Dr. Methods 2.1. Chemicals and Reagents Butyrolactone I and other compounds were provided by Dr. Jongheon Shin (Seoul National University, Seoul, Republic of Korea). The extraction and isolation were conducted as previously reported [25]. Butyrolactone I Appearance: Pale yellow amorphous solid, []+95 (1.0, EtOH), FT-IR (KBr, cm?1): 3179, 1763; 1H-NMR (DMSO-= 10.52 (brs, 1H), 9.92 (s, 1H), 9.12 (s, 1H), 7.50 (d, = 8 Hz, 2H aromatic H), 6.88 (d, = 8 Hz, 2H aromatic H), 6.53 (d, = 7.5 Hz, 1H), 6.47 (dd, = 8 Hz, 2 Hz, 1H), 6.37 (m, 1H), 5.01 (t, = 7.1 Hz, 1H), 3.74 (s, 3H), 3.36 (m, 2H), 3.00 (t, = 7 Hz, 2H), 1.62 (s, 3H), 1.53 (s, 3H); 13C-NMR (DMSO-= 169.8, 167.9, 157.8, 153.7, 138.0, 131.3, 130.8, 128.7, 128.3, 126.4, 123.1, 122.3, 121.0, 115.7, 114.0, 84.7, 53.4, 38.0, 27.5, 25.4, 17.4; HR-ESI-MS: strain. The cells were grown at 37 in Luria Broth media containing 30 g/mL kanamycin and induced by 0.5 mM isopropyl 1-thio–d-galactopyranoside at an OD600 of 0.6 and then incubated for additional 20 h at 20 . The cells were harvested Parsaclisib by centrifugation at 6000 for 10 min and lysed by sonication in buffer A (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 5 mM imidazole, 10% glycerol, and 1 mM TCEP) containing 1 mM phenylmethanesulfonylfluoride. The lysates were centrifuged at 35,000 for an hour and the supernatants were filtered with a 0.45 m syringe filter device (Sartorius, G?ttingen, Germany). For affinity chromatography, they were loaded onto 5 mL HiTrap chelating HP column (GE Healthcare, Chicago, IL, USA) that was charged with Ni2+ and equilibrated with buffer A. Upon eluting with linear gradient of buffer B (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 300 mM imidazole, 10% glycerol, and 1 mM TCEP), PPAR LBD was Parsaclisib eluted at an imidazole concentration of 50C100 mM. After the eluted protein was desalted using HiPrep Desalting column 26/10 (GE Healthcare) to buffer C (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 10% glycerol, and 1 mM TCEP), the protein was treated with thrombin (Sigma-Aldrich) for the cleavage of His6-tag at 1 unit/mg and incubated at 4 overnight. The His6-tag-cleaved PPAR LBD was purified by passing through the Ni2+ charged HiTrap chelating HP column (GE Healthcare) to remove His6-tag or uncleaved His6-tagged target proteins, followed by gel Parsaclisib filtration chromatography column, HiLoad 16/600 Superdex 200 pg (GE Healthcare), that was previously equilibrated with buffer C. For crystallization, the PPAR LBD was concentrated to 15.5 mg/mL using an Amicon Ultra-15 Centrifugal Filter Unit (Merck Millipore, Darmstadt, Germany). 2.6. Crystallization The ligand-free PPAR LBD crystals were grown by the sitting-drop vapor diffusion method at 22 by mixing 0.5 Rabbit polyclonal to DYKDDDDK Tag L each of the purified protein sample and a crystallization solution containing 1.4 M sodium citrate tribasic dihydrate (Hampton Research, Aliso Viejo, CA, USA) Parsaclisib and 0.1 M HEPES pH 7.5. The crystals suitable for data collection were grown in the presence of micro-seeds that were made from the initial crystals using Seed Bead Kits (Hampton Research) according to the manufacturers instructions. The cubic-shaped crystals with a dimension of approximately 0.2 mm 0.2 mm 0.2 mm were obtained within a few days. For butyrolactone I-bound PPAR LBD, butyrolactone I was completely dissolved in 100% DMSO at 100 mM concentration and was soaked into ligand-free PPAR LBD crystals with 1:5 molar ratio containing 1% ([36]. The structures were refined by iterative manual buildings in [37] and [38] in the CCP4 program suite. All refinement steps were monitored using an Rfree value [39] based on the independent reflections and the reliability of refined models was evaluated using [40]. The statistics of data collection and refinement are summarized in Table 1. Table 1 Statistics for the data collection and model refinement. = hi|I(h)iC|/hiI(h)i, where I(h) is the intensity of reflection h, h is.